final-23rd biotech colloquium 2015 tentative programme · 2020-03-14 · 23rd biotech colloquium...

23rd Biotech Colloquium 2015

Faculty of Biotechnology & Biomelecular Sciences

Universiti Putra Malaysia

43400 UPM Serdang

Selangor Darul Ehsan


Faculty of Biotechnology & Biomolecular Sciences

23232323rdrdrdrd Biotech ColloquiumBiotech ColloquiumBiotech ColloquiumBiotech Colloquium

Semester 2 (2014/2015)Semester 2 (2014/2015)Semester 2 (2014/2015)Semester 2 (2014/2015)

Organized by:

Deputy Dean of Research and Postgraduate Studies Office

& Department of Biochemistry, FBSB

8th & 10th June 2015

Biotech 1, FBSB


Abstracts printed here have not been edited. The contents of

abstracts are the sole responsibility of the authors.


Memo



Description of the images on the frontcover:

Main circle: Disciplines of Biotechnology

1st circle: Foods science & nutritions in biotechnology

2nd circle: Marine microalgae (Chaetoceros calcitrans) extract induces apoptosis

3rd circle: Candida rugosa lipase immobilised on mica-clay as biocatalyst

4th circle: Sensitivity test of plant cell culture (Citrullus lanatus) against herbicide

“Every human being is a logician: Some use logic well & some use it

poorly. The Qur’an asks us to use logic: To think, reflect, reason &

understand”

– Hamza Yusuf -


Editorial BoardEditorial BoardEditorial BoardEditorial Board

AdvisorAdvisorAdvisorAdvisor Assoc. Prof. Dr. Norazizah Shafee

CoordinatorCoordinatorCoordinatorCoordinator Dr. Uswatun Hasanah Zaidan

Working CommitteeWorking CommitteeWorking CommitteeWorking Committee Pn. Norazian Zainal

Pn. Suryani Ahmad

i


66

A COMPARATIVE EVALUATION OF THE CHARACTERISTICS AND APPLICATION POTENTIAL OF BIOLOGICALLY AND CHEMICALLY

SYNTHESIZED SILVER AND MAGNETIC NANOPARTICLES

Patricia Jayshree Samuel Jacob1, Mas Jafri Masarudin1, Prof. Dr. Mohd Zobir Hussein2

Department of Cell and Molecular Biology, Faculty of Biotechnology and

Biomolecular Sciences1, Universiti Putra Malaysia, Institute of Advanced Technology, Universiti Putra Malaysia2.

It is undeniable that nanotechnology has made great strides in the recent decade find-ing application in nearly every pertinent facet of life. Nanoparticles, the workhorses of nanotechnology have established a comfortable niche in therapeutics, diagnostics, electronics and communication and in environmental remediation. Currently, these nanoparticles are generated using chemical methods of synthesis, which has high cost implications and leave behind toxic residues, making it undesirable in biomedical applications. Biological synthesis would be a welcome alternative to this end. Thus, this project is proposed to produce magnetic nanoparticles using locally isolated mi-croorganisms and to compare its effectiveness with chemically synthesized ones in selected biomedical applications. Soil and sediment samples will be collected from various sites and screened for microorganisms that demonstrate the extracellular pro-duction of silver or magnetic nanoparticles. These biosynthesized nanoparticles will then be characterized for their structural and optical properties using FESEM, HRTEM, X-Ray Diffraction, FTIR, UV spectrophotometer and DLS. Biosynthesized silver and magnetic nanoparticles would be investigated for cytotoxicity and designed as a therapeutic carrier in cell targeting and drug delivery systems using the core-shell design. The results of this investigation would provide valuable data on the effective-ness of biosynthesized magnetic and silver nanoparticles in both in vivo and in vitro drug delivery systems and the possibility of replacing their chemical counterparts which are detrimental to the environment and incur a high cost, in these biomedical applications. Keywords: Magnetic silver nanoparticles, biosynthesis, microorganisms, drug delivery systems


65

DEVELOPMENT OF A GENETICALLY ENGINEERED GAMMA-TERPINENE SYNTHASE OF Plectranthus amboinicus (BANGUN-BANGUN) FOR

THYMOQUINONE PRODUCTION

Nur Suhanawati Ashaari1,4, Raha Abdul Rahim1, Suriana Sabri2, Lai Kok Song1, Adelene Song Ai Lian1, Mohd Hairul Ab. Rahim3, Janna Ong Abdullah1

Department of Cell & Molecular Biology1, Department of Microbiology2, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; Faculty of Industrial Sciences and Technology3,

Universiti Malaysia Pahang, 26300 Gambang, Kuantan, Pahang, Malaysia; Malaysia Genome Institute4, Jalan Bangi, 43000 Kajang, Malaysia.

Plectranthus amboinicus is an aromatic medicinal plant, whose essential oils contain gamma-terpinene, a monoterpene compound that serves as a precursor for thymoquinone synthesis. Thymoquinone is the bioactive constituent of the black seed, Nigella sativa, oil with a wide range of medical applications and has been reported as a promising anti-cancer drug. Despite the huge applications of thymoquinone, extraction of this compound from black seed remains a technical challenge for large scale production. Alternatively, microbial heterologous biosynthesis of thymoquinone precursor, the gamma-terpinene, catalysed by P. amboinicus gamma-terpinene synthase would be a promising alternative as it may readily be scaled up for thymoquinone commercial production. To investigate the biosynthetic pathway and regulation of P. amboinicus gamma-terpinene, identifica-tion, characterisation and heterologous expression of gamma-terpinene synthase will be conducted. Full-length cDNA of gamma-terpinene synthase will be isolated using Rapid Amplification of cDNA Ends (RACE) followed by cloning and functional expression of the gamma-terpinene synthase in the Pichia pastoris system. The recombinant gamma-terpinene synthase will be characterised using enzyme assay and gas chromatography-mass spectrometry (GC-MS), and the assay products will be identified by comparing the mass spectra to those in the National Institute of Standards and Technology (NIST) library database. The overproduction of gamma-terpinene will be conducted by co-overexpression of gamma-terpinene synthase and 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in eukaryote mevalonate pathway. The expected outcomes of this research would be the full-length transcript of P. amboinicus gamma-terpinene synthase and development of Pichia pastoris cell factory for the production of gamma-terpinene compound.

Keywords: Plectranthus amboinicus, gamma-terpinene, gamma-terpinene synthase, thymoquinone, Pichia pastoris


ii

ContentsContentsContentsContents

Editorial Board ……………………………………i

Tentative Programme …………………iii - xvii

Abstracts ………………………………………1 – 66


iii

TENTATIVE TENTATIVE TENTATIVE TENTATIVE

PROGRAMMEPROGRAMMEPROGRAMMEPROGRAMME


64

USING TOBACCO CELL SUSPENSION CULTURE TO FUNCTIONALLY EXPRESS AN ORCHID TERPENOID BIOSYNTHESIS PROTEIN

Siti Nabilah Yusoff, Mohd Puad Abdullah, Parameswari Namasivayam,

Janna Ong Abdullah

Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,

Selangor Darul Ehsan, Malaysia.

Plants generally produce terpenoids via two pathways: the mevalonate pathway (MVA) and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Terpenoids are vital for plants as they are a large and diverse group of metabolites that has different functions and roles such as membrane biogenesis, respiration, control of growth and development and act as defence system in plants. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) has been identified as a rate limiting step in terpenoid biosynthesis and a key regulator in the mevalonate pathway. In previous studies, a cDNA transcript of this HMGR (designated as VMPHMGR) was isolated from Vanda Mimi Palmer (Orchidaceae), and successfully expressed in two prokaryotic systems. However, it is unknown whether it is functional when express in a heterologous eukaryotic system. Thus, the aim of this study is to functionally express this terpenoid biosynthesis protein, designated as VMPHMGR, in Nicotiana tabacum cell suspension culture. This VMPHMGR will be cloned into the expression vector pMDC32 by using Gateway Technology through BP/LR recombination. This pMDC32 vector contains a kanamycin resistance gene, a hygromycin resistance gene, a nos terminator and a doubled CaMV35S promoter driving the VMPHMGR expression. This pMDC32::VMPHMGR will then be introduced into Agrobacterium tumefaciens LB4404 for subsequent co-cultivation with N. tabacum cell suspension culture induced from internode explant. Functional characterization of the expressed VMPHMGR will be carried out via enzymatic assay, gas chromatography-mass spec-troscopy, and Western blot. Keyword: Mevalonate pathway, Vanda Mimi Palmer, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, Agrobacterium tumefaciens, tobacco cell suspension culture


63

ESTABLISHMENT AND ENHANCEMENT OF BETALAINS PRODUCTION IN Hylocereus polyrhizus CALLUS CULTURE

Nor Fadzliana Abu Faizal1, Rogayah Sekeli3, Noor Azmi Sahaluddin2, Janna Ong Abdullah1

Department of Cell & Molecular Biology1, Department of Biochemistry2,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,43400 UPM Serdang,Selangor Darul Ehsan, Malaysia;

Malaysian Agricultural Research and Development Institute (MARDI)3, Kuala Lumpur.

Advancement in technologies makes it possible to synthesize synthetic colorants to impart colors in food to make them more attractive and appetizing. Self-consciousness of consumers on healthcare causes a shift of using synthetic to natural colorants in the food and beverages industries. Betalain is a secondary metabolite pigment, which is water-soluble and contains clusters of nitrogen that play a role in producing betacyanin and betaxathin pigments. Betalain has become a promising food colorant in food industries. Maximizing betalain yield in food industries is crucial since it is highly unstable during processing and storage. The aim of this study is to enhance betalain production in red pitaya (Hylocereus polyrhizus) callus through elicitation. Pigmented calli were induced on Murashige and Skoog basal salt medium supplemented with different combinations of desig-nated phytohormones [α-naphthalene acetic acid (NAA), thidiazuron (TDZ) and 6-benzylaminopurine (BAP)] and elicitors [L-tyrosine, methyl-jasmonate (MeJa) and ascorbic acid]. Calli produced were reddish and friable. Spectrophotometry read-ings of the extracts from the induced pigmented calli revealed that addition of L-tyrosine and MeJa helped to boost the betalain content to about 5.4-fold higher than the control (calli grown without elicitors or phytohormones). HPLC analysis of the calli extracts showed four compounds, namely betaxanthin, betacyanin, phenolic acid and flavonoid. High contents were detected in the pigmented calli with betacya-nin at 1.2-fold, betaxanthin at 2.3-fold, flavonoids at 5-fold and phenolic acids at 3-fold higher than the control. This study shows the callus culture induced from the red dragon fruit.

Keywords: Betalains, red pitaya, callus culture, elicitors, phytohormones.


iv

8th June 2015

Room : Bilik Latihan 1.3

Chairperson: Siti Nurbaya Oslan

Evaluators : Abu Bakar Salleh Amir Syahir Amir Hamzah

Mohd Arif Syed Mohd Shukuri Mohamad Ali

Norhani Abdullah Noor Azmi Shaharuddin

Maziah Mahmood Siti Aqlima Ahmad

Syahida Ahmad Uswatun Hasanah Zaidan

Nur Adeela Yasid

Time Name/ Matric No./

Course Title/Supervisor

Page

No.

10.00 - 10.30 Suria bt. Johari GS39767 (BBS5903)

Antioxidant properties in different plant parts of a wild orchid Dendrobium crumenatum (Maziah Mahmood)

1

10.30 - 11.00

Tanko AbuBakar Sadiq GS38166 (BBS5903)

Antioxidative and antidiabetic potentials of commercial and traditional bran extracts of some Malaysian rice varieties (Mohammed Nazrim Marikkar)

2

11.00 - 11.20

Nur Iznida bt. Mahyon GS41304 (SPS5903)

The use of alcohol oxidase promoter in the expression of recombinant proteins without the presence of methanol (Siti Nurbaya Oslan )

3

11.20 - 11.40

Amirah Nor bt. Kamarudin GS41473 (SPS5903)

Endophytic colonisation of Hendersonia toruloidea and its effect on the expression of thiamine biosynthesis genes (THI1/THI4 and THIC) in oil palm (Elaies guineensis) (Zetty Norhana Balia Yusof)

4

11.40- 12.00 Abu Mary Ladidi GS41882 (SPS5903)

Enhancing Pichia guilliermondii strain so as a host for heterologous proteins production (Abu Bakar Salleh)

5


v

Time Name/ Matric

No./Course Title/Supervisor

Page

No.

2.00 - 2.20

Nurul Izzati bt. Mohd Rosli GS42501 (SPS5903)

Deciphering protein-solvent interaction of a cold adapted-organic solvent tolerant lipase(Abu Bakar Salleh)

6

2.20 - 2.40

Muhammad Afiq b. Abdul Halim GS42622 (SPS5903)

Functional characterization of oil palm’s gibberelic acids (GA) genes in Arabidopsis thaliana via RNAi and overexpressor technologies (Noor Azmi Shaharuddin)

7

2.40 - 3.00

Adekola Khadijat Adetola GS42950 (SPS5903)

Study of anti-amylase, anti-glucosidase, anti-glycation and antioxidant potentials of coconut testa (mature and tender) and varieties of beans seed coat (Mohammed Nazrim Marikkar)

8

3.00 - 3.30 Chan Seow Neng GS33884 (BBS6904)

Molecular cloning and recombinant expression of cysteine protease inhibitor (phytocystatin) and kunitz-trypsin inhibitor (KTI) from turmeric, Curcuma longa (Noor Azmi Shaharuddin)

9

3.30 - 4.00

Arilla Sri Masayu bt. Abd. Rahim GS34152 (BBS6904)

Use of mini protein that mimics uricase in the development of nanowire based biosensor (Abu Bakar Salleh)

10

8th June 2015


Chairperson: Uswatun Hasanah Zaidan

Evaluators : Abu Bakar Salleh Amir Syahir Amir Hamzah




Syahida Ahmad Siti Nurbaya Oslan

Nur Adeela Yasid


62

DEVELOPMENT OF PAPAYA (CARICA PAPAYA L. CV. ‘EKSOTIKA’) WITH POTENTIAL RESISTANCE TO DIEBACK DISEASE

Roslinda A. Razak1 , Rogayah Sekeli2, Noor Azmi Shaharuddin3 ,

Janna Ong Abdullah 4*

1 Department of Microbiology, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, 43400 UPM Serdang, Malaysia

2 Malaysian Agricultural Research and Development Institute (MARDI), P.O. Box 12301, 50774 Kuala Lumpur, Malaysia

3 Department of Biochemistry, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, 43400 UPM Serdang, Malaysia

4Department of Cell & Molecular Biology, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan,

43400 UPM Serdang, Malaysia Papaya is a commercially valuable fruit crop in Malaysia. There are many varieties of papaya grown in Malaysia such as ‘Eksotika’, ‘Sekaki’, ‘Hong Kong’ and ‘Solo’. One of the major diseases that have potential to cripple the Malaysian papaya industry is the papaya dieback disease. This disease could destroy the whole papaya plant and result in 100% fruit production losses. Papaya dieback was re-ported in 2003 in Batu Pahat, Johore and eventually spread to Perak, Selangor, Penang, Kedah and Pahang. The causal agent has been determined as Erwirnia mallotivora of the family Enterobacteriaceae. Currently the only solution to control the disease is to destroy the infected plant. Alternative way is via genetic engineer-ing to develop an ‘Eksotika’ papaya variety that is resistant to the dieback. Hence, the main objective of this study is to genetically transformed papaya calli tissue with a gene cassette harboring the acyl-homoserine lactonase (AHL-lactonase) gene and phosphomannose isomerase (pmi) as selectable marker via Agrobacterium-mediated transformation. A total of 1090 one-month-old embryogenic calli of ‘Eksotika’ papaya were transformed with the above target genes and the transformed calli were selected on medium containing 30 g/L mannose for five months. After 5 months on selection, the putative transformed calli were regenerated on De Fossard salts medium supplemented with 0.2 mg/L NAA and 0.2 mg/L BAP. Presence of the transgenes in the regenerated transformed calli was verified using PCR. Putative transgenic plants were further analyzed by challenging them with the Erwinia mallotivora. Verification of putative transgenic plants via real-time PCR is still on going. Successful establishment of the non-antibiotic selection system, mannose, is useful in eliminating antibiotic usage for future transformation of ‘Eksotika’ papaya targeting at other commercial viable traits besides resistance to dieback disease.


61

ISOLATION OF THE PHENYLALANINE AMMONIA-LYASE PROMOTERS FROM OIL PALM AND ANALYSIS OF THE PROMOTER ACTIVITIES IN ARABIDOPSIS

Yusuf Yu Lok Chong1, Janna Ong Abdullah1, Noor Azmi Shaharuddin2, Idris Abu

Seman3, Mohd Puad Abdullah1

Department of Cell and Molecular Biology1, Department of Biochemistry2, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia.

Malaysia Palm Oil Board (MPOB), No.6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor2

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) is encoded by a multigene family with different copy number in different species. It catalyzes the deamination of phenylalanine to trans-cinnamic acid, the rate limiting step of the phenylpropanoid pathway. The pathway is largely responsible for the synthesis of phenylpropanoid precursors required for plant structural development and defense. As a family, the expression of the PAL genes is expected to be coordinately regulated through their promoter activities. To get insight into the gene regulation in oil palm, this study was initiated. To date, there is no report on the PAL promoter activities in oil palm, preventing in-depth studies of oil palm structural development and adaptation. Therefore, this study was carried out to isolate the promoter of EgPAL1 and EgPAL5, and analyze their activities using transgenic approach. The promoter of EgPAL1 and EgPAL5 were isolated using inverse-PCR and approximately 2.3 kb of the 5’-flanking region of EgPAL1 and EgPAL5 were fused to the gusA gene and transformed into Arabidopsis thaliana through agrobacteria-mediated method. Several cis-acting elements related to lignin biosynthesis and stress response were identified in the pro-moters of EgPAL1 and EgPAL5. The 5’-flanking regions of EgPAL1 and EgPAL5 drove the expression of the gusA gene in several organs of the transgenic plants including root, leaf, stem, bud, flower and sillique. Although the expression of gusA can be detected in various organs, but the magnitude was negatively correlated with the growth stage of the organs. The promoters also responded differently towards different chemical treatments. The results reflected the involvement of PAL in plant development and in response to environmental factors. Such a role is reflected in the way the various PAL isoforms operate in oil palm during plant development and in response to different environmental conditions.

KEYWORDS: Phenylalanine ammonia-yase, Promoter study, Oil palm


vi

10th June 2015


Chairperson: Amir Syahir Amir Hamzah

Evaluators : Abu Bakar Salleh Uswatun Hasanah Zaidan




Syahida Ahmad Siti Nurbaya Oslan

Nur Adeela Yasid



Page

No.

10.00 - 10.30 Teh Chui Yao GS34772 (BBS6904)

Biochemical and physiological responses of selected Malaysian rice cultivars treated with exogenous osmoprotectants under salinity stress (Maziah Mahmood)

11

10.30 - 11.00

Karamba Kabiru Ibrahim GS37491 (BBS6904)

Biodegradation of cyanide by free and immo-bilized cells of Serratia marcescens isolate AQ07 (Siti Aqlima Ahmad)

12

11.00 - 11.20

Mayaki Fatima Gogo GS41883 (SPS5903)

Promoter prediction system for recombinant lipase expression in Pichia pastoris (Siti Nurbaya Oslan)

13

11.20 - 11.50

Alafiatayo Akinola Adekoya GS34365 (BBS6904)

Total antioxidant and flavonoid profiling of selected Zingiberaceae (ginger) rhizomes and anti-skin aging effects (Maziah Mahmood)

14

(Presented

earlier)

Ibrahim Yusuf GS42950 (BBS6904)

Biotechnological application of newly isolated feather degrading Bacillus sp khayat in keratinase, protein hydrolysate production and biodegradation of feather wastes (Siti Aqlima Ahmad)

15


vii

8th June 2015


Chairperson: Mohamad Faizal Ibrahim

Evaluators : Mohd Ali Hassan Phang Lai Yee

Suraini Abd. Aziz Arbakariya Ariff

Rosfarizan Mohamad Norhayati Ramli

Murni Halim Mohamad Faizal Ibrahim

Hidayah Ariffin Umi Kalsom Binti Md Shah

Ahmad Muhaimin Roslan Helmi Wasoh @ Mohamad Isa

Lai Oi Ming Mohd Shamzi Mohamed

Time Name/ Matric


Page

No.

10.00 - 10.30

Muhammad Azman b. Zakaria GS36199 (BBS5903)

yqiG Pseudogene of Escherichia coli influenced hydrogen production (Mohd Ali Hassan)

16

10.30 - 11.00

Muhammad Nazmir b. Mohd Warid GS36250 (BBS5903)

Optimization of superheated steam treatment for surface modification of oil palm biomass fibers prior to biocomposite production (Hidayah Ariffin)

17

11.00- 11.30

Mohamad Farhan b. Mohamad Sobri GS37445 (BBS5903)

Molecular cloning of codon optimized β-glucosidase gene into Saccharomyces cerevisiae for enhancement of bioethanol production from oil palm empty fruit bunch hydrolysates (Norhayati Ramli)

18

11.30 - 11.50 Reema E. Elabid GS41058 (SPS5903)

Optimisation of bacteriocin production by isolated lactic acid bacteria grown in milk (Nor'Aini Abdul Rahman )

19


60

MUTAGENESIS AND IMPACT OF THE SIGNAL PEPTIDE SPK1 FOR SECRE-TION OF HETEROLOGOUS PROTEINS IN LACTOCOCCUS LACTIS

Nur Aqlili Riana Alias1, Adelene Song Ai Lian1, Sieo Chin Chin2,

Raha Abdul Rahim1

Department of Cell Biology and Molecule1, Department of Microbiology2, Faculty of Molecular Bioscience and Biotechnology, Universiti Putra Malaysia.

Lactococcus lactis, a generally regarded as safe (GRAS) probiotics which has been widely used in fermentation and dairy industry, are recently been utilized as cell factories for heterologous protein production and vaccine delivery. Secretion of heterologous proteins is mostly preferred than intracellular protein production due to its simpler downstream purification, better interactions with targets and better quality of protein due to cell wall associated chaperone for correct folding. However, the yields of secreted heterologous proteins were often reported to be insufficiently low. Selection of signal peptide is crucial for protein production and secretion optimization. Currently, native USP45 is the most successful signal peptide (SP) used for secretion system in L. lactis. Our recent study has proven that a novel SP from Pediococcus pentasaceus, SPK1 elicits higher secretion efficiency (SE) of 49% compared to that of USP45. Therefore, we aim to conduct site-directed mutagenesis on the signal peptide SPK1 with the objective of increasing SE. Furthermore, combination of other strategies including optimizing the propeptide region of SPK1 and creating HtrA mutant will be performed. The improved secretion system in L. lactis will be used for antigen production and presentation of a K-ras peptide epitope against metastatic colorectal cancer (mCRC).Ten SPK1 mutants with different amino acid mutations in N, H and C regions will be targeted. Vectors with different SPK1 mutants/propeptides fused to Staphylococcus nuclease (Nuc1) gene with His-tag sequence at the N-terminal will be constructed and the NUC-1 secretion will be quantified by zymogram and ELISA. The best SPK1mutant/propeptide combination with the highest SE will be fused to mCRC K-ras antigen and expressed before orally administered into mice for in vitro and in vivo immuno-genicity assessment. The outcome of the study would be an efficient GRAS lactoc-cocal host for targeted high immune response as a biological therapy alternative for metastatic colon cancer treatment. KEYWORDS: Signal peptides, Lactococcus lactis, secretion, mCRC K-ras epitope


59

TRANSCRIPTOME PROFILING AND GENERATION OF GENIC-SSR MARKERS FOR CYNOMOLGUS MACAQUE (MACACA FASCICULARIS) FROM

PENINSULAR MALAYSIA

Joey Ee Uli1, Tan Soon Guan1, Christina Yong Seok Yien2, Yeap Swee Keong3, Noorjahan Banu Mohamed Alitheen1, Nurulfiza Mat Isa1

Department of Cell and Molecular Biology1, Faculty of Biotechnology and

Biomolecular Sciences, Department of Biology2, Faculty of Science, Laboratory of Vaccines and Immunotherapeutics3, Institute of Bioscience,

Universiti Putra Malaysia The cynomolgus macaque (Macaca fascicularis) is an extensively and critically utilized nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic and genetic information of the cynomolgus macaques are useful to further expand its use for research in various fields, however, there is presently a shortage of genomic information and intergenic-SSR markers for the Malaysian population of the cynomolgus macaque, and also no genic-SSR markers based on transcriptomic data have been developed for this population yet. This study aims to collect and interpret transcriptomic data, to identify microsatellite regions within the transcriptomic dataset of the cynomolgus macaque, as well as to develop genic-SSR markers from the transcriptomic data. These markers will then be tested and validated for their utility in population studies. Total RNA will be extracted and converted to complementary DNA which will be subjected to next-generation sequencing to obtain whole transcriptomic data. The raw transcriptomic data will be filtered and assembled, and further analyses will be carried out. From the transcriptomic data, microsatellite regions will be identified using bioinformatics tools and genic-SSR markers will be developed based on unique microsatellite regions. Potential primers will be synthesized, and their effectiveness will be screened and validated on selected wild cynomolgus macaque samples from several Malaysian populations. Novel transcriptomic data will potentially enrich the presently incomplete cynomolgus macaque genomic database. The large amount of transcriptomic data also provides future research potentials, such as novel gene discovery and gene mapping. The screening of newly developed genic-SSR markers will identify primers that have the potential to be utilized in future population studies and management of wild cynomolgus macaques of Peninsular Malaysia that are facing habitat destruction due to human development. Up-to-date genetic information will ensure the maintenance of the genetic diversity of wild cynomolgus macaque populations.


viii

8th June 2015


Chairperson: Ahmad Muhaimin Roslan




Mohd Zulkhairi Mohd Yusoff Mohamad Faizal Ibrahim

Hidayah Ariffin Umi Kalsom Binti Md Shah

Helmi Wasoh @ Mohamad Isa Murni Halim




Page

No.

2.00 - 2.30 Fairuzana bt. Jaafar GS37717 (BBS5903)

Effects of mono and diunsaturated fatty acids on production of rhamnolipids by locally isolated Pseudomonas aeruginosa RS6 (Helmi Wasoh @ Mohamad Isa)

20

2.30 - 3.00 Nur Idayu bt. Zahari GS38037 (BBS5903)

Xylanase production by Penicillium oxalicum T3.3 using rice staw as substrate for biobleaching of bamboo pulps (Umi Kalsom Md Shah)

21

3.00 - 3.30

Farrah bt. Mohamad Hatta GS39032 (BBS5903)

Aeration requirements for the cultivation of oil palm (Elaeis guineensis jacq.) cell suspension cultures in stirred-tank bioreactor (Arbakariya Ariff)

22

3.30 - 3.50

Nurul Hanisah bt. Badrul Hisham GS40893 (SPS5903)

Characterization and optimization of biosurfactant from used cooking oil by local isolates for heavy metals removal (Suraini Abd. Aziz)

23

(Presented earlier)

Dhurga Devi A/P Rajaratanam GS38734 (SPS5903)

Control led hydrolysis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for oligoester production (Hidayah Ariffin)

24


ix

10th June 2015


Chairperson: Mohd Zulkhairi Mohd Yusoff




Mohd Zulkhairi Mohd Yusoff Murni Halim

Hidayah Ariffin Umi Kalsom Md Shah

Helmi Wasoh @ Mohamad Isa Ahmad Muhaimin Roslan




Page

No.

10.00 - 10.20 Yong Xiou Shuang GS40329 (SPS6903)

Development of new catanionic surfactant vesicles for drug delivery applications(Phang Lai Yee)

25

10.20 - 10.40

Nahrul Hayawin bt. Zainal GS42267 (SPS6903)

Simultaneous carbonization and activa-tion of oil palm kernel shell for activated carbon production (Suraini Abd. Aziz)

26

10.40 - 11.00

Nurshazana bt. Mohamad GS42909 (SPS5903)

Enzyme-assisted extraction of essential oil from pineapple peels using cellulase (Suraini Abd. Aziz)

27


58

DEVELOPMENT OF EXPRESSED MARKERS FOR GRACILARIA SPECIES WITH HIGHER YIELD AND QUALITY OF AGAR

Wei-Kang Lee 1, Janna Ong Abdullah 1, Parameswari Namasivayam 1,

Chai-Ling Ho1

Department of Cell and Molecular Biology 1, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM-Serdang,

Selangor, Malaysia. Agar is a sulfated polysaccharide produced by red algae (Rhodophyta), with the genus Gracilaria contributing more than half of the global agar production. In Malaysia, G. changii was reported to have a higher agar content and good gel strength compared to other Gracilaria species, making it a potential commercial source in agar industry. The main objective of this project is to develop expressed markers for the screening of Gracilaria species with good agar yield and quality. G. changii and G. salicornia from Morib, Selangor, were collected during dry and raining seasons, and subjected to sulfate deprivation for 5 days. Slight increments of agar yield, gel strength, gelling and melting temperatures were recorded in seaweeds upon treatments. mRNA sequencing of G. changii and G. salicornia sam-ples produced 10.45 x 107 and 7.27 x 107 paired end reads, respectively. These short reads were de novo assembled into 14,605 and 22,639 unigenes for G. changii and G. salicornia, respectively. By mapping mRNA reads to the unigenes, 1636 up-regulated (>2-fold) and 2185 down-regulated (>2-fold) genes were identi-fied in sulfate-starved G. changii, compared to that in the untreated G. changii. In G. saliconia, 5439 genes were up-regulated (>2-fold) and 4611 genes were down-regulated (>2-fold) in sulfate-starved samples compared to that in the untreated sample. Some of these differentially expressed genes (DEGs) were found to have a major role in sulfur assimilation, biosynthesis of sulfur-containing metabolites, carbon metabolism, photosynthesis and synthesis of extracellular/cell wall proteins. A total of 17 DEGs were verified with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), with 16 of them having similar expression profiles to those demonstrated by mRNA sequencing. Based on literature search and DEGs from sulfate deprivation treatments, 18 candidate markers were selected and vali-dated on different G. changii and G. salicornia samples, with 10 of them showing significant correlation (P<0.05) between gene expression and agar yield/gel strength. These expressed markers can be used to estimate agar yield and gel strength in Gracilaria samples by assessing the level of gene expression of the marker genes. KEYWORDS: Agar, agar yield, expressed markers, gel strength, Gracilaria


57

REGULATION OF PROTEIN S-NITROSYLATION FOR EFFECTIVE CONTROL OF FUSARIUM WILT IN BANANA

Nurul Amirah Mahamad1, Noor Azmi Shaharuddin2, Amalia Mohd. Hashim1,

Noor Baity Saidi1

Department of Cell and Molecular Biology1; Department of Biochemistry2; Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia.

Nitric oxide (NO) has been shown to be an effector for defense signaling in plant response to pathogen infection. Protein S-nitrosylation, a redox-related modification of cysteine thiols by NO became an important aspect in a broad spectrum of plant defense. S-nitrosylation is a precisely targeted and rapidly reversible post-translational modification that serves as an on/off switch for protein function during cell signaling. The emerging picture shows that, in many cases, disease develop-ment correlates with hypo- or hyper-S-nitrosylation of specific protein targets. Until today, the knowledge of S-nitrosylated target proteins in banana are totally unknown. This research will embark on the analysis of proteins undergoing S-nitrosylation following pathogen challenge, specifically during banana-Fusarium oxysporum interaction. In the absence of pathogen challenge, treatment of protein extracts from banana with an S-nitrosylating agent, S-nitrosoglutathione resulted in an increased level of S-nitrosothiols in a concentration-dependent manner compared to untreated sample. This result provides a strong basis for further analysis on S-nitrosylated proteins in banana plants challenged with virulent and avirulent Fusarium oxysporum where a number of proteins are expected to be S-nitrosylated. Subsequent identification of specific plant protein being a target for S-nitrosylation is important for effective control of plant disease specifically Fusarium wilt disease in banana. KEYWORDS: Nitric oxide; S-nitrosylation; banana; Fusarium oxysporum


x

8th June 2015


Chairperson: Mohd Termizi Yusof

Evaluators : Khatijah Mohd Yusoff Muhajir Hamid

Tan Wen Siang Norazizah Shafee

Sieo Chin Chin Nurhidayah Roslan

Raja Noor Zaliha Raja Abd Rahman

Janna Ong Abdullah Shuhaimi Mustafa

Asilah Tajudin Amalia Mohd. Hashim



Page

No.

10.00 - 10.30 Jiivittha Veno GS37107 (BBS5903)

Crystal structural analysis of concise chalcone synthase from Boesenbergia rotunda (BRCHS) (Raja Noor Zaliha Raja Abd Rahman)

28

10.30 - 11.00 Lee Pei Lee Angel GS37211 (BBS5903)

The potential of endophytic Trichoderma as a biofungicide for ganoderma basal stem rot infection (Mohd Termizi Yusof)

29

11.00 - 11.20

Fairuz Nabila bt. Zulkifli GS42430 (SPS5903)

The in-vitro and in-vivo study of stingless bee honey (Heterotrigona itama) on learning and memory development in mice (Nurhidayah Roslan)

30

11.20- 11.40 Bashirat Bello GS42276 (SPS5903)

Extraction and characterization of soluble non-digestible polysaccharides from coconut kernel cake (ckc) and palm kernel cake (pkc) as potential prebiotics (Shuhaimi Mustafa)

31

11.40 - 12.10

Roya Biabanik-hankahdani GS29863 (BBS6904)

Application of hepatitis B virus nano-particles as a nanocarrier for drug delivery (Tan Wen Siang)

32


xi

8th June 2015


Chairperson: Nurhidayah Roslan



Sieo Chin Chin Asilah Tajudin


Mohd Termizi Yusof Suriana Sabri

Amalia Mohd. Hashim Shuhaimi Mustafa



Page

No.

2.00 - 2.30

Mariam Dayana bt

Mohd Taha

GS37103

(BBS5903)

Discovering antagonistic biological agents from Carica papaya for potential control of papaya dieback disease (Amalia bt. Mohd. Hashim)

33

2.30 - 3.00

Nadila bt. Hanafee

GS38912

(BBS5903)

Investigation and growth optimization of locally isolated phenol-degrading fungi (Mohd Termizi Yusof)

34

3.00 - 3.20

Hartini bt. Ahmad

Sani

GS41249

(SPS5903)

Structural investigation of the C-terminal residues in thermostable L2 lipase (Fairolniza Mohd Shariff)

35

3.20 - 3.40

Siti Norhasmah bt.

Ishak

GS40877

(SPS6903)

Redesign of thermostable T1 lipase based on space-grown crystal structure (Raja Noor Zaliha Raja Abd Rahman)

36


56

NMR STRUCTURE, INTERACTION AND DYNAMICS OF HYPOTHETICAL CSOR

PROTEIN OF GEOBACILLUS ZALIHAE

Ashwaani Mangavelu1, 2, Yahaya M. Normi1, 2, Adam Leow Thean Chor1,2

1Department of Cell and Molecular Biology, 2Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Malaysia

Copper sensor regulator (CsoR) protein which is widespread in most Gram positive bacteria and are categorized under metalloregulatory protein that regulates tightly the in and out of copper (I) ions via homeostasis to maintain the cell viability and metabolism preventing cell toxicity. However, the absence of three-dimensional (3D) structure of native CsoR protein provides limited understanding of its binding capability and dynamics. Understanding the protein dynamics provide an important connection between protein function and structure. CsoR-like hypothetical protein which exists in Geobacillus zalihae (CsoRGz) share highly significant (up to 100%) sequence identity with hypothetical proteins of various bacterial strains. It also contains the domain of unknown function (CsoR-DUF). Remarkably, CsoRGz lacks significant similarity structurally-characterised CsoR proteins, sharing only 22-36% of similarity. This highlights the possibility of structural novelty of CsoRGz. Therefore, this study aims to investigate the structure, dynamics and interaction of CsoRGz with various metals via Nuclear Magnetic Resonance (NMR). The approach involve is to map out the functional metal-binding residues and sites of CsoRGz and their possible structural novelty. CsoRGz will be expressed using recombinant pET-28b:CsoRGz in E. coli Rosetta Gami 2 (DE3) minimal media (M9) supplemented with isotopically labelled 13C-glucose and 15N-ammonium sulphate. Tertiary structure prediction and protein dynamics determination will be carried out using 15N – 13C three-dimensional (3D) solution state NMR spectroscopy. Analysis will be carried on structure of CsoRGz without the presence of metal, how it folds and forms the cavity in order to bind with copper (I) and whether the cavity changes upon binding with different metals. The structure, dynamics and interaction of CsoR protein is expected to reveal its functional interacting metal-binding residues and its metal binding capability. This allows the possibility of miniaturizing CsoRGz to be used as a mini-metal sensor protein in sensor applications.

Keywords: Copper Sensor Regulator Protein (CsoR), Nuclear Magnetic Resonance

(NMR)


55

MODULATORY EFFECTS OF SELECTED FUNCTIONAL AMINO ACIDS ON THE TRANSCRIPTS ASSOCIATED WITH THE PROTECTIVE GENES AND IMMUNITY

IN POULTRY

Lee Chai Yan 1, Sieo Chin Chin 2,4 , Loh Teck Chwen 3,5 and Raha Abdul Rahim 1,4,*

1 Department of Cell and Molecular Biology, 2 Department of Microbiology,

Faculty of Biotechnology and Biomolecular Sciences, 3 Department of Animal Science, Faculty of Agriculture

4 Institute of Bioscience, 5 Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

The poultry industry is one of the most advanced livestock industry in Malaysia and its feed supplement consist of 65-70% amino acids. Deficiency of any amino acids will affect the immunity of the animal. The mucin glycoprotein and natural resistance-associated macrophage protein 1 (NRAMP1) confers genetic resistance to pathogens in chickens. This study investigates the effects of providing selected amino acids on the gene expression of mucin 2 (MUC2) and SLC11A1 (encodes NRAMP1) in broiler chickens by monitoring the transcript levels of these genes. Chickens are fed with commercial amino acids (lysine, threonine and methionine) as feed supplement. Intestine, spleen and liver samples were collected from chickens and RNA was isolated. RNA was converted to complementary DNA (cDNA) by reverse transcription polymerase chain reaction. The cDNA would be used in real-time PCR (qPCR) to determine the levels of gene expression. Gene expression of both MUC2 and SLC11A1 may differ when provided with lysine, threonine and methionine and may also be affected by the quality of amino acids fed to the chickens. The objective of the study is to determine if MUC2 and SLC11A1 gene expression is increased when chicken feed is supplemented with amino acid supplement. KEYWORDS: mucin, NRAMP1, amino acids, poultry, gene expression


xii

10th June 2015


Chairperson: Asilah Tajudin



Sieo Chin Chin Nurhidayah Roslan


Mohd Termizi Yusof Suriana Sabri

Wan Zuhainis Saad Amalia bt. Mohd. Hashim

Shuhaimi Mustafa



Page

No.

10.00 - 10.30

Liew Sien Yei GS39281 (BBS6904 & SPS6903)

Development of a high-throughput cell-based assay for screening of hypoxia-inducible factor activities (Norazizah Shafee)

37

10.30 - 10.50

Joanne Chng Yu Rou GS40859 (SPS5903)

Discovering the potential of nitric oxide treatment on erlotinib resistant non-small cell lung cancer cells (Nurhidayah Roslan)

38

10.50 - 11.10

Nur Fadhliah bt. Rusmii GS40927 (SPS5903)

Exploring the role of N-terminal of L2 lipase by functional and structural approaches (Fairolniza Mohd Shariff)

39

11.10 - 11.40

Mohamad Khairil b. Radzali GS37593 (BBS5903)

Antibacterial activites of locally isolated fungi from peninsular Malaysia (Wan Zuhainis Saad)

40

11.40 - 12.10 Wong Li Yin GS33577 (BBS6904)

Bioretting of kenaf using microbial enzymes (Wan Zuhainis Saad)

41


10th June 2015


Chairperson: Saila Ismail



Sieo Chin Chin Asilah Tajudin


Mohd Termizi Yusof Shuhaimi Mustafa

Nurhidayah Roslan Wan Zuhainis Saad



Page

No.

2.00 - 2.20 Siti Hajar Yusoff GS42186 (SPS5903)

Engineering Escherichia coli utilizing sucrose for production of lipase (Suriana Sabri)

42

2.20 - 2.50

Wan Nurhaida Azlin bt. Wan Hassan GS37560 (BBS5903)

Suppression of anthracnose disease on chili by locally isolated fungi from soil sample (Wan Zuhainis Saad)

43

2.50 - 3.20

Laavanya A/P M. Kumar GS37986 (BBS5903)

Antibacterial potential of biofilms of locally isolated lactic acid bacteria (Wan Zuhainis Saad)

44

3.20 - 3.50

Muhammad Hariadi b. Mohd Nawawi GS37235 (BBS5903)

Biobleaching of kenaf pulp using xylano-pectinolytic enzymes from Bacillus subtilis strain ADI1 (Wan Zuhainis Saad)

45

xiii


54

THE ANTIOBESITY EFFECT OF STINGLESS BEE HONEY ON OBESE RAT INDUCED BY FORMULATED HIGH FAT DIET

Ahmad ZulkifliMohd Rafie1, Mariatulqabtiah Abdul Razak2,3, Nurhidayah Roslan4,

Department of Cell and Molecular Biology1, Department of Microbiology2 ,

Faculty of Biotechnology and Biomolecular Sciences,Institute of Bioscience3, Universiti Putra Malaysia

Obesity prevalence has increased at worrying rates in developed and underdeveloped countries around the world. Around 3.4 million adults became overweight each year and more than 40 million children under age of five were obese in year 2012 world-wide. In Malaysia, obesity problem has become common among children that lead to metabolic disease. Due to that case, honey as natural product that consumed by the local population in Malaysia will make an alternative to treat obesity as it has medici-nal properties. It was shown through in vivo or pre-clinical studies that stingless bee honey has good antioxidant, anti-mutagenic and antibacterial activities which can help in lowering risk of severe disease. This study will involve on formulation of high fat diet in order to know the best composition of food pellet that can induce obesity. Three different concentrations of stingless bee honey from Heterotrigona itama species will be used. This study will consist of 4 objectives : 1) Formulation of high fat diet; 2) De-termination of food intake weekly on body weight; 3) Determination of adiposity index; 4) Biochemical analysis of blood and histophatology evaluation of heart, liver , lung and pancreas. The method to formulate high fat diet is through the usage of ghee, normal pellet diets with low calories and fat concentration, and tablets that contain calcium and vitamin D3 only. The amount of food intake and body weight changes of rat will be calculated once per week. The adiposity index will be determined from the epididymal, visceral, and retroperitoneal fat. Biochemistry analysis which are lipid pro-file, liver function test and diabetes screen will be tested using ILAB 300 Plus Clinical Chemistry analyser. Histophatology evaluation will be examined microscopically. This study expects to show a decrease of weight and adiposity index of rats administered with stingless bee honey and a reduced level of cholestrol, triglycerides, ALT, AST and glucose in rats administrated with stingless bee honey compared to other groups. KEYWORDS: Obesity, Stingless bee honey


53

DEVELOPMENT OF NEW VARIETIES OF STEVIA REBAUDIANA WITH HIGH PARTICULAR TYPES OF STEVIOLGLYCOSIDES SUITABLE FOR GROWING

IN MALAYSIA

Siti Nadhirah Sidi Ahmad1, Norihan Mohd Saleh1, Sobri Hussein2, Janna Ong Abdullah1

Department of Cell & Molecular Biology1, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;

Agrotechnology & Biosciences Division2, Malaysian Nuclear Agency, 43000 Bangi, Selangor, Malaysia.

Stevia rebaudiana is a commercially valuable plant due to the high demand for its steviolglycoside (SG) compounds present in the leaves. The purified form of steviolglycosides particularly rebaudioside A is 300 times sweeter than the commer-cially available cane sugar. There are many existing varieties of Stevia. Unfortu-nately, the growth of Stevia and its steviolglycosides content are greatly affected by climatic conditions, such as temperature, day length and intensity of photoperiod. A major setback to the Stevia industry in Malaysia is lack of varieties that can grow well in Malaysia climatic conditions. Many studies have shown that the synthesis of steviol-glycosides is reduced at or just before flowering, thus reduces the steviolglycosides yield production. Mutation induction such as gamma irradiation has proven to be effective in generating genetic variations in many plants from which desired mutants were successfully selected. Hence, the aims of this study is to create new varieties of S. rebaudiana with delayed flowering to increase the accumulation of particular types of sweet steviolglycosides, and are suitable to grow in Malaysia. Seeds and in vitro plantlets will be treated with acute irradiation at different doses of gamma rays (ranging from 5 to 30 Gy) while seedlings will be exposed to chronic irradiation at different irradiation doses ranging from 0.01 Gy/hour to 3 Gy/hour. The germination pattern and phenotypic changes (leaf size, leaf color, stem length, onset of flowering) of the irradiated mutants will be observed. It is expected that gamma irradiation will induce variability of S. rebaudiana with higher SGs contents. Desired mutants of S. rebaudiana screened based on delayed flowering and SGs contents will be selected for commercial cultivation via micropropagation. Keywords: Stevia rebaudiana, Steviolglycosides, gamma irradiation, delayed flowering


8th June 2015


Chairperson: Parameswari Namasivayam

Evaluators : Raha Abdul Rahim Janna Ong Abdullah

Normi Mohd Yahaya Nurulfiza Mat Isa

Adam Leow Thean Chor Tan Soon Guan

Mariatulqabtiah Abdul Razak Mas Jaffri Masarudin

Noorjahan Banu Mohammed Alitheen



Page

No.

10.00 - 10.20

Ummu Afiqah bt. Hassan GS42314 (SPS5903)

Development of a nanoparticle-mediated delivery system to study release of fluores-cently-labeled amino acids encapsulated in chitosan nanoparticles (Mas Jaffri Masarudin)

46

10.20 - 10.40

Syazaira Arham bt. Yahya Ariff GS42313 (SPS5903)

Investigating the enhanced functionality of mirna-encapsulated chitosan nanoparticles as an anti-metastatic agent for cancer therapy. (Mas Jaffri Masarudin)

47

10.40- 11.10 Nur Rizi bt. Zamberi GS37170 (BBS5903)

The effects of malaysian kefir culture towards antioxidants and anti-metastasis in breast cancer cells (Noorjahan Banu Mohammed Alitheen)

48

11.10- 11.40

Innanurdiani bt. Koko GS38058 (BBS5903)

Engineering food-grade integrative vectors based on bacteriophage site specific recombination mechanism for Lactococcus lactis (Raha Abdul Rahim)

49

11.40 - 12.00 Seif-Eddine Naadja GS40014 (SPS6903)

Promoter activities of the AtPAL gene family in Arabidopsis thaliana (Mohd Puad Abdullah)

50

xiv


Time Name/ Matric


Page

No.

2.00 - 2.30 Tay Chee Chun GS39033 (BBS5903)

Determination of genetic diversity and inbreeding level in deli dura and avros advanced breeding materials in oil palm (Elaeis guineensis jacq.) using microsatellite markers (Tan Soon Guan)

51

2.30 - 3.00

Nurul Elyani bt. Mohamad GS34177 (BBS6904 & (SPS6903)

Evaluation on the biological activities of coconut and pineapple vinegar using murine model (Noorjahan Banu Mohammed Alitheen)

52

3.00 - 3.20

Siti Nadhirah bt. Sidi Ahmad GS42046 (SPS5903)

Development of new varieties of Stevia rebaudi-ana with high particular types of steviolglycosides suitable for growing in Malaysia (Janna Ong Abdullah )

53

3.20 - 3.40

Ahmad Zulkifli b. Mohd Rafie GS42748 (SPS5903)

The antiobesity effect of stingless bee honey on obese rat induced by formulated high fat diet (Mariatulqabtiah Abdul Razak)

54

3.40 - 4.10 Lee Chai Yan GS37118 (BBS5903)

Modulatory effects of selected functional amino acids on the transcripts associated with the pro-tective genes and immunity in poultry (Raha Abdul Rahim)

55

xv

8th June 2015


Chairperson: Mariatulqabtiah Abdul Razak

Evaluators : Raha Abdul Rahim Janna Ong Abdullah

Noor Baiti Saidi Normi Mohd Yahaya

Nurulfiza Mat Isa Adam Leow Thean Chor

Tan Soon Guan Mohd Puad Abdullah


Siti Sarah Othman


52

EVALUATION ON THE BIOLOGICAL ACTIVITIES OF COCONUT AND PINEAPPLE VINEGAR USING MURINE MODEL

Nurul Elyani Mohamad1, Shaiful Adzni Sharifuddin2,Kamariah Long2,

NoorjahanBanuAlitheen1*

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang,

Selangor 43400, Malaysia 2Biotechnology Research Centre, Malaysian Agricultural Research and

Development Institute (MARDI), Serdang, Selangor 43400,Malaysia Improper handling of work stress, diet and lifestyle cause imbalance to the biological system in our body and have led to various diseases nowadays. The number of patients with chronic diseases such as cardiovascular disease and cancer have increased greatly and becoming a major public health concern. Obesity and liver inflammation are indirect risk factors that can lead to cardiovascu-lar diseases. The current treatment approaches are unfavorable due to the unwanted side effect observed. Natural resources such as fruits and vegetables contain antioxidants, minerals and vitamins that may help to prevent diseases and enhance the immune system. Vinegar produced from carbohydrate sources, such as fruits and grains, contains not only acetic acid, but also other bioactive compounds such as polyphenolics, volatile compounds, and organic acids. Production of vinegar involves alcoholic and acetic fermentations. Yeast converts sugar into alcohol during alcoholic fermentation, which is then transformed into acetic acid by Acetobacter bacteria during acetification. The present study was carried out to investigate several biological activities such as hepatoprotective activity, anti-tumor activity and anti-obesity activity of coconut and pineapple vinegar using murine model. Assays such as anti-oxidant, real-time PCR and western blot analyses revealed the underlying mechanism of both vinegar samples in all in vivo study. Notably, oral administration of tested concentrations in all in vivo studies exhibited that both coconut and pineapple vinegar showed hepatoprotective, anti-tumor and anti-obesity effects in dose dependent manner. This study signifies the potential of coconut and pineapple vinegar as a functional food for therapeutic pur-poses. Keywords: disease, coconut vinegar, pineapple vinegar, biological activities


51

DETERMINATION OF GENETIC DIVERSITY AND INBREEDING LEVEL IN DELI DURA AND AVROS ADVANCED BREEDING MATERIALS IN OIL PALM (ELAEIS

GUINEENSIS JACQ.) USING MICROSATELLITE MARKERS

Tay Chee Chun1, Tan Soon Guan1

1 Department of Cell and Molecular Biology, Faculty of Biotechnology and

Biomolecular Sciences, University Putra Malaysia (UPM),Serdang, Selangor, Malaysia

Oil palm has a narrow genetic diversity due to intensive selection in breeding. Loss of diversity in breeding material can lead to many consequences, despite uniformity is critical to breeders. Thus, an understanding on diversity of advanced breeding parental material at the molecular level is crucial for crop improvement and seed production. The objectives of the study are to evaluate the genetic diversity in Deli dura and AVROS populations, and to determine the levels of inbreeding in the advanced parental materials. The materials involved are 186 palms from 8 DxD/D-selfs Deli dura and 188 palms from 8 TxT/T-selfs AVROS progenies sourced from Agency 1 and Agency 2. Ekona population was included as a control. Seventy four CIRAD’s simple sequence repeat (SSR) markers were screened for polymorphism and only 32 markers were shortlisted for genotyping using capillary electrophoresis. Genotyping results showed that 203 alleles were found among the 17 progenies and the number of alleles scored per SSR ranged between 2 and 7. The average number of alleles per locus (A) was 6.3438. The expected heterozygosity (He) and observed heterozygosity (Ho) among the populations were 0.7063 and 0.5270, respectively. The genetic distance value was based on Nei et al. (1972). Dendogram from Unweighted Pair-Group with Arithmeric Average (UPGMA) cluster analysis revealed that the 17 populations were grouped into three main clusters namely Deli dura, AVROS and Ekona. The AVROS cluster was further grouped into two sub-clusters according to the two Agencies. Deli clusters were further branched out based on the cross type i.e. DxD and D-selfs. Two different generations of AVROS (same original source) have shown an overview of the inbreeding level among the Agency. AVROS from Agency 2 had inbreeding coefficient of 0.0283 as compared to AVROS from Agency 1 which was -0.0863. The inbreeding value indicated that Agency 2 AVROS had lower heterozygosity as compared to Agency 1. Findings from this study will enable breeders to exploit the variable collection for selection of traits of interest.

Keywords: Elaeis guineensis Jacq. , simple sequence repeat (SSR), Inbreeding coefficient




Page

No.

10.00 - 10.20

Aswaani A/P Man-gavelu GS42467 (SPS5903)

NMR structure, interaction and dynamics of hypothetical csor protein of Geobacillus zalihae (Normi Mohd Yahaya)

56

10.20 - 10.50

Nurul Amirah bt. Mahamad GS38980 (BBS5903)

Regulation of protein S-nitrosylation for effective control of fusarium wilt in banana (Noor Baiti Saidi)

57

10.50 - 11.20 Lee Wei Kang GS35690 (BBS6904)

Development of expressed markers for Gracilaria species with higher yield and quality of agar (Ho Chai Ling)

58

11.20 - 11.40 Joey Ee Uli GS40165 (SPS6903)

Transcriptome profiling and generation of genic-SSR markers for cynomolgus ma-caque (Macaca fascicularis) from peninsu-lar Malaysia (Tan Soon Guan)

59

11.40 - 12.00

Nur Aqlili Riana bt. Alias GS41732 (SPS6903)

Mutagenesis and impact of the signal peptide SPK1 for secretion of heterologous proteins in Lactococcus lactis (Raha Abdul Rahim)

60

xvi

10th June 2015


Chairperson: Noor Baiti Saidi

Evaluators : Raha Abdul Rahim Siti Sarah Othman

Normi Mohd Yahaya Mohd Puad Abdullah

Nurulfiza Mat Isa Adam Leow Thean Chor

Ho Chai Ling Tan Soon Guan


Mariatulqabtiah Abdul Razak


xvii

10th June 2015


Chairperson: Nurulfiza Mat Isa

Evaluators : Raha Abdul Rahim Siti Sarah Othman

Noor Baiti Saidi Normi Mohd Yahaya

Adam Leow Thean Chor Tan Soon Guan

Mohd Puad Abdullah Ho Chai Ling


Mariatulqabtiah Abdul Razak



Page

No.

2.00 - 2.30

Yusuf Chong Yu Lok GS35905 (BBS6904)

Isolation of the Phenylalanine ammonia-lyase promoters from oil palm and analysis of the promoter activities in arabidopsis(Mohd Puad Abdullah)

61

2.30 - 3.00

Roslinda bt. A Razak GS37880 (BBS5903)

Development of papaya (Carica papaya l. Cv. ‘eksotika’) with potential resistance to dieback disease (Janna Ong Abdullah)

62

3.00 - 3.30

Nor Fadzliana bt. Abu Faizal GS37531 (BBS5903)

Establishment and enhancement of betalains production in Hylocereus polyrhizus callus culture (Janna Ong Abdullah)

63

3.30 - 3.50

Siti Nabilah bt. Yusoff GS42537 (SPS5903)

Using tobacco cell suspension culture to functionally express an orchid terpenoid biosynthesis protein (Janna Ong Abdullah)

64

3.50 - 4.10

Nur Suhanawati bt. Ashaari GS42723 (SPS6903)

Development of a genetically engineered gamma-terpinene synthase of Plectranthus amboinicus (Bangun-bangun) for thymo-quinone production (Janna Ong Abdullah )

65

4.10 - 4.30 Patricia Jayshree Samuel Jacob (SPS6903)

A comparative evaluation of the characteris-tics and application potential of biologically and chemically synthesized silver and magnetic nanoparticles (Mas Jaffri Masarudin)

66


50

PROMOTER ACTIVITIES OF THE AtPAL GENE FAMILY IN ARABIDOPSIS

THALIANA

Seïf-Eddine Naadja1, Nor Aini Ab. Shukor2, Kok Song Lai1, Dhilia Udie Lamasudin1

and Mohd. Puad Abdullah1

1 Department of Cell & Molecular Biology, Faculty of Biotechnology & Biomolecular

Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,

Selangor Darul Ehsan, Malaysia. 2 Department of Forest Management, Faculty of Forestry, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.

Phenylalanine Ammonia-Lyase (PAL; EC 4.3.1.5) is the rate-limiting enzyme of the

phenylpropanoid pathway (PPP). This enzyme regulates many aspects of cellular

functions in plants during growth, development and adaptation. PAL is encoded by

many genes and often called as the PAL gene family. Our understanding on how

each member of the PAL gene family executes its functions is limited and relies

only on the expression studies of the PAL gene family in Arabidopsis thaliana. In

this plant, PAL is encoded by four different genes namely AtPAL1, AtPAL2, AtPAL3

and AtPAL4. AtPAL1 and AtPAL2 are closely related functionally and phylogeneti-

cally, whereas AtPAL3 is with AtPAL4. AtPAL1 and AtPAL2 are largely involved in

plant development. Previously, AtPAL2 has also been reported to be important dur-

ing plant structural adaptation to a pathogen challenge. However, the promoter ac-

tivities of the complete members of the family during development and adaptation

are still unknown. Therefore, this study will be carried out to analyse the activities of

each individual AtPAL gene promoter. Knowing the activities of all members of the

family will give clues on how they work as a team. One way of studying this is

through a transgenic approach; in this study, four transgenic plants carrying the

AtPAL1, AtPAL2, AtPAL3 and AtPAL4 gene promoters will be developed. The ac-

tivities of all AtPAL gene promoters in different organs and at different developmen-

tal stages of the plants will be evaluated. The plants will be exposed to various bi-

otic and abiotic stress elicitors to investigate how the gene promoters behave under

such conditions. This study is expected to provide insight into the roles of individual

members of the AtPAL gene family in plant development and adaptation.

Keywords: Arabidopsis thaliana plant, AtPAL gene promoters, Biotic & abiotic stress

elicitors, Phenylalanine ammonia-lyase, Phenylpropanoid pathway, Plant develop-

ment, Plant growth, Plant structural adaptation, Promoter activities.


49

ENGINEERING FOOD-GRADE INTEGRATIVE VECTORS BASED ON BACTERIOPHAGE SITE SPECIFIC RECOMBINATION MECHANISM FOR

LACTOCOCCUS LACTIS

Innanurdiani Koko1, Chin Chin Sieo2, 3 and Raha Abdul Rahim1, 3

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Bio-

molecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia

Integrative vector allows stable expression of foreign genes by integration of foreign DNA into the host chromosome for recombinant protein production. Lactococcus lactis have been engineered for various industrial applications for human and ani-mal consumption and vaccine production. The demand of food-grade biological platforms constructed from these particular bacteria has led to the development of food-grade vectors to express genes of interest. The Integrative vectors for cloning and expression of heterologous protein in Lactotoccus lactis have been successfully constructed. The integration was based on the site-specific recombination system of temperate lactococcal phage TP901-1. The integrating system was constructed with two vectors; (1) the helper vector, integrase (int) carrying plasmid (pNZint) which integrates attP carrying plasmid into L. lactis genome, (2) the integrative vectors, plasmid carrying phage attachment site (attP); pNZattS and pNZattD, were equipped with P170 auto-inducible promoter, multiple cloning sites (MCS), signal peptide-encoding sequence usp45 (), and proteinase anchor domain (PrtP) which can be applied for secretion and surface display of recombinant protein respec-tively. The vectors also harboured a small heat shock protein (sHsp) as food-grade selection marker. The engineered synthetic integrative vectors can be used for het-erologous protein production in lactococcal expression system for research or in-dustrial purposes. KEYWORDS: Integrative vector, Site-specific recombination, Food-grade vector, Secretion and Surface display of recombinant protein, Lactococcus lactis.


ABSTRACTSABSTRACTSABSTRACTSABSTRACTS


1

ANTIOXIDANT PROPERTIES IN DIFFERENT PLANT PARTS OF A WILD ORCHID DENDROBIUM CRUMENATUM

Suria Johari and Maziah Mahmood

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Dendrobium crumenatum or known as “Pigeon Orchid” is a tropical epiphytic orchid which can be found abundantly grown on mature trees. D. crumenatum has been used as folklore medicine since ages by the indigenous community. The present study was undertaken to investigate the potential antioxidant activity as well as to determine the total flavonoid, total phenolic acid and total polyphenol content from different parts of D. crumenatum (pseudobulb, leaf and stem. In this study, the whole plant of D. crumenatum was collected around Universiti Putra Malaysia (UPM) campus. The antioxidant activities were determined using two methods, namely 1,1-diphenyl-2-picrylhydrazil (DPPH) free scavenging assay and ferric reducing antioxi-dant potential (FRAP) assay. The total antioxidant content was found to be signifi-cantly different depending on extracting solvents, plant parts, drying temperatures and shaking duration of extraction. The result indicated that methanol was more efficient as an extraction solvent compared to aqueous and ethanol. Both methods showed all plant parts of D.crumenatum exhibited high antioxidant activities using plant materials dried at 45°C. The same trend was also observed for total flavonoid, total phenolic acid and total polyphenol content from different parts of D. crumenatum. The current study showed that D. crumenatum has great potential to be developed into herbal products, which may be applicable for food and nutraceutical industries. Due to the potential of this species as source of antioxidant, further studies were undertaken to investigate the antibacterial activity of different plant parts of D. crumenatum against four pathogenic bacteria using disc diffusion assay (DDA) method to give an idea of the presence or absence of substances with antimicrobial activity. KEYWORDS: epiphytic orchid, folklore medicine, antioxidant, DPPH, FRAP


48

THE EFFECTS OF MALAYSIAN KEFIR CULTURE TOWARDS ANTIOXIDANTS AND ANTI-METASTASIS IN BREAST CANCER CELLS

Nur Rizi Zamberi1, Yeap Swee Keong2, Md Zuki Abu Bakar2,

Nik Mohd Afizan Nik Abd Rahman1, Noorjahan Banu Alitheen1 1Department of Cell and Molecular Biology,

Faculty of Biotechnology and Biomolecular Science, 2 Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Kefir is a unique cultured product and reports have shown that microbial interactions in kefir culture have contributed to health-promoting benefits such as giving rise to antioxidants and anti-tumor properties. Metagenomics analysis of kefir culture is es-sential to understand the genetic diversity of microbial populations in the kefir grains especially in evaluating its probiotic effectiveness. Extensive reports have been made on the microbial diversity in kefir, which contained some known probiotics bac-teria that may contribute to antioxidant properties and hold potency in cancer ther-apy. Although the Malaysian kefir grains are regularly consumed, no report has been made on its microbial profile and its contributions to antioxidant and anti-tumor po-tency. This study aims to detect microbial diversity present in kefir culture using 16S metagenomic approach, evaluate its antioxidant potential and assess the anti-tumor effects of kefir towards murine breast cancer model in vitro and in vivo. The microbial diversity in kefir cultures were determined using Next-Generation-Sequencing (NGS) and antioxidant assays were performed to evaluate the antioxidant potentials of kefir culture. In vitro and in vivo anti-metastasis study of 4T1 murine breast cancer cells were further investigated. Lactobacillus genus was the dominant genus detected in the sample where the predominant species was L. kefiranofaciens (80% to 91%) while L. kefiri (2% to 2.5%) was the second abundance. Kefir culture displayed prominent antioxidant activity values and it also reduced metastasis significantly in breast cancer in vitro and in vivo. These findings suggested that Malaysian kefir cul-tures contained largely Lactobacillus species and the members of the microbial com-munity have contributed to the source of antioxidant activities in kefir grains and its potential towards overcoming metastasis in breast cancer. These results require further research on the specific detected species, which have contributed to overall effects on the antioxidants and anti-metastasis in breast cancer cells. Keywords: kefir, Lactobacillus kefiranofaciens, anti-metastasis


47

INVESTIGATING THE ENHANCED FUNCTIONALITY OF miRNA-ENCAPSULATED CHITOSAN NANOPARTICLES AS AN ANTI-METASTATIC

AGENT FOR CANCER THERAPY

Syazaira Arham Yahya Ariff1, Khatijah Mohd Yusoff2, Mas Jaffri Masarudin1

Department of Cell and Molecular Biology1, Department of Microbiology2,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia.

The uncontrolled growth and metastasis of tumors caused approximately 8.2 million deaths worldwide in 2012.Cancer treatment have varying effectiveness among patients, causing unwanted side effects and killing normal cells. MicroRNAs (miRNA) have been utilized as a repressor for metastasis of tumor cells as the molecule inhibits fundamental processes related to cellular and physiological pathway at the mRNA level. However, application of miRNAs is impaired by their premature degradation in the extracellular environment by endonucleases. Therefore, the successful administration of miRNAs is contingent upon effective relocation into the nuclear membrane of targeted nuclear cells. This research describes the design of an effective, non-efficacious, nanoparticle-mediated delivery system for enhanced delivery of miRNA-186, a tumor-suppressor in the development and progression of non-small cell lung carcinoma. Through ionic gelation methods, miRNA-186 will be encapsulated in chitosan nanoparticles, a drug carrier with high particle stability, low cellular toxicity, and a robust preparation method. Physiochemical and morphological characterization of the synthesized nanoparticles will be conducted using the TNBS assay, CHNS analysis, DLS, FESEM, TEM and FTIR. Additionally, in-vitro nanoparticle efficacy in cancer cells will be evaluated through the MTT cell cytotoxicity and cellular invasion assays. It is expected that the implementation of a nanoparticle-mediated delivery system will be able to protect miRNA-186 from extracellular enzymatic degradation, due to the association of the miRNA within the nanoparticle core. The encapsulated miRNA-186 is expected to be released in proximity to the cell nucleus, due to changes in physiological pH between miRNA-186 encapsulated chitosan nanoparticles with the cell membrane. With the increased of cellular uptake and protection from degradation, it is expected that the invasiveness of cells treated with the nanoparticles will be greatly disrupted. Successful implementation of the re-search project will lead to potential therapies preventing invasiveness in cancers, and towards future aversion of cancer progression in patients. KEYWORDS: Chitosan nanoparticles, miRNA-186, anti-metastatic agent, miRNA, cancer therapy.


2

ANTIOXIDATIVE AND ANTIDIABETIC POTENTIALS OF COMMERCIAL AND

TRADITIONAL BRAN EXTRACTS OF SOME MALAYSIAN RICE VARIETIES

Tanko Abubakar Sadiq1, Nazrim Marikkar1, Abubakar Salleh1, Azrina Azlan2

Department of Biochemistry1, Faculty of Biotechnology and Biomolecular Sciences Department of Nutrition and Dietetics2, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia

Malaysian traditional rice are believed to have more nutraceutical values than the im-

proved rice varieties. This study compared the anti-diabetic potential, total polypheno-

lic content and antioxidant activities of bran extracts(RBEs) of seven Malaysian tradi-

tional rice varieties (Adan halus, Adan kasar, Salleh halus, Salleh Kasar, Beras

merah, Beras hitam and Nanung) against those of three improved varieties collected

from Selangor, Kedah and Perlis. 10 grams of the dried rice bran samples were

extracted successively with 70% ethanol (3x100 mL) by overnight soaking to obtain

ethanolic extracts, which were subsequently concentrated under reduced pressure

using a rotary evaporator. α-amylase and α-glucosidase inhibitory potentials of the

rice bran extracts (RBEs) were studied in vitro. The total poly phenolic content (TPC)

and antioxidant activities (FRAP, DPPH) of the crude extracts were also determined in

vitro. The results show that RBEs of Beras merah and Beras hitam displayed signifi-

cant enzyme inhibitory and antioxidant activities having alpha glucosidase inhibition of

96.56 % ± 0.58 and 81.52 % ± 0.96, alpha amylase inhibition of 88.44 % ± 3.41 and

84.27 % ± 3.02, and total poly phenolic content of 14.94 ± 0.53 and 10.22 ± 0.25 mg

gallic acid equivalent /g of dry rice bran respectively. These two varieties showed

significantly (<0.05) higher enzyme and antioxidant activities in comparison to the

brans of the improved rice varieties and therefore would be used to investigate their

antidiabetic potential in vivo on alloxan induced Sprague Dawley rats. Hence, brans of

traditional rice varieties such Beras merah, and Beras hitam could have a higher

potential as raw materials for formulation of nutritional supplements as well as good

sources of natural anti-diabetic agents.

Key Words: alpha glucosidase inihibition, alpha amylase inihibition, antioxidant

activities, anti-diabetic potential, sprague Dawley rats, anti-diabetic agents.


3

THE USE OF ALCOHOL OXIDASE PROMOTER IN THE EXPRESSION OF RE-COMBINANT PROTEINS WITHOUT THE PRESENCE OF METHANOL

Nur Iznida Mahyon1,2, Siti Nurbaya Oslan1,2*, Suriana Sabri1,3, Abu Bakar Salleh1,2

Enzyme and Microbial Technology Research Center1, Department of Biochemistry2, Department of Microbiology3, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Alcohol oxidase promoter (AOX) is a tightly regulated methanol inducible promoter in methylotrophic yeast. Pichia guilliermondii strain SO is a locally isolated yeast from spoilt orange; has been developed as an expression host. It has the capability to express recombinant bacterial lipase in the presence and absence of methanol as inducer. The ability to express the recombinant protein without methanol is a very significant finding in this new yeast system. The use of methanol is a vital factor in AOX promoter regulation. Methanol is a chemical derived petroleum which highly toxic, unfit for consumption and unsuitable to be used in protein production particularly for food product. Therefore, the main objective of this project is to understand the regulation of AOX without the presence of methanol in P. guilliermondii strain SO. This research will cover the expression of recombinant proteins from different classes of enzymes (hydrolase, transferase and oxidoreductase) driven by AOX promoter in P. guilliermondii strain SO. Expression of recombinant proteins in this host will be verified using enzymatic assay and western blot. This study is important in order to prove that this strain can express various recombinant proteins without methanol induction. Moreover, comparison of AOX gene will be made between P. guilliermondii strain SO and Pichia pastoris via bioinformatics analysis. To isolate AOX gene from this strain, inverse PCR will be performed using degenerate primer. It is expected that this study will reveal the different feature(s) of AOX gene presented in P. guilliermondii strain SO as compared to AOX gene in P. pastoris which responsible for AOX regula-tion in the absence of methanol. KEYWORDS: Alcohol oxidase, promoter, methanol induction, Pichia sp.


46

DEVELOPMENT OF A NANOPARTICLE-MEDIATED DELIVERY SYSTEM TO STUDY RELEASE OF FLUORESCENTLY-LABELED AMINO ACIDS

ENCAPSULATED IN CHITOSAN NANOPARTICLES

Ummu Afiqah Hassan1, Mohd Zobir Hussein2, and Mas Jaffri Masarudin1

Department of Cell and Molecular Biology1,

Faculty of Biotechnology and Biomolecular Sciences, Institute of Advanced Technology2, Universiti Putra Malaysia.

Application of nutraceuticals as an alternative to current modern medicine for treat-ment and prevention of diseases has garnered great research attention. Though more bioactive compounds are identified to promote physiological benefits, their bioavailability and efficacy remain limited post oral administration. Developing meaningful therapeutic doses is challenging, owing to the nature of gastrointestinal system. Presence of degradative enzymes along with constant pH changes degrade the chemical structure of nutrients, causing loss of bioactivity. Moreover, large-size nutrients are difficult to permeate through intestinal epithelia leading to inefficient ab-sorption by the digestive system thus reduces concentration of the nutrients reaching systemic circulation. Nanotechnology-based approaches for nutraceutical applications have been proposed to tackle this issue. Previous studies report that formulation of nutraceuticals such as amino acids into nanoparticle systems protect the nutrients from enzymatic degradation, whilst its nanometer size enhance cellular membrane permeability. However, scientific studies on nutraceutical release and intercellular uptake from nanoparticle systems remain lacking. This research proposes develop-ment of an effective, non-efficacious nanoparticle-mediated system for delivery of amino acids. Chitosan-based nanoparticle delivery system will be used for encapsula-tion of amino acids. Morphological features will be analyzed using DLS, FESEM, and TEM before and following encapsulation. Chemical characterization will be conducted by FTIR, CHNS and TNBS assays to confirm amino acid loading. For visualization of amino acid release, FITC-labelled amino acids will be used for visualization under confocal microscopy. Amino acid uptake and release will be compared in cells treated both with and in absence of the nanoparticle system. Findings from this study will pro-vide a scientific report on release profiles of nutraceuticals, and the successful imple-mentation of this delivery system will lead to potential enhanced delivery of functional food ingredients in the nutraceutical field, as well as drug delivery in pharmaceutical field. KEYWORDS: nutraceuticals, chitosan nanoparticles, nanobiotechnology, amino acid, bioavailability.


45

BIOBLEACHING OF KENAF PULP USING XYLANO-PECTINOLYTIC ENZYMES FROM BACILLUS SUBTILIS STRAIN ADI1

1Muhammad Hariadi Nawawi, 2Rosfarizan Mohamad, 2Paridah Md Tahir and 1

Wan Zuhainis Saad

1Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, 2Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia. Biobleaching has been carried out using either lignolytic or hemicellulolytic enzymes. The hemicellulolytic enzymes depolymerize hemicellluloses precipitated on the surface of the fiber. Thus it increases the access of bleaching chemicals to the lignin layer by opening the pulp structure. Xylanase treatment substantially reduced the xylose content present in pulp. In other word, xylan content decreased when pulp was bleached. Pectinase are known to degrade pectin, by incorporating pectinases in the bleached or alkaline treated pulp, such harmful pectins in the aqueous phase of the pulp are degraded and thus rendered harmless to paper making process by lowering the cationic demand of the pulp. Therefore, it was hypothesized that xylano-pectinolytic enzymes, which can degrade pectin and xylan present on the pulp fibers by minimizing the damage to the cellulose pulp, can be used to evaluate for the prebleaching of kenaf pulp. Therefore, the main objective of this study is to investigate the synergistic action of xylano-pectinolytic enzymes from the bacterial isolate for effective biobleaching of kenaf pulp. Methods that will be employed include isolation of local thermophilic bacteria, enzymes assay such as pectinase, xylanase and cellulase assay, optimization of various parameters for enzymes production, optimization of enzymatic pretreatment reaction conditions, and biobleaching of kenaf pulps. Information that will be obtain from this study will be invaluable for understanding the xylano-pectinolytic synergism in paper and pulp industry will ultimately help in making the process not only economically feasible but also eco-friendly. Keywords: Alkalothermostable, Biobleaching, Kenaf, Pectinase, Xylanase.


4

ENDOPHYTIC COLONISATION OF HENDERSONIA TORULOIDEA AND ITS EFFECT ON THE EXPRESSION OF THIAMINE BIOSYNTHESIS GENES (THI1/

THI4 AND THIC) IN OIL PALM (ELAIES GUINEENSIS)

Amirah Nor Kamarudin1,Idris Abu Seman2,Zetty Norhana Balia Yusof1

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia1, Ganoderma and Diseases Research Oil Palm Unit,

Biological Division, Malaysian Oil Palm Board (MPOB)2

Vitamin B1 (thiamine) has been shown to have a role in plant adaptation to biotic and abiotic stress. Recently, THI1/THI4 and THIC, the first two enzymes in the thiamine biosynthesis pathways were identified and isolated in oil palm. In the present study, the expression of thiamine biosynthesis genes in response to endophytic colonisation of endophytic fungus Hendersonia toruloidea was examined. The colonisation of endophytic fungus in the oil palm root was studied by histological analysis and dilution plate count. Biochemical analysis on the induction of plant defence enzymes i.e peroxidase, phenyl-alanine lyase (PAL) in response to endophytic colonisation will also be explored. Total RNA extraction, reverse transcriptase PCR will be carried out and the expression of thiamine biosynthesis genes (THI1/THI4 and THIC) will be examined using real-time PCR. Thiamine and its intermediates’ accumulation will be analysed by HPLC. It is expected that the transcripts of the first two enzymes in the thiamine biosynthesis pathway, THI1/THI4 and THIC will be upregulated in oil palm treated with endophytic fungus compared to untreated oil palm. Defense enzymes are also expected to be upregulated in treated than untreated oil palm. It is hoped that this study will unravel the role of endophytes in boosting thiamine levels in oil palm which could lead to the production of a more stress-tolerant oil palm variety.

KEYWORDS: thiamine; vitamin B1; endophytes; endophytic fungi; induced systemic resistance; oil palm


5

ENHANCING PICHIA GUILLIERMONDII STRAIN SO AS A HOST FOR HET-EROLOGOUS PROTEINS PRODUCTION

Mary Ladidi Abu, Abu Bakar Salleh, Siti Nurbaya Oslan

Enzyme and Microbial Technology Research Centre,

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences. Universiti putra Malaysia. 43400 Serdang Selangor.

Yeast is considered a good expression system for production of heterologous proteins. However, current yeast models such as; Pichia pastoris and Saccharomyces cerevisiae are not without their drawbacks. The high methanol demand which is a potential fire hazard by alcohol oxidase promoter (AOX) and high level of expression which could result in misfolding and inefficient processing of certain proteins are of great concern. Low and inefficient secretion by S. cerevisiae and its high mannose glycosylation process limit its applications. Putting into consideration the growing demand for heterologous proteins, it therefore, becomes necessary to enhance and develop new yeast isolate Pichia guilliermondii - a methylotroph, as a host for production of heterologous proteins becomes necessary to overcome these limitations. This research focuses on the modification of the commercial vector pPiczαB for compatibility with P. guilliermondii. New promoter system can be introduced by replacing the AOX present in pPiczαB with methanol oxidase (MOX) promoter can be induced either by methanol moderately as high level methanol induction inhibits its activity or its induction can be by glycerol. These are to serve as tools to achieving an improved and efficient expression system. Keywords: Pichia pastoris; Pichia guilliermondii; heterologous protein; promoter.


44

ANTIBACTERIAL POTENTIAL OF BIOFILMS OF LOCALLY ISOLATED LACTIC ACID BACTERIA

Laavanya M Kumar1, Rosfarizan Mohamad2, Raha Abdul Rahim3,

Wan Zuhainis Saad1

Department of Microbiology1, Department of Bioprocess Technology2, Department of Cell and Molecular Biology3,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor.

The threat of pathogenic bacteria is a major health concern in the food industry. Accordingly, numerous studies have shown that the foodborne diseases are largely caused by the biofilms formed by the persistent food-borne bacteria. By omitting the options of chemical preservatives, bacteriocin producing lactic acid bacteria (LAB) have shown great potential in inhibiting food pathogens. However, majority studies on antimicrobial properties of bacteriocins only use the conventional planktonic forms of LAB. This study evaluates the potential application of LAB biofilms to inhibit formation of biofilms by pathogenic bacteria. It is thus proposed that the biofilms formed by LAB could be a promising tool for the control of pathogen biofilm formation. In this study, biofilm-forming LAB exhibiting antimicrobial properties were isolated from local fruits and dairy products. From the total of 21 distinguished LAB isolates, four isolates were chosen for their range of antimicrobial activity and comparable biofilm strength. The antibacterial activity in biofilms by two bacteriocin producers LAB (Lactobacillus casei Y1, Lactobacillus plantarum Y5) and two non-producers (Lactobacillus casei G5, Lactobacillus plantarum KF) will be evaluated against two common foodborne pathogen, Salmonella spp. E5 and Lis-teria monocytogenes L10. The small scale model of LAB biofilms will be developed in 24-well microtiter plates and upon pathogen incubation, the cells attached to the wells (biofilms) will be enumerated. The statistical significance of results will be determined using a t-test (95% confidence interval). The results from this study could highlight the importance of analyzing biofilms of bacteriocinigenic LAB to enhance their antimicrobial efficacy. Preferably, these protective biofilms could be a better alternative to control the formation of biofilms by deadly pathogens in the food industry.

KEYWORDS: bacteriocins, biofilms, food-borne pathogen, lactic acid bacteria


43

SUPPRESSION OF ANTHRACNOSE DISEASE ON CHILI BY LOCALLY ISOLATED FUNGI FROM SOIL SAMPLE

Wan Nurhaida Azlin Wan Hassan1, Wan Zuhainis Saad1, Mohd Termizi Yusof1

Department of Microbiology1, Faculty of Biotechnology & Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor.

Anthracnose disease is one of the major constraints to chili production worldwide in-cluding in Malaysia. Ineffectiveness of commercial fungicides and harmful effect to human health and soil fertility lead to the application of biological control agents (BCAs). In this study, selected thermophilic and mesophilic fungi from local soil sam-ple as BCAs was investigated. The preliminary result shows that the extract of ther-mophilic fungi at 1 mg/ml did not show any significant inhibition zone when tested with disc diffusion method. Among the antagonistic microorganisms, Trichoderma har-zianum, Aspergillus species and isolate U1 from paddy soil had proved their effective-ness as BCAs. The inter-fungal interaction studies by dual culture method observed in isolate fungiU1 showed zone of inhibition indicated there is antibiosis, competition, exploitation or vice versa. In present study, fungi agentsinvolved in infected chili types of Cili Kulai and Cili Kecil, with anthracnose symptoms, was investigated. Twenty iso-lates were isolated from infected chili fruit and only 6 isolates suspected as Colleto-trichum sp. at first were subject to morphological character, colony growth and conidia shape. Pathogenicity test was carried out by wound drop injection method and the percentage of the infected plants was calculate after a define time from injection de-pending on the fungal species. Among the selected isolates, one isolate B17 and an-other isolate Ch2 showed high pathogenicity effect on Cili Kulai types. Both isolates were able to induce familiar dark ring symptom and reduce in fruit size of width and length. Meanwhile, one isolate L10 showed very low on the native cultivar. Keyword: Anthracnose, Colletotrichum sp., Pathogenicity


6

DECIPHERING PROTEIN-SOLVENT INTERACTION OF A COLD ADAPTED-ORGANIC SOLVENT TOLERANT LIPASE

Nurul Izzati Mohd Rosli. Abu Bakar Salleh. Mohd. Shukuri Mohamad Ali.

Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and

Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, Department of Biochemistry, Faculty of Biotechnology and Biomolecular

Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Psychrophilic enzyme have attracted attention in industrial applications due to their low optimum temperature and high activity at low temperature. Utilizing computer-aided software, the structure of the enzyme lipase AMS8 (LipAMS8) (isolated from psychrophilic Pseudomonas sp. obtained from Antarctic soil) with and without pres-ence of organic solvent will be studied. In this study, 12 ns MD simulation will be performed at different temperatures and concentrations of organic solvents for activity, structural flexibility and stability analysis. From the result, the structure of LipAMS8 will be modeled to explore the possible effect of critical point mutation towards activity, structure flexibility and stability of the enzyme. To validate the computational data, the critical point mutation will be conducted using site-directed mutagenesis experimentally. The information obtained could be a useful tool to design specific catalyst for industrial applications and molecular engineering purposes, in the near future.


7

FUNCTIONAL CHARACTERIZATION OF OIL PALM’S GIBBERELIC ACIDS (GA) GENES IN ARABIDOPSIS THALIANA VIA RNAI AND OVEREXPRESSOR

TECHNOLOGIES

Muhamad Afiq Abdul Halim1,2, Noor Azmi Shaharuddin.1, and Zubaidah Ramli2 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia1, Advanced Breeding and Biotechnology Centre, Malaysian Palm Oil Board2

Palm Oil has been bestowed with high prices with buoyant demand globally. How-ever, the economical lifetime of oil palm tree itself is short in the industry as tall palms are particularly difficult to manage and costly to maintain. Therefore, the plant height regulation will significantly extend the economic cropping cycle. Gibberellic acids-related genes are among the factors that regulate the height of the oil palm. In the present study, Paclobutrazol (PBZ) treatment on the oil palm plantlets has been inves-tigated and the effect of the treatment will be analyzed. Genes associated with gibberellic acids from oil palm are being isolated and characterized. Gateway® cloning system will be used to construct overexpression and RNAi-silencing vectors of the isolated genes associated with height regulation. Transformation of the constructs will be performed in the model plant Arabidopsis thaliana. The phenotypic changes of the A. thaliana of the overexpressor and RNAi lines will be analyzed and characterized. The changes of phenotypic characteristic will elucidate the physiologi-cal and biological function mechanism of the genes in the oil palm. Homology analysis through BLAST against the isolated genes from the oil palm shows the association of the genes with plant height regulation in many other plants species. This study sug-gests a green route for the identification and molecular characterization of the genes encoding member of gibberellic acids pathways which may add variations to the complex developmental and agronomic trait of this important Malaysian commodity. KEYWORDS: Oil palm, Gibberellins, Paclobutrazol


42

ENGINEERING ESCHERICHIA COLI UTILIZING SUCROSE FOR PRODUCTION

OF LIPASE

Siti Hajar Yusoff1,2, Suriana Sabri1,2, Adam Thean Chor Leow1,3 and Raja Noor

Zaliha Raja Abd Rahman1,2

1Enzyme and Microbial Technology Research Group, 2Department of Microbiology, 3Department of Cell and Molecular Biology,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor.

Lipase is an important enzyme for various industrial applications. Currently, this enzyme (and many others) is expressed in plasmid-based system. Plasmid-based expression has a number of disadvantages that could lead to reduction of recombi-nant protein productivity. One way to encounter this problem is by expression of recombinant protein from the genome. Another aspect that should be considered in production of recombinant protein industrially is the carbon source. This is because carbon contributes significantly to the overall production cost. Many attempts have been made to produce recombinant proteins in E. coli from molasses, a cheap car-bon source. However, E. coli cannot utilize sucrose which is the major sugar in mo-lasses. The aim of this study is to produce a stable recombinant lipase expression from the genome of E. coli strain that utilizes sucrose. The objectives of this study are to clone and integrate lipase gene into E. coli utilizing sucrose genome, to ex-press the recombinant lipase and to optimize the production in lab scale fermenter. The lipase gene will be cloned into the integrative KIKO plasmids. The gene cas-sette will be designed in such a way that different promoters can be cloned for ex-pression optimization. The gene cassette will then be integrated into the E. coli ge-nome using Red recombinase system. The expression of the different constructs will be determined in shake flasks and the optimization of the production will be done in lab scale fermenter. A combination of the approaches described above may permit the industrial scale utilization of E. coli for bioconversion of low-cost starting materials (sucrose-molasses) into industrially important enzymes. Furthermore, production of recombinant lipase from the stable expression in the genome can be a model for production of other industrially important recombinant proteins.


41

BIORETTING OF KENAF USING MICROBIAL ENZYMES

Li Yin Wong1, Wan Zuhainis Saad1,3, Rosfarizan Mohamad2, Paridah Md Tahir 3

1Department of Microbiology, 2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,

3Institute of Tropical Forestry and Forestry Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Retting is a process of fibre separation from the non-fibrous component. Bioretting using microbial enzymes offers an alternative to the current method of water retting as it is environment-friendly, shorter retting time, and controllable fibre quality. Kenaf fibre has vast application in paper and pulp industry, textile industry and automobile indus-try. The objectives of this study are: isolation of a locally pectinolytic fungus, optimiza-tion of the pectinases production, characterization of the pectinases and characteriza-tion of pectinases-retted kenaf fibre. A potential strain of pectinolytic fungus isolated from kenaf retting tank, identified as Aspergillus fumigatus R6 was chosen based on the plate screening test and quantitative pectinases assay. Rice bran was found to be a good low-cost fermentation substrate for pectinases production by A. fumigatus in solid state condition. The production of pectinases was also evaluated at different initial moisture level, pH, temperature and time using one-factor-at-a-time approach. Central composite design was further applied to optimize the condition for high pro-duction of pectinases enzyme production. Maximum productivity of pectinases was under optimum condition of 49.6% initial moisture level, 33°C and 5.37 days of incu-bation. Interestingly, the pectinases production of A. fumigatus was in a broad range of acidity. The pectinases enzymes produced by the studied strain obtained its high-est activity at 65°C, however, not heat stable. pH 5 was optimum for pectinases hy-drolysis, pH stability was found to be in wide range of acidity. Samples of kenaf were treated with crude enzymes solution of A. fumigatus R6. Fibre surface morphology and physical-chemical properties were analyzed. A. fumigatus R6 pectinases sepa-rated the fibre from non-fibre component effectively. A retting time of 24 hours was able to produce good quality kenaf fibre. Different enzymes formulation and retting condition on the fibre quality were further investigated to improve the fibre quality. Keywords: Retting; Kenaf; Aspergilus fumigatus; Fermentation; Pectinases.


8

STUDY OF ANTI-AMYLASE, ANTI-GLUCOSIDASE, ANTI-GLYCATION AND

ANTIOXIDANT POTENTIALS OF COCONUT TESTA (MATURE AND TENDER)

AND VARIETIES OF BEANS SEED COAT

Adekola K.A.1, Marikkar M.N.1, Salleh A.B.1, Azlan A.2

Department of Biochemistry1, Faculty of Biotechnology and Biomolecular Sciences,

Department of Nutrition and Dietetics2, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia.

Coconut testa and beans seed coats are significant sources of antioxidants from phenolic compounds. Due to the correlation between phenols and carbohydrate enzyme inhibitory activities, this study will investigate the inhibitory potential of the coconut testa and beans seed coat on alpha amylase, alpha glucosidase, glycation and free radicals. The Anti-hyperglycaemic and antidiabetic effects of the extracts on animals will also be studied. The results will give a better understanding on the efficacy, safety and efficiency of the extracts in relation to diabetes. It is expected that ethanolic extracts of coconut testa and beans seed coat will show inhibitory potential on alpha amylase, alpha glucosidase and glycation, thus could be used as a natural source of antidiabetic therapy.

KEYWORDS: Diabetes, hyperglycaemia, α-amylase, α-glucosidase, glycation, anti-oxidants, coconut testa, beans seed coat


9

MOLECULAR CLONING AND RECOMBINANT EXPRESSION OF CYSTEINE PRO-TEASE INHIBITOR (PHYTOCYSTATIN) AND KUNITZ-TRYPSIN INHIBITOR (KTI)

FROM TURMERIC, CURCUMA LONGA.

Chan Seow Neng1, Norliza Abu Bakar2, Maziah Mahmood1, Ho Chai-Ling3 and Noor Azmi Shaharuddin1*,

1Department of Biochemistry, 3Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

2Biotechnology Research Center, Malaysian Agricultural Research and Development Institute (MARDI).

Protease inhibitors (PIs) are biochemical compounds commonly found in plants to regulate proteases activities and act as defense mechanism against pathogens and insects. Well-known PIs such as cysteine protease inhibitor (CYP) and kunitz-type trypsin inhibitor (KTI) have been reported to show anti-pathogenic properties and plant stress tolerance. Hence, numerous plant PIs genes have been identified and their recombinant proteins were produced as active anti-pathogenic compound. In order to overcome the resistance continuously developed by pathogens, it is important to discover new PIs gene from novel sources like turmeric (Curcuma longa) where there are no PIs have been characterized yet from the plant. In this experiment, cDNA fragments of CYP and KTI were identified from leaf samples of the turmeric by using degenerate primers designed from conserved regions of respective PIs from different plant species. The full-length cDNA, designated Cypcl and ClKTI, were obtained using rapid amplification of cDNA ends method (RACE) with complete open reading frames detected. The cDNA sequences have been deposited in NCBI database with the accession number of KF545954.1 (Cypcl) and KF889322.1 (ClKTI). ORFs of both PIs had been subcloned into expression vectors and the recombinant proteins were overexpressed in bacteria systems. Optimum conditions for the recom-binant protein expression were determined and the detection of expressed recombi-nant proteins was performed with western blotting. A large-scale recombinant protein expression was conducted and followed up by affinity-chromatography purification. The partially purified recombinant protein will be subjected to inhibitory and anti-pathogenic test.

KEYWORDS: Protease inhibitors, cysteine protease inhibitor, kunitz-trypsin inhibitor, turmeric, anti-pathogenic


40

ANTIBACTERIAL ACTIVITES OF LOCALLY ISOLATED FUNGI FROM PENINSULAR MALAYSIA

Mohamad Khairil Radzali1, Sieo Chin Chin1, Syahida Ahmad2, Wan Zuhainis Saad1*

1Department of Microbiology, 2Department of Biochemistry Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia 43400 UPM Serdang, Selangor, Malaysia

To date, there is data paucity of thermophilic fungi on natural products. Abundant of journals published on thermophilic fungi worldwide focussing more on its thermosta-ble enzymes for industrial applications despite on drug discovery. The emergences of drug-resistant microorganisms (superbugs) have caused certain antibiotics that available in markets nowadays, are irrelevant to be used. Fungi acts as reser-voirs of bioactive compounds for biological activities exploitation, and an increasing number of metabolites are being isolated. Thus, this research is conducted to iso-late local thermophilic fungi from Hulu Langat Hot Springs and to assess their anti-bacterial activitiy against four Gram-negative as well as four Gram-positive test bac-teria. The methanolic fungal extracts was screened for its antimicrobial properties using disc diffusion method against test microorganisms at 500 µg/disc and 1000 µg/disc after the isolates, HHS01 and HHS02 isolates were cultivated at 11th days and 20th days, respectively based from the growth profiles. The extracts of HHS01 isolate showed highest zone of inhibition (18 mm) against Bacillus subtilis ATCC 6633 and has broad spectrum antibacterial properties to all Gram-positive test bac-teria (Methicillin-resistant Staphylococcus aureus S547, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633 and Listeria monocytogenes L10) with minimum inhibitory concentration (MIC, 0.097– 12.5 mg/ml) and minimum bacteri-cidal concentration (MBC, 0.195-25 mg/ml) ranges. No surviving cells were de-tected at 15 h after treatent with 2MIC of the extracts for time-kill assays. HHS02 methanolic extracts has shown weak zone of inhibition against S. aureus ATCC 6538 yet none to the rest of test microorganisms. The isolate may provide such potentially promising sources of new compounds in drug discovery. KEYWORDS: antibacterial activity, bioactive compounds, thermophilic fungi


39

EXPLORING THE ROLE OF N-TERMINAL OF L2 LIPASE BY FUNCTIONAL AND STRUCTURAL APPROACHES.

Nur Fadhliah Rusmi1, Fairolniza Mohd Shariff1,2 Raja Noor Zaliha Raja Abd

Rahman1,2, Abu Bakar Salleh1,3,

1Enzyme and Microbial Technology Research Center, 2Department of Microbiology, 3Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science,

Universiti Putra Malaysia, Serdang, Selangor 43400 Malaysia Proteins are made up of linear sequences of amino acids involved in many physio-logical processes in all organisms. Knowledge of the 3D structure is crucial in order to understand the protein functions and this information is important in designing new proteins with specific functions. This sequence of protein is important for func-tionality; even the single amino acids mutation could contribute to disruption of the entire folding or the stability of protein. However, until recently no experimental evi-dence was available concerning the role played by N-terminal residues in protein stability. For this reasons, it is an advantage to study the function of amino acids residues at N-terminal of protein through rational protein engineering. Thus, in this study, the role of N-terminal residues in thermostable L2 lipase will be investigated. The 3D crystal structure of the L2 lipase will be used and the mutation point will be selected via in silico method. Site-saturation mutagenesis method will be used to substitute the amino acids. The expression of the best mutants will be optimised while the enzymatic activity and the physiochemical properties will be studied. The 3D structures of the mutated and wild type L2 lipase will be elucidated by X-ray crystallography method and the stuctures will be analysed and compared to the recombinant wild-type of L2 lipase. Keywords : N-terminal, L2 lipase, site-saturation mutagenesis, protein structure, crystallisation, thermostable


10

USE OF MINI PROTEIN THAT MIMICS URICASE IN THE DEVELOPMENT OF NANOWIRE BASED BIOSENSOR

Arilla Sri Masayu Abd Rahim1,2, Abu Bakar Salleh1,2, Normi Mohd Yahaya1,3,

Mohd Adzir Mahdi4

Enzyme and Microbial Technology Research Center, Universiti Putra Malaysia, 43400 Serdang, Selangor 1

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor 2

Department of Cell Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor 3

Department of Computer and Communication Systems Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor 4.

There is an Increase in demand for enzyme as bioreceptors for detection of metabolites. Native enzyme may not be convenient because of the instability issue. Uricase is an enzyme that participates in catalyzing the oxidative breakdown of uric acid to allantoin. The increasing amount of uric acid causes pathological conditions such as gout. Therefore, the existence of small size of enzymes, active and function can be a good improvement in the biosensor field. Five mini proteins comprising 20, 40, 60, 80 and 100 amino acids were constructed from the active site clefts of the two subunits and the structures modelled by YASARA software. The best evaluated structures of each mini protein were simulated with molecular dynamics (MD) simula-tions using YASARA in periodic box filled with water at long simulation time (20 ns). Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Solvent Accessible Surface Area (SASA) and radius of gyration were used in MD simulations analysis to study protein folding and stability. To validates, five recombi-nants of mini proteins (20, 40, 60, 80 and 100-residue) were constructed in pET32a vector. All recombinant mini proteins were successfully transformed into expression host (E.coli Bl21 (DE3)). The smallest mini protein (20 amino acids) was fused with his-tag at N-terminal and over expressed heterogeneously. Interestingly, this novel 20 amino acids mini protein showed positive binding interaction towards uric acid via circular dichroism (CD) spectra and isothermal titration calorimetry (ITC). Direct elec-trochemistry of mini protein immobilized on modified screen printed carbon electrode (SPE/NWs/Cys/GA/mini protein) and its application as a disposable sensor were measured by electrochemical measurements with cyclic voltammetry (CV) using AUTOLAB potentiostat/galvanostat. Keywords: uricase, mini protein, homology modeling, molecular dynamics, CD spec-tra, ITC, CV


11

BIOCHEMICAL AND PHYSIOLOGICAL RESPONSES OF SELECTED MALAYSIAN RICE CULTIVARS TREATED WITH EXOGENOUS OSMOPROTECTANTS UNDER

SALINITY STRESS

Teh Chui Yao1, Noor Azmi Shaharuddin1, Ho Chai Ling2 Maziah Mahmood ¹´³

¹Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, ²Department of Cell and Molecular Biology, Faculty of Biotechnology and

Biomolecular Sciences ³Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Rice (Oryza sativa L.) is one of the most important food crops in the world and also a popular model plant for monocot studies. Salinity is considered as one of the most brutal environmental problem that limits plant growth and productivity. Osmoprotec-tants or compatible solutes are low molecular weight, freely soluble organic compounds that are non-toxic even present at a high concentrations in cytosol. They serve to raise osmotic pressure in the cytoplasm and protecting the cellular component from dehydration under stress. To date, there is limited information on the effect of salt stress and the effectiveness of exogenous osmoprotectants in mitigating the salt-induced damages in Malaysian rice cultivars. Hence, the in vitro multiple shoot regeneration system was developed using MR 220 and MR 253 rice cultivars as a model to study the growth performance. The results showed that KIN (Kinetin) at 4-6 mg/L proved to be more effective as compared to BAP (6-benzylaminopurine) in pro-ducing relatively high number of quality shoots. Evaluation on the effect of salinity (0-300 mM NaCl) on the growth of rice shoot apices revealed that the susceptible concentration for both cultivars was 150 mM NaCl resulted in reduction in all growth parameters measured. Proline and glutathione showed promising effects in alleviating salt-induced damages as compared to glycine betaine and ascorbic acid. Further study on the nitrogen metabolism enzyme activities as affected by exogenous proline showed that shoot GS glutamine synthetase (GS, EC 6.3.1.2) and root NR nitrate reductase (NR, EC 1.6.6.1) activities were greatly reduced by salt-stress. Improved nitrate ion content, NR and GS activities were observed under salt-stress through the external supply of proline. It was concluded that exogenous proline was able to mitigate salt-induced damages through improving the overall plant growth perform-ance and the alteration in few key nitrogen metabolism enzymes activities.

Keywords: nitrogen metabolism, proline, salinity, shoot apices


38

DISCOVERING THE POTENTIAL OF NITRIC OXIDE TREATMENT ON ER-LOTINIB RESISTANT NON SMALL CELL LUNG CANCER CELLS

Joanne Ch'ng Yu Rou, Mashytah Abdul Karim, Nurhidayah Roslan

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia In 2012, there were an estimated 1.8 million newly diagnosed lung cancer cases, and around 1.5 million deaths due to this disease. Although advances have been made in available and affordable treatments for patients, the overall five year sur-vival rate remains at 15%. About 70% of all lung cancer cases are caused by non-small cell lung cancer (NSCLC). NSCLC cells with epidermal growth factor receptor (EGFR) mutations are particularly sensitive to erlotinib, a Tyrosine Kinase inhibitor (TKI). Patients with the mutation show drastic improvement within weeks of starting treatment. Erlotinib inhibits EGFRs, which regulate cell proliferation, by binding to the tyrosine kinase site and preventing signaling pathways from being activated. However, drug resistance to erlotinib in NSCLC cells typically occur within 12 months of the initial treatment, causing metastasis and tumour progression. Since Nitric Oxide (NO) has been shown to play a role in apoptosis, its potential as a main or complementary treatment should be explored. H1299 NSCLC cells were ex-posed to increasing concentrations of erlotinib over a period of 4 months until they were resistant to concentrations of 10 µM of erlotinib. After drug resistance was established, both non-resistant and resistant cells were treated with varying concen-trations of DETANONOate, an NO donor with a half life of 20 h at 37oC. DETA-NONOate compensates for the short half life of free NO radicals by producing a steady concentration of NO. Assays for cell viability, cell cytotoxicity, and reactive oxygen species were carried out and recorded. The IC50 of the erlotinib-resistant NSCLC cells towards NO was determined. The effect of a steady concentration of NO on resistant and non-resistant NSCLC cells were examined. The feasibility of using NO as a complementary or main treatment for erlotinib resistant NSCLC cells was analysed. Keywords: erlotinib, drug resistance, lung cancer, nitric oxide, epidermal growth factor receptors


37

DEVELOPMENT OF A HIGH-THROUGHPUT CELL-BASED ASSAY FOR SCREENING OF HYPOXIA-INDUCIBLE FACTOR ACTIVITIES

Sien-Yei Liew1, Eric J. Stanbridge2, Khatijah Yusoff1, Norazizah Shafee1

1Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Salangor, Malaysia.

2Department of Microbiology and Molecular Genetics, School of Medicine, Univer-sity of California at Irvine, Irvine, California, 92717.

In recent decades, advancements in developing small scale yet high-throughput preclinical cell-based bioassay systems tremendously accelerate the screening and identification of new therapeutic drugs. These cell-based assays are preferred due to more efficient, cost-effective and biological disease-relevant in drug screening. Hypoxia, low oxygen tension, stabilizes the hypoxia-inducible factor (HIF). HIF is a transcription factor that is responsible for cellular adaptation to hypoxia. HIF has been shown to be involved in various cellular regulations in normal cell functions as well as disease development and progression. This discovery has raised the need for a more robust cell-based assay system to measure HIF activities. Unfortunately, the currently available systems are costly and lack sensitivity. Therefore, the aim of the present study is to develop a highly sensitive and robust cell-based assay sys-tem that can be used to evaluate HIF activities in drug discovery. We have gener-ated a genetically-modified osteosarcoma cell line, Saos-2, which stably expressing a quadruple tandem repeat of HRE in erythropoietin (EPO) gene, fused to a firefly luciferase reporter gene in hypoxia. This reporter cell line was extremely sensitive in response to a true hypoxic condition by at least 500-fold of increment in comparison to a normoxic signal. The high intensity signal remained over at least 45 in vitro passages. The specificity, linearity, precision, accuracy and robustness of the assay were all found to be statistically significant (p<0.05). The assay quality was as-sessed and graded as excellent assay at Z-factor of 0.7. Findings from this study indicated the assay is applicable to the high throughput screening format of drug candidates. This finding helps to contribute towards accelerating identification of HIF-related molecules in drug discovery. Keywords: High-throughput cell-based assay, Hypoxia-inducible factor (HIF) activi-ties


12

BIODEGRADATION OF CYANIDE BY FREE AND IMMOBILIZED CELLS OF SERRATIA MARCESCENS ISOLATE AQ07

Kabiru Ibrahim Karamba1, 2, Moh’d Arif Syed1, Moh’d Yunus Shukor1,

Siti Aqlima Ahmad*

Faculty of Biotechnology and Bio-molecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia1,

Faculty of Science, Department of Microbiology, Bauchi State University, Gadau Main Campus, Bauchi State, Nigeria2.

Cyanide is a very toxic chemical and is one of the environmental pollutants in sewage. Serratia marcescens isolated from soil sampled in Universiti Putra Malaysia was found to have cyanide degrading capability. Spectrophotometric method was used to examine the biodegradation ability of the bacteria in free and immobilized forms using cyanide incorporated buffer medium. Factors affecting cyanide biodegradation such as carbon and nitrogen sources, pH of medium, inoculums size, cyanide concentration and temperature were optimized using one factor at time and response surface methods. Cyanide tolerance and effect of heavy metals (silver, arsenic, cadmium, cobalt, chromium, cupper, mercury, nickel, lead and zinc) were investi-gated. The results illustrates that glucose at 5.5 g/L, yeast extract at 0.55 g/L, pH 6, 20% inoculums size, 200 mg/L cyanide concentration and 32.5 ºC are the optimum biodegradation conditions required by the bacteria. Immobilized form of the bacteria showed better biodegradation in terms of time duration as it degrades the cyanide in 24 hours compared to free cells that degrades in 72 hours. The bacteria can tolerate 700 mg/L cyanide concentration in free cell and 900 mg/L in immobilized forms. Heavy metals tested at 1 ppm illustrates that the bacteria can stand their effect with the exception of mercury which degrades only 24.7% in free cell and 61.6% in immobilized forms. Serratia marcescens isolate AQ07 has the ability to degrade cyanide and suitable growth and biodegradation conditions were obtained using the optimization methods. It demonstrates that immobilized cells of the bacteria have greater ability for cyanide biodegradation compared to free cell of the bacteria which can be applied for cyanide treatment in sewage. It has been registered in the gene bank as isolate AQ07 with assigned accession number KP213291. KEYWORDS: Serratia marcescens, cyanide biodegradation, free cell, immobilized


13

PROMOTER PREDICTION SYSTEM FOR RECOMBINANT LIPASE EXPRESSION IN PICHIA PASTORIS

Fatima Gogo Mayaki1,2, Siti Nurbaya Oslan1,2*, Arpah Abu4, Abu Bakar Salleh1,2,3

1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia;

2Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia;

3Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia; 4Institute of Biological sciences, Faculty of Sciences, Universiti Malaya, Malaysia.

In industrial enzyme production, many genes are cloned and expressed in various hosts. Most of the time, the choice of promoter and host are by trials and error requiring a lot of time and effort. Therefore, an effective prediction system is necessary to predict the expression of an enzyme in a host. In this study, a promoter prediction system for recombinant lipase expression in Pichia pastoris will be developed using bioinformatics tools. In order to develop the prediction system data of lipases expressed in P. pastoris will be collected from different sources mostly published data like the lipase name, source, level of expression, promoter and strain used for expression. This data will be used to develop a database in MySQL which will be used for the algorithm of the prediction system. The prediction system will be validated experimentally in the laboratory. KEYWORDS: Pichia pastoris, level of expression, lipase, database


36

REDESIGN OF THERMOSTABLE T1 LIPASE BASED ON SPACE-GROWN CRYSTAL STRUCTURE

Siti Nor Hasmah Ishak1, Mohd Shukuri Mohamad Ali1,4, Adam Leow Thean Chor 1,3,

Nur Hafizah Kamarudin 1,3, Raja Noor Zaliha Raja Abd Rahman1,2.

1Enzyme and Microbial Technology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia. 2Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 3Department of Cell and Molecular Biology, Faculty of Biotechnology and

Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

4Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

The lack of sedimentation and convection in microgravity environment has become a favorable condition for growing protein crystals. Microgravity environment has been found to provide an effective condition for growing high quality protein crystals with improved in size and better internal orders. The improvement of the quality of protein crystals lead to a better understanding of the structural mechanism and their biologi-cal activity for the production of high quality enzymes. Under collaboration of UPM-JAXA protein crystal growth (PCG) #2 Flight program in 2013, crystallization of ther-moalkalophilic T1 lipase derived from Geobacillus zalihae has been succeeded under microgravity condition using counter diffusion crystallization method. Therefore, the structure obtained will be employed as model in this study to design new enzyme with improve characteristics. Molecular modeling program and mutagenesis will be applied to manipulate the structure of enzyme. The mutants of T1 lipase obtained in this study will be purified via affinity chromatography and ion exchange chromatography. The effects of temperature, substrates, surfactants, metal ions, organic solvents tolerance and pH on enzyme activity and stability will be studied. The new enzyme will be crys-tallized using modified capillary counter diffusion method. The crystals obtained will be used for X-Ray diffraction analysis and followed by structure determination. This study will enhanced our understanding of enzyme structure and their potential in biotechnol-ogy industry. KEYWORDS: microgravity, crystallization, Geobacillus zalihae, T1 lipase


35

STRUCTURAL INVESTIGATION OF THE C-TERMINAL RESIDUES IN THERMO-STABLE L2 LIPASE

Hartini Ahmad Sani 1,2, Fairolniza Mohd Shariff 1,2, Raja Noor Zaliha

Raja Abd. Rahman 1,2, Abu Bakar Salleh1,3 and Adam Leow Thean Chor1,4

Enzyme and Microbial Technology Research Center1, Department of Microbiology2, Department of Biochemistry3, Department of Cell and Molecular Biology4,

Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;

The substitutions of the amino acid residue at the predetermined of the critical point at the C-terminal of the native L2 lipase may cause increases on its thermostability and enzymatic activity, or even otherwise may speed up the unfolding of the protein structure. Prediction of the critical point of the stability of the L2 lipase structure will be done by analyzing 20 amino acids of its C-terminal residues by using I-Mutant2.0 software. The effects of the substitution of the amino acids at the critical point will be analyzed with Molecular Dynamics (MD) simulation by using Yet Another Scientific Artificial Reality Application (YASARA) software. The best predicted mutants will be generated in the wet lab by site-directed mutagenesis and the over expression of the enzymes from the successful mutants will be optimized. The best mutant will be chosen for purification and characterization. The physico-chemical properties of the best mutant will be explored and the thermostability of the mutated lipase will be studied. Highly purified mutated lipase will undergo crystallization screening and optimization. The best crystals will be diffracted and analyzed to obtain the best 3D structure of the mutated lipase. The 3D crystal structures of the mutated L2 lipase 3D structure will be compared to the structure of the native L2 lipase.

Keywords: L2 lipase, thermostability, site-directed mutagenesis, Molecular Dynam-

ics (MD) simulation


14

TOTAL ANTIOXIDANT AND FLAVONOID PROFILING OF SELECTED ZINGIBERACEAE (GINGER) RHIZOMES AND ANTI-SKIN AGING EFFECTS

1Alafiatayo Akinola A., 1Noor Azmi Shaharuddin, 1Syahida Ahmad,

2Lai Kok Song and 1, 3 Maziah Mahmood*

1Department of Biochemistry, 2 Department of Cell and Molecular Biology, 3 Institute of Tropical Agriculture,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Plants are source of precursors of many natural products and secondary metabo-lites with pharmacological and therapeutic potentials. Zingiberaceae are plants with both anti-oxidative and anti-skin aging properties. Skin aging is the gradual building up of molecular damages as a result of production of superoxide from cellular oxi-dative phosphorylation and the vulnerability of the skin to external damaging factor such as solar Ultraviolet radiation which lead to loss of skin structural integrity and degradation of extra cellular matrix (ECM) by certain enzymes. Therapeutic ap-proach to the management of skin aging is to induce the proliferation of dermal fi-broblast cells for the production of procollagen and subsequent inhibition of ECM degrading enzymes. In this study some selected species of Zingiberaceae were screened by DPPH and FRAP methods for Total antioxidant capacity and with HPLC flavonoids content were identified and quantified. Biochemical profiling was carried out using Total Protein, Lipid, Total Hydrolysable and reducing sugar, beta carotene and Ascorbic acids and with GC-FID fatty acids methyl esters was also profiled. The ability of extracts to proliferate human dermal fibroblast cells (ATCC 210-012®) was determined using MTT cell viability assay and anti-wrinkle potentials was assessed by the ability of extracts to inhibit ECM degrading enzymes (Elastase, Hyaluronidase and Collagenase) while toxicity effect was evaluated with embryos and larval of Zebrafish (Danio rerio). The results revealed high antioxidant capacity in Methanol extract of Curcuma xanthorrhiza and Curcuma longa of 245.40±0.5b mg/g/TE and 270.40±1.6e mg/g/TE respectively. These two extracts were found to be embryo toxic with similar teratogenic effects on the Zebrafish at concentration of 62.5µg/mL and above. The results provide an insight into the anti-aging potential of C. xanthorrhiza and C. longa and also support their folkloric us-age as anti-aging agent. These plants can be used in the prevention and manage-ment of premature skin-aging. Keywords: Antioxidant, Flavonoids, Skin-aging, Zingiberaceae, Zebrafish


15

BIOTECHNOLOGICAL APPLICATION OF NEWLY ISOLATED FEATHER DEGRADING BACILLUS SP KHAYAT IN KERATINASE, PROTEIN

HYDROLYSATE PRODUCTION AND BIODEGRADATION OF FEATHER WASTES

Ibrahim Yusuf1, Mohd Yunus Shukor1, Mohd Arif Syed1, Phang Lai Yee2 and Siti Aqlima Ahmad1*.

1Department of Biochemistry, Faculty of Biotechnology and Bio-molecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;

2Department of Bioprocess, Faculty of Biotechnology and Bio-molecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Slaughter and poultry houses generated large amount of both coloured and non col-oured chicken feathers which constitute environmental waste, but due to their cheap availability are being used in biotechnological removal of heavy metals from surface water. Heavy metal-laden and melanin containing feather wastes can be hardly used in the biological process of protease and protein productions despite their accumula-tion due to combined tough structure of keratin and toxic nature of heavy metals. Bio-degradation of the duo wastes is needed to replace the non-eco-friendly incineration method. Conditions influencing growth, keratinase production and feather degradation by a novel feather degrading bacterium Bacillus sp. khayat were studied. Free living and gellan gum immobilised cells of the bacillus were used to degrade all chicken feather wastes, mixture of different coloured feathers and feather laden heavy metals for the production of keratinase and protein rich hydrolysates. Statistical optimisation resulted in 11-fold increase in keratinase production (97.1 U/ml) over the initial 8.85 at pH 8, 2% inoculum, temperature 27 ºC and 36 h of incubation. The free bacterium grew well, produced higher keratinase activity and protein hydrolysates in black feather medium compared to brown and white feather media while the immobilised cells resulted in an increase in keratinase production from 90.9 to 117.5, 85.5 to 115.6 and 86.5 to 101.5 U/ml in black, brown and white feather media, respectively. About 90% of 5 g/l mixed coloured feathers were degraded in 36 h and both free and immo-bilised cells secrete higher keratinase enzyme in the presence of high concentration of some heavy metals, with higher tolerance in immobilised cells. The relatively high tolerance of the bacillus to heavy metals and its ability to degrade different types and mixture of feathers, demonstrated potential usage of the bacterium not only in biodeg-radation of highly recalcitrant melanised and heavy metal laden feathers, but also in producing keratinase enzymes and valuable soluble proteins for possible industrial usage. Keywords: Keratinase, melanin, heavy metals, immobilisation, gellan gum, optimiza-tion, Bacillus sp khayat, feather


34

INVESTIGATION AND GROWTH OPTIMIZATION OF LOCALLY ISOLATED PHENOL-DEGRADING FUNGI

Nadila Hanafee1, Siti Aqlima Ahmad2, Wan Zuhainis Saad1, Mohd Termizi Yusof1

Department of Microbiology1, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Department of Biochemistry2, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Phenol commonly present in the environment due to natural and chemical proc-esses. This compound exists in effluents of many industrial processes including petrochemical, pesticides, textiles and coal industries. Due to its toxicity, disposal of phenol gives a great impact to the environment and human beings. Even at low concentration, it can be toxic and lethal to organisms. Therefore, it is necessary to find solutions in removing phenol from the environment. Biodegradation is one of the potential and costs saving method for phenol removal. Many microorganisms have shown their ability to degrade phenol efficiently. In this study, 141 fungal strains were isolated and tested for phenol degrading ability. Only 16 fungal isolates gives positive result when grown on liquid mineral salt medium (LMS) supple-mented with phenol. Different concentration (100 mg/L, 300 mg/L, and 500 mg/L) of phenol were used to test the ability of each isolates to degrade phenol. Two isolates (N7P1C1 and N5P2C2) were shown to have the highest percentage of phenol deg-radation; 99% of phenol degradation in 5 days. Both isolates, N7P1C1 and N5P2C2 were isolated from coal industry at Dungun, Terengganu. The macroscopic, micro-scopic, and molecular analyses were done for identification purposes. The optimal condition including the effect of nitrogen source, concentration of nitrogen source, pH, agitation and temperature were determined followed by Response Surface Methodology (RSM) analysis. This study shows that fungal isolates N7P1C1 and N5P2C2 have the ability to degrade phenol.

KEYWORDS: Biodegradation, fungi, phenol


33

DISCOVERING ANTAGONISTIC BIOLOGICAL AGENTS FROM CARICA PAPAYA FOR POTENTIAL CONTROL OF PAPAYA DIEBACK DISEASE

Mariam Dayana Mohd Taha1, Noor Baity Saidi1, Raha Abdul Rahim1,

Umi Kalsom Md Shah2 and Amalia Mohd Hashim1

1Department of Cell and Molecular Biology, 2Department of Bioprocess Technology,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Papaya (Carica papaya) is an economically significant fruit crop grown in Malaysia. Nevertheless, the outbreak of Papaya Dieback disease has caused significant threats to the papaya plantation for almost a decade as effective treatment found to date has been limited. Endophytic bacteria have been used as biological control agent against several plant diseases. This study proposed that bacterial endophytes isolated from papaya plant might be used as biological alternative to synthetic bactericide to restrain this disease. The aim of this study is to find an effective microorganism to suppress Papaya Dieback disease. In this study, a total of 230 bacterial endophytes with antagonistic activities against Erwinia mallotivora were isolated from seeds and sarcotesta of two Carica papaya collected from Sabak Bernam (PPS) and Perak (PPK) respectively through rapid screening using Agar Overlay method. Twelve and twenty one pure isolates from the respective PPS and PPK showed significant inhibitory effect against Erwinia mallotivora as revealed by Agar Disc Diffusion technique. PPKSD19 and PPKST37 showed the highest value of inhibition zone at 21.7 mm and 15.7 mm, respectively during in vitro screening on MRS agar. Based on morphological and biochemical tests, all of the tested isolates were Gram-positive coccobacilli, catalase - negative and positive in acidity test, suggesting that they are potentially Lactic Acid Bacteria. All potent bacterial endophytes will be identified through 16S rRNA gene sequencing. The selected high-performing endophytic bacteria will be investigated for their synergis-tic activities and their effects on infected papaya plant will be tested through in vitro pre-colonization of endophytes in host plants. Our findings suggest that Carica pa-paya seed-borne bacterial endophytes could potentially be applied as biological control agent to inhibit Papaya Dieback disease. KEYWORDS: Antagonism, Biological control, Carica papaya, Endophytic bacteria, Erwinia mallotivora, Papaya dieback


16

yqiG PSEUDOGENE OF ESCHERICHIA COLI INFLUENCED HYDROGEN PRODUCTION

Muhammad Azman Bin Zakaria1, Mohd Zulkhairi Mohd Yusoff1, Mohd Ali Hassan1,

Mohd. Rafein Zakaria1, Toshinari Maeda2

Department of Bioprocess Technology1, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;

Department of Biological Functions and Engineering2, Graduate School of Life Sciences and System Engineering, Kyushu Institute of

Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan. Hydrogen is an attractive energy source since it is renewable, clean and high energy value. Modification of Escherichia coli genome has been extensively reported for hydrogen production due to accessibility of its genome information and well characterized protein functions. Hydrogen can be produced from glucose through glycolytic pathway. Glucose converted into phosphoenolpyruvate, pyruvate, and formate. Pseudogenes known as defunct genomics loci or junk genes whereas the sequence is quite similar to functional genes but lacking of coding potential. Pseudogene from active gene which evolutionarily lost their function as a result from mutation. Based on Eco gene database, 182 ORF’s listed as pseudogenes and 116 of them considered as uncharacterized genes (y genes). In this study, the role of yqiG pseudogene was studied to elucidate its contribution in hydrogen metabolism. Complex glucose and formate were used as substrate during fermentation analysis. Addition of IPTG into medium depends on the strains used and initial OD for inoculum was controlled appropriately. Deletion of yqiG pseudogenes in E. coli has shown lower hydrogen production compared to wild type. However, opposite effect observed with addition of complex formate as carbon source. Knockout of yqiG pseudogene reduces glucose consumption about 50% compared to wild type. From transcriptional analysis, high repression observed in pykF and pflB gene in absence of yqiG gene. Based on current finding, It is shows that yqiG is important in glycolytic pathway of hydrogen production. Hence, yqiG pseudogene in E. coli have been identified that plays an essential role towards hydrogen production. Keywords: hydrogen, yqiG pseudogene, Escherichia coli, glycolytic pathway


17

OPTIMIZATION OF SUPERHEATED STEAM TREATMENT FOR SURFACE MODIFICATION OF OIL PALM BIOMASS FIBERS PRIOR TO

BIOCOMPOSITE PRODUCTION

Muhammad Nazmir bin Mohd Warid 1, Hidayah Ariffin 1, Mohd Ali Hassan 1, Yoshihito Shirai 2

1 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, 43400 UPM Serdang, Selangor, Malaysia.

2 Department of Biological Functions and Engineering, Graduate School of Life Science and System Engineering, Kyushu Institute of

Technology, 2-4 Hibikino, Wakamatsu, Fukuoka 808-0196, Japan.

Incompatibility between hydrophilic lignocellulose fiber and hydrophobic polymer has become the resistance in producing biocomposite with good mechanical properties. Recent research has demonstrated that a green, non-chemical superheated steam (SHS) treatment is an effective method for surface modification of lignocellulose, whereby removal of hemicellulose from fiber can be achieved, making the fiber more hydrophobic, thus improving its compatibility with polymer. Nevertheless, the current retention time and temperature used for SHS treatment causing degradation of cellu-lose, which is an important component to provide high crystalline fiber. Therefore, this study was aimed at optimizing SHS treatment conditions for oil palm biomass fiber in such a way hemicellulose can be removed while retaining cellulose content. Three types of oil palm biomass fiber were used: Oil Palm Mesocarp Fiber (OPMF), Oil Palm Empty Fruit Bunch (OPEFB) and Oil Palm Frond (OPF). Our results showed that the optimum SHS treatment temperature and time for pretreatment of OPMF was 265°C / 5 mins with 63% and 2% hemicellulose and cellulose removal, respectively while for pretreatment of OPEFB was 280°C / 5 mins with 68% and 6% hemicellulose and cel-lulose removal, respectively. OPF had higher optimal pretreatment temperature at 300°C / 8 mins, with higher hemicellulose removal (84%), however retaining the level of cellulose removal (2%). Overall, it was shown that rapid removal of hemicellulose up to 84% from OPMF, OPEFB and OPF can be achieved with the use of high SHS treatment temperature, without affecting cellulose content much. Fiber with low hemi-cellulose and high cellulose content can be a good prospect as more compatible filler in biocomposite.

Keywords: Oil Palm Biomass Fiber; Pretreatment; Superheated steam; Surface modi-fication; Biocomposite


32

APPLICATION OF HEPATITIS B VIRUS NANOPARTICLES AS A

NANOCARRIER FOR DRUG DELIVERY

Roya Biabani khankahdani1, Noorjahan Banu Mohamed Alitheen2,3, Kok Lian Ho4, Wen Siang Tan 1,3

Department of Microbiology1, Department of Cell and Molecular Biology2, Faculty of Biotechnology and Biomolecular Sciences, Institute of Bioscience3,

Department of Pathology4, Faculty of Medicine and Health Sciences Universiti Putra Malaysia

Nanoparticles are rapidly being developed to overcome several limitations of tradi-tional drug delivery systems and are coming up as a distinct therapeutics carrier for cancer treatments. Self-assembled empty hepatitis B virus (HBV) nanoparticles with 35 nm diameter can be used as a potential nanocarrier for the development of a drug delivery system. In order to mediate internalization of HBV nanoparticles to the target cell, folic acid which interacts specifically with cancer cells was displayed on HBV capsid. Folic acid conjugated HBV nanoparticles were then loaded with doxorubicin and used to transfect different human cell lines. Translocated nanoparticles were detected by an antibody conjugated to Alexa Fluor® 488 and observed under a fluorescence microscope. Cellular uptake of doxorubicin was visualized by live cell imaging microscopy and quantified by using spectrophotometer. The result indicated that the conjugated HBV nanoparticles increased the uptake and cytotoxicity of doxorubicin in the colon cancer cells. On the other hand, our research group recently displayed Hexa-histidine on HBV nanoparticles. The His-tag can be linked to bioactive compounds via metal ions and mediates high efficiency delivery of the compounds to specific cells. Prior to the development of such linked nanocarrier, it is of importance to study the interactions between transition metal ions and His-tagged HBV nanoparticles. The thermody-namic and binding property of His-tagged HBV nanoparticles with metal ions were determined by nano Isothermal titration calorimetry (NanoITC). The results revealed that Zn2+, Ni2+, Co2+ and Cu2+ bound to His-tagged HBV nanoparticles with nanomo-lar affinity. The binding of divalent metal ions to His-tagged HBV nanoparticle was exothermic and spontaneous. The results showed the ability of His-tagged HBV nanoparticles to bind to various divalent metals with high affinities. This property can be used to link bioactive compounds to His-tagged HBV nanoparticles.


31

EXTRACTION AND CHARACTERIZATION OF SOLUBLE NON DIGESTIBLE POLYSACCHARIDES FROM COCNUT KERNEL CAKE (CKC) AND PALM KER-

NEL CAKE (PKC) AS POTENTIAL PREBIOTICS

Bashirat Bello and Shuhaimi Mustafa

Department of Microbiology, Faculty Biotechnology and Biomolecular Sciences,

The knowledge of consumers regarding healthy foods suggested that the basic role of diet is to provide nutrients to accomplish metabolic requirements and to regulate vari-ous functions in the body. This leads to the needs to produce new sources of prebiot-ics that are relatively low cost for public consumption. We hypothesis that PKC and CKC may have high yield of soluble non digestible polysaccharides that could be use as potential prebiotics. To test this hypothesis we will 1. Extract soluble polysaccha-rides from PKC and CKC. 2. The extracts obtained will be characterize and, 3. In-vitro testing of the soluble polysaccharide(s) on microflora. This study will focused on the extraction and characterization of soluble polysaccharides from CKC and palm kernel cake PCK as potential source of prebiotic. The extraction methods include: using three different solvents (Ethanol, n-hexane and Citric acid) to extract the soluble poly-saccharides from PKC and CKC. Proximate analysis will be carried out to see weather the samples meets up the specification that will be establish during the formation.The molecular weight of the soluble polysaccharides will be estimated using gel chroma-tography. Fourier Transform Infra-Red (FTIR) will be use to determine the degree of esterifiction and functional group of the soluble polysaccharides. Resistance towards digestibility of the extract obtain will be tested by calculating the degree of its hydroly-sis when administered to artifical human gastric juice. Prebiotic potential will be tested using fructooligosaccharides as control and soluble polysaccharrides under investiga-tion as the carbon source using B. longum, B. animalis and L. acidophilis in MRS broth medium as probiotics. Prebiotic activities will be tested using the pH meter and optical density to emulate the survivability of the bacteria. The outcome of the study will hope to explore new prebiotics that will meet the demand of consumers at lower cost. Key words: Prebiotics, soluble polysacharides, extraction, characterization


18

MOLECULAR CLONING OF CODON OPTIMIZED Β-GLUCOSIDASE GENE INTO SACCHAROMYCES CEREVISIAE FOR ENHANCEMENT OF BIOETHANOL PRODUCTION FROM OIL PALM EMPTY FRUIT BUNCH HYDROLYSATES

Mohamad Farhan Mohamad Sobri1,2, Norhayati Ramli2, Suraini Abd-Aziz2,

Farah Diba Abu Bakar3

1 School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysia

2 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

3 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

Environmental and economic concerns related to the use of fossil fuel have driven the need for alternative renewable fuel such as bioethanol. In Malaysia, abundance of oil palm empty fruit bunch (OPEFB) as lignocellulosic waste following palm oil production offers great potential as sustainable substrate. Consolidated biomass processing (CBP) involves biomass conversion into final product in a single step, possibly mediated by a single organism. For bioethanol production, engineering of common ethanologen Saccharomyces cerevisiae has resulted in heterologous expression of cellulase enzymes, allowing for simultaneous saccharification and fermentation. Enhanced bioethanol production was achieved following improved cellulase expres-sion, of which codon optimization is an applicable approach. A cellulase constituent, β-glucosidase, in particular is significant for preventing end product inhibition via cellobiose hydrolysis. As such, this research aims to improve the expression of rate limiting enzyme, β-glucosidase in Saccharomyces cerevisiae via codon optimization, to hypothetically lead to enhanced ethanol production. Work is currently underway on β-glucosidase gene isolation and characterization from locally isolated strain T richoderma asperellum UPM1. Following this, yeast will be transformed with shuttle vectors harbouring the codon optimized gene and the wild type sequence, respectively. Extent of cellobiose degradation, glucose liberated and the resulting bioethanol yield is then to be quantified via gas chromatography (GC) and high performance liquid chromatography (HPLC), which then will be compared to the wild type producer. In short, this research represents progression in enhancement of bio-ethanol production from oil palm empty fruit bunch hydrolysates.

KEYWORDS: β-Glucosidase, Saccharomyces cerevisiae, oil palm empty fruit bunch, bioethanol, codon optimization


19

As an attachment


30

THE IN-VITRO AND IN-VIVO STUDY OF STINGLESS BEE HONEY (HETEROTRIGONA ITAMA) ON LEARNING AND MEMORY DEVELOPMENT

IN MICE

Fairuz Nabila Zulkifli1, Nurhidayah Roslan1, Mohd Zulkifli Mustafa2

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia1. Department of Neurosciences, School of Medical Sciences,

Universiti Sains Malaysia2.

Honey is an insect-derived natural product with therapeutic and nutritional value. In addition to having excellent nutritional value, honey is a good source of physiologically active natural compounds, such as polyphenols and flavonoids, which act as antioxi-dants. Unfortunately, there are very few current research projects investigating the nootropic and neuropharmacological effects of honey, especially for stingless bee honey such as from Heterotrigona Itama. Raw honey possesses nootropic effects such as memory-enhancing effects and is belief to help in improving brain functions. However, most research that has been done to prove this memory enhancing effects are only from social study. Therefore, in order to prove this more scientifically, an in vitro study using Human Astrocyte (HA) cell line which is one of the brain glial cells that helps in memory and brain development will be done to investigate its synapto-genesis proses after the cells have been treated with the stingless bee honey. This study will also investigate the in vivo effects of the stingless bee honey supplementa-tion to the learning and memory development of four group of treated C57 / BL mice as compared to the normal controlled group through Intellicage Behaviour Test. In this study, it is hypothesized that the stingless bee honey will increase the learning and memory development of the brain by increasing the synaptogenesis process that oc-cur within the treated cells. It is also hypothesized that the honey supplementation to the four groups of C57 / BL mice will increase their potential in learning and memory development as compared to other control mice. The findings from this study will help in proving the scientific effect of this honey from in vitro and in vivo view. Therefore, it will also help in investigating the potential of this local breed honey and can further increase their commercial value. KEYWORDS: Heterotrigona Itama, Human Astrocyte (HA), synaptogenesis, Intellicage


29

THE POTENTIAL OF ENDOPHYTIC Trichoderma AS A BIOFUNGICIDE FOR Ganoderma BASAL STEM ROT INFECTION

Lee Pei Lee Angel1,2, Mohd Termizi bin Yusof1, Intan Safinar Ismail2,3, Bonnie Tay

Yen Ping4, Shamala Sundram5

Department of Microbiology1, Faculty of Biotechnology and Biomolecular Science; Department of Chemistry2, Institute of Bioscience3, Universiti Putra Malaysia,

43400 UPM Serdang, Selangor, Malaysia. Advanced Oleochemical Technology Division4, Biology Research Division5;

No. 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia.

Ganoderma boninense has been identified as the pathogen that causes basal stem rot (BSR), a devastating disease affecting the oil palm tree. Trichoderma has been recognized as a potential antagonistic biological control agent (BCA) against patho-genic fungi that are responsible for several plant diseases. The success of Trichoderma isolates as a potent BCA is due to the antagonistic characteristics such as mycoparasitism and antibiosis. Therefore, this study attempts to investigate the effects of endophytic Trichoderma isolated from roots of oil palm on G. boninense. Two potential endophytic Trichoderma isolates were assessed through simple in vitro assays, namely Trichoderma virens 7b and Trichoderma virens 159c. Compounds with different polarity from culture filtrates were extracted using n-hexane, ethyl acetate and n-butanol, respectively and tested against G. boninense (PER 71) for their antifungal activity. The active fraction was further fractionated with chromatographic methods and the composition of active fraction was analyzed with gas chromatography-mass spectrometry detector (GC/MSD). Siderophore detection was conducted with universal chrome azurol S (CAS) blue agar. The extracts from hexane (T. virens 7b) and ethyl acetate (T. virens 159c) were found to be most active against PER 71 with percentage of inhibition radial growth (PIRG) of 62.60 % ±6.414 and 78.39 % ±5.396, respectively. The activity of each extract was further proven with scanning electron microscope (SEM) where severe deformation of G. boninense mycelia observed at the inhibition region. T. virens 7b active fractions constituted of volatile compounds such as ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides and free fatty acids. T. virens 159c produced ketones, aldehydes, acetamide, alcohol, lac-tones and free fatty acids. Both Trichoderma strains were active in producing siderophores as CAS blue agar turned pale yellow. This study deciphers the poten-tial mechanisms involved in the suppression by endophytic Trichoderma on G. boninense. KEYWORDS: Oil palm, Ganoderma boninense, endophytic Trichoderma, volatile compounds, antifungal activity, siderophore


20

EFFECTS OF MONO AND DIUNSATURATED FATTY ACIDS ON PRODUCTION OF RHAMNOLIPIDS BY LOCALLY ISOLATED

PSEUDOMONAS AERUGINOSA RS6

Fairuzana Jaafar1, Helmi Wasoh @ Mohamad Isa1, Lai Oi Ming1, Murni Halim1

Department of Bioprocess Technology,

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia1

Pseudomonas aeruginosa is a primary rhamnolipids producer which able to produce rhamnolipids by utilization of both hydrophobic and hydrophilic substrates. Vegetable oils are the most studied hydrophobic substrate effectively promoting rhamnolipids production and they contain various fatty acids with different chain lengths and saturation level. In this study, palm olein (rich in oleic acid/C18:1/monounsaturated fatty acids) or sunflower oil (rich in linoleic acid/ C18:2/diunsaturated fatty acids) was blend with palm oil /palm stearin / coconut oil with glucose as co-substrate to enhance the rhamnolipids production. To this end the rhamnolipids produced from all blends were compared with media using only glucose as sole carbon source. The substrate combination of O:PO:Glu (3:3:3) and SFO:PO:Glu (1:1:3) was successfully produced 14.16 (15.57% of carbon conversion) and 13.62 g/l (32.50% of carbon conversion) of rhamnolipids respectively. The rhamnolipids production was estimated based on yields, surface tension reduction and emulsification index (E24). The cell free supernatants of this bacterium successfully reduced the surface tension ranging from 26 to 28 mN/m by using DeNouy ring tensiometer. The biosurfactant emulsified diesel and palm oil and showed stability in the face of salinity, exposure to high temperature and ex-treme pH. These physicochemical properties demonstrated the potential use of this rhamnolipids in various applications. Rhamnolipids were characterized by using High Performance Liquid Chromatography. The most abundant rhamnolipids con-gener obtained was identified as Rha-C10-C10 and Rha-C8-C10.This study suggested the difference production aspect to find suitable and economic substrate and develop new strategies to increase the volumetric productivity by using mono or diunsaturated fatty acids in dual substrates combination involving hydrophilic and hydrophobic substrate.


21

XYLANASE PRODUCTION BY PENICILLIUM OXALICUM T3.3 USING RICE STAW AS SUBSTRATE FOR BIOBLEACHING OF BAMBOO PULPS

Nur Idayu Zahari1, Umi Kalsom Md Shah1,2,3, Ainun Zuriyati Mohamed Asa’ari 2,

and Rosfarizan Mohamad 1,2,3

Department of Bioprocess Technology1, Institute of Tropical Forestry and Forest

Products2, Bioprocessing and Manufacturing Research Centre3, Universiti Putra Malaysia

Recently, xylanase raised high attention in pulp and paper industry due to their bleach-boosting properties, which reduces chemical consumption during bleaching process. Thus, the aims of this research are to determine the optimum cultural condition which enhance the production of xylanase enzyme and to evaluate the performance of the xylanase enzyme on biobleaching of bamboo pulps. Four fungi species namely Penicillium oxalicum T3.3, Aspergillus niger ATCC 6275, Colletotrichum gliosporoides and Pycnoporus sanguineus were screened for xylanases production based on the observation of clear zone formation on Malt Extract Agar (MEA) plate containing xylan. P. oxalicum T3.3 and A. niger ATCC 6275 showed a larger clear zone forma-tion on the agar plate. These fungi were grown in liquid media containing rice straw as substrate. P. oxalicum T3.3 showed highest xylanase activity (65.89 U/mL) with low-est carboxymethyl cellulase (CMCase) (1.88 U/mL) and filter paperase (FPase) activity (0.16 U/mL) after 4 days fermentation at 30 °C. By optimizing substrate concentration at 1 % and initial pH at 6, temperature 35 °C, agitation speed at 150 rpm, yeast extract as the nitrogen sources, and additional of Tween 80 as surfactants, it yields high xylanase production (79.12 U/mL). The xylanase enzyme was characterized and was found more stable at pH range of 4.0 to 7.0 and temperature ranging from 45 °C to 55 °C. The biobleaching of bamboo pulps with xylanase was the most effective at an enyme dose of 5 U/g oven dried pulp at pH 7 and incubate in water bath at 50 °C. Under the optimized conditions, xylanase pretreatment increased paper brightness by 7.71 points. Thus, cellulase poor xylanase produced from P. oxalicum T3.3 grown on rice straw has high potential for biobleaching applica-tion in pulp and paper industry in terms of technical and biological performance and economical aspects.


28

CRYSTAL STRUCTURAL ANALYSIS OF CONCISE CHALCONE SYNTHASE FROM BOESENBERGIA ROTUNDA (BRCHS)

V. Jiivittha1, Mohd Shukuri Mohammad Ali2, Raja Noor Zaliha Raja Abd. Rahman1

Department of Microbiology1; Department of Biochemistry2 ,

Faculty of Biotechnology and Biomolecular Sciences, Enzyme and Microbial Technology Research Group, Universiti Putra Malaysia,

Selangor 43400, Malaysia Chalcone synthase is a member of type III PKS family that catalyses the decarboxy-lative condensation of malonyl-CoA with a specific starter CoA to produce chalcone intermediate, a precursor for the synthesis of various classes of flavonoid. Flavon-oids are one of the natural therapeutic drugs that mainly derived from plants. The efforts made to understand enzyme mechanisms which involve in flavonoid biosyn-thesis pathway are expected to be resulted in discovery or production of enzymes or engineered enzymes with floricultural, biopharmaceuticals, food and chemical industries potential. Crystal structure of complete CHS was derived only from Medi-cago sativa. Therefore, there are no studies reported on expression and crystal structure elucidation of concise CHS from Boesenbergia rotunda, a herbal plant that has been used commonly as a food ingredient in Southeast Asia. In this study, a gene encoding concise CHS was cloned from B.rotunda and overexpressed by introducing recombinant pGEX-4T-1/ BrCHS into E. coli. Optimization of expression was resulted in high yield of concise BrCHS. Activity of concise BrCHS was deter-mined by HPLC assay when BrCHS produces a pyrone compound using hexanoyl-CoA as a starter CoA reacted with malonyl-CoA. Subsequently, BrCHS was purified to homogeneity prior to crystallization. Then, BrCHS crystal structure will be eluci-dated by using X-ray crystallography and potential active sites are expected to be visualized. Current research revealed that, concise BrCHS is equally active as com-plete CHS in spite of some missing amino acid residues at both ends of sequence. Crystal structure analysis will report on molecular properties of concise BrCHS that are responsible for their catalytic function. This study will be focused on BrCHS structure elucidation which is important to compare concise CHS structure from B.rotunda with complete CHS structure from M.sativa. This will provide insights into better understanding of CHS enzyme mechanisms and functionality. Additionally, this approach will enable selective modifications of structure for function enhance-ment. Keywords: chalcone synthase; crystal structure ; concise CHS ; expression ; flavonoid


27

ENZYME-ASSISTED EXTRACTION OF ESSENTIAL OIL FROM PINEAPPLE PEELS USING CELLULASE

Nurshazana Mohamad, Mohamad Faizal Ibrahim, Suraini Abd-Aziz

Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang.

The pineapple canning industry produces a substantial amount of solid waste like peels, cores, stems, crowns and pulp. Pineapple waste disposal can cause to micro-bial spoilage and environment problems due to the waste material containing high moisture and sugar content. Therefore this study is proposed to utilize the pineapple waste, focusing on the peels, to produce a high value added product of essential oil. The volatile compounds of pineapple peels will be extracted through a green approach using hydrodistillation method. In order to improve the essential oil yield, pretreatment of the substrate using commercial cellulase will be applied prior to the hydrodistillation extraction. The cellulase will act on the substrate’s cell wall and hydrolyse them and thus will increase the permeability, expecting to release more essential oil. In addition, several factors will be optimized to determine the optimum condition of enzymatic pretreatment and extraction process. The factors that will be involved are: enzyme loading (FPU/g), hydrolysis time (minutes), substrate to solvent ratio, extraction time (hour) and extraction temperature (ºC). With the optimized process condition, it is expected that a higher yield of essential oil will be obtained. The fatty acid and aromatic compounds of the obtained essential oil will be analyzed using GC and GC-MS, respectively. Moreover, the cell wall of the enzymatic pretreated substrate will be observed under Scanning Electron Microscopy (SEM) as compared to the untreated substrate. Overall, this study will be promoting a proper way for pineapple waste management by producing a high value added product of essential oil through an optimized green method of enzyme-assisted extraction. Keywords: Pineapple peels, essential oil, hydrodistillation, enzyme-assisted extraction


22

AERATION REQUIREMENTS FOR THE CULTIVATION OF OIL PALM (ELAEIS GUINEENSIS JACQ.) CELL SUSPENSION CULTURES IN STIRRED-TANK

BIOREACTOR

Farrah Mohamad Hatta1, Murni Halim1,2 and Arbakariya B. Ariff1,2

1Bioprocess and Biomanufacturing Research Centre,

2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor

Shake flask cultivation method has been successfully used for the propagation of oil palm cell suspension cultures. However, the monitoring and controlling of culture conditions in shake flask system are difficult. In addition, scaling up of cultivation using shake flask is not possible. Proper process control and monitoring of factors influencing the growth of plant cells may be achieved with cultivation in bioreactor system. The effect of four DOT levels (20%, 40%, 60% and 80% saturation) on growth of oil palm cells during batch cultivation, in terms of biomass production, sugar con-sumption, and germination response, was investigated in cultivation using 1 L stirred tank bioreactor (STB). The DOT level was controlled by the manipulation of air flow and gas blending system while the agitation speed, provided by marine type impeller, was fixed at 100 rpm. The DOT level cannot be controlled at DOT level higher than 80%, due to formation of excessive foaming at high air flow rate. Cell suspensions were established from immature leaf explants-induced embryogenic calli of oil palm inoculated in MS liquid medium supplemented with 1 mg litre-1 1-napthaleneacetic acid (NAA), 1 mg litre-1 2,4-dichlorophenoacetic acid (2,4-D) and 3% (w/v) sucrose as the carbon source. Inoculum size of 0.75%, which gave high cultivation performance in shake flask was selected as inoculum size for the cultivation using STB. Biomass production was increased almost linearly with increasing DOT level up to 80%. The biomass concentration obtained in cultivation where DOT was controlled at 80% was increased by about 14.1-times after 42 days as compared to biomass concentration after inoculation. The highest efficiency of sucrose hydrolysis to its monosaccharides, fructose and glucose, was also observed at DOT level of 80% saturation. The oil palm cells behaved in similar aggregation pattern throughout the cultivation in STB. Keywords: Oil palm, plant cell suspension cultures, dissolved oxygen tension, stirred-tank bioreactor, oxygen requirement


23

CHARACTERIZATION AND OPTIMIZATION OF BIOSURFACTANT FROM USED COOKING OIL BY LOCAL ISOLATES FOR HEAVY METALS REMOVAL

Nurul Hanisah Md Badrul Hisham, Mohamad Faizal Ibrahim and Suraini Abd-Aziz

Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular

Sciences, Universiti Putra Malaysia (UPM), 43400 UPM Serdang, Selangor, Malaysia

Soil pollution by heavy metals from industrial effluents and sewage creates serious problems in most developing countries. Biological methods for the removal of heavy metals from the industrial wastes using biosurfactants may provide an alternative solution to the problems. The eco-friendly and biodegradable nature of biosurfactants make their usage more favourable over chemical surfactants. Biosurfactants are surface-active compounds that exhibit the properties of reducing surface tension, stabilizing emulsions, promoting foaming, and are able to be synthesized from renew-able resources. This study aims to produce biosurfactant from renewable feedstock which is used cooking oil (UCO) by local isolates for heavy metals removal. Microor-ganism that is able to produce the highest amount of biosurfactant from UCO and possess the lowest surface tension will be selected as biosurfactant producer. Chemical characterization and structural analysis of the biosurfactant will be carried out. The ability of the selected microorganism to produce biosurfactant in extreme conditions also will be investigated since the compounds will be applied in harsh envi-ronmental conditions. The test will be conducted by using one factor at time approach in which wide ranges of temperature, pH and salinity will be studied. Further, statistical analysis using artificial neural network (ANN) will be adopted to screen five parameters namely substrate concentration, inoculum size, agitation speed, incuba-tion temperature, and pH of the medium for the optimization process of biosur-factant production. Finally, the potential of environmentally compatible biosurfactant produced by the selected microorganism to remove heavy metals specifically for chromium, cadmium, lead and zinc from artificially contaminated soils will be investi-gated. The present study will suggests a promising alternative to alleviate heavy met-als in the environments as well as recycling used cooking oil through the production of biosurfactant. KEYWORDS: Biosurfactant, used cooking oil, bioremediation, heavy metals


26

SIMULTANEOUS CARBONIZATION AND ACTIVATION OF OIL PALM KERNEL SHELL FOR ACTIVATED CARBON PRODUCTION

Nahrul Hayawin Zainal1, Astimar Abd Aziz2, Juferi Idris3,4, Mohamad Faizal

Ibrahim1, Mohd Ali Hassan1 and Suraini Abd-Aziz1

1Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

43400 Serdang, Selangor, Malaysia. 2 Biomass Technology Unit, Engineering & Processing Division,

Malaysian Palm Oil Board (MPOB), P.O Box 10620, 50720 Kuala Lumpur Ministry of Plantation Industries & Commodities, 6,

Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia 3 Faculty of Chemical Engineering, Universiti Teknologi MARA (UiTM) Sarawak,

94300, Kota Samarahan, Sarawak, Malaysia. 4 Faculty of Chemical Engineering, Universiti Teknologi MARA (UiTM)

Malaysia,40450, Shah Alam, Selangor, Malaysia. Malaysia is the second largest producers of palm oil in the world. Huge biomass is produced during oil palm processing. The availability of the oil palm biomass through-out the year as a source of materials is for bioconversion for biotechnological produc-tion. Due to huge abundant of oil palm kernel shell (OPS) (4.44 million tonnes in 2014), the production of activated carbon (AC) from oil palm biomass can be sustained. Production of activated carbon (AC) requires high energy and undergoes complicated method. Currently, the production of AC involved two steps process i.e carbonization and activation separately. For instance, 1) the carbonization of carbona-ceous precursor below 800°C, in the absence of oxygen, and (2) the activation of car-bonized product (char), and either use physical or chemical activation methods. To date, biochar activated carbon has been shown viable with the simultaneous activa-tion process (two in one) using oil palm shell (OPS) as substrate. In this study, a si-multaneous carbonization and activation process to be introduced and compared un-der self-sustained carbonization brick reactor and microwave carbonization. The ob-jectives of this study are to evaluate and characterize biochar activated carbon from oil palm shell at different activation temperature under simultaneous self-sustained carbonization and activation reactor and to evaluate and characterize biochar acti-vated carbon of oil palm shell at optimum activation temperature under simultaneous microwave carbonization and activation reactor. The biochar activated carbon will be characterized for proximate and ultimate analysis, surface area, calorific value, SEM and yield. The technology under simultaneous carbonization and activation can re-duce the cost and time production, hence suitable for oil palm industry for value added palm biomass waste. KEYWORDS: activated carbon, oil palm shell, self-sustained carbonization, micro-wave carbonization, simultaneous


25

DEVELOPMENT OF NEW CATANIONIC SURFACTANT VESICLES FOR DRUG DELIVERY APPLICATIONS

Xiou Shuang Yong1, 2, Lai Yee Phang1, Wen Huei Lim2

Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.1

Advanced Oleochemical Technology Division (AOTD), Malaysia Palm Oil Board (MPOB), 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor,

Malaysia.2

Catanionic surfactant is a relatively new class of surfactants. It is produced from an equimolar mixture of two oppositely charged of ionic surfactants, resulting a compound with no net charge. This type of surfactant exhibits extraordinary surface properties as compared to their individual parent surfactant. The rich diversity of phase behavior is due to co-adjustment of electrostatic effects and surfactant molecular geometry of different combination of ionic surfactants. Catanionic surfactant has the ability to self-aggregate into various structures such as vesicles, micelles, microemulsion, etc. in aqueous solution. These structures have attracted interests of many researchers. In this study, several new series of catanionic surfactants are syn-thesized by mixing equimolar ratio of different chain length of both alkyltrimethylam-monium bromide and carboxylic acid, respectively. Spectroscopy analysis such as Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FTIR), CHNS analyzer and Differential Scanning Calorimetry (DSC) are employed to identify and characterize the formation of new catanionic surfactant, alkyltrimethylammonium-alkylcarboxylate. As catanionic surfactant exhibits different behaviour as compared to their parent surfactant, investigation into their physicochemical properties and surface activities are conducted. Anti-microbial activities and irritancy properties of the catanionic surfactant are tested as well. The unique surface properties including low critical micelle concentration (CMC) and spontaneous formation of vesicles exhibited by the new catanionic surfactants indicated their potential use in various industrial applications. One of which the catanionic surfactant can be explored is as a vehicle to carry active ingredients (e.g. vaccine, anti-cancer chemotherapy, drug delivery, gene delivery, etc.).This study suggests the formation of a new catanionic surfactant for drug delivery applications.


24

CONTROLLED HYDROLYSIS OF POLY(3-HYDROXYBUTYRATE-CO-3-

HYDROXYHEXANOATE) FOR OLIGOESTER PRODUCTION

Dhurga Devi Rajaratanam1,3, Hidayah Ariffin1,2, Haruo Nishida4, Mohd Ali Hassan1

Department of Bioprocess Technology1, Faculty of Biotechnology and Biomolecular Sciences; Laboratory of Biopolymer and Derivatives2, Institute of Tropical Forestry

and Forest Product (INTROP), Universiti Putra Malaysia. Graduate School of Life Science and Systems Engineering3; Eco-Town Collaborative

R&D Center for the Environment and Recycling4, Kyushu Institute of Technology, Japan.

Polyhydroxyalkanoate (PHA) is a type of biodegradable plastic that being produced by many types of bacteria as an intracellular energy reserve material. However, the in-tracellularly produced PHAs have high molecular weight (200 - 3000 kDa) which makes them undesirable for the production of specialty polymers that require a low, specific range of molecular weight (1 - 5kDa). Therefore, an effective degradation method is necessary to produce oligoesters with desired low range of molecular weight. In the present study, controlled degradation of PHA by superheated steam (SHS) will be studied using two types of PHA, poly(3-hydroxybutyrate), PHB and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBHHx. Prior to hot-pressed films preparation, purified PHA samples will be characterized. The thermal properties, crys-talline properties and molecular weight will be determined using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and gel permeation chromatography (GPC) respectively. The percentage of HHx unit will be determined using proton nuclear magnetic resonance (1H-NMR). Then, controlled degradation of PHA films by SHS hydrolysis will be studied by monitoring the mass and molecular mass changes at different treatment temperatures (130⁰C, 150⁰C, 170⁰C and 190⁰C). The produced oligoesters will be analyzed using 1H-NMR and carbon-13 nuclear magnetic reso-nance (13CNMR) to determine the chemical structure and chain-ends formed during SHS treatment. On the other hand, the molecular weight reduction will be determined using GPC. Kinetic parameters of PHA hydrolysis will be evaluated using non-autocatalytic and autocatalytic hydrolysis mechanism. The influence of HHx unit com-position towards the SHS hydrolysis also will be evaluated based on the diad se-quence distribution. The current research results revealed that the controlled SHS condition caused the hydrolysis to occur specifically at the ester bond to produce PHA oligoesters. This study suggests a green route for controlled degradation and depoly-merization of PHA for the production of PHBHHx oligoesters for various packaging and medical applications. KEYWORDS: Polyhydroxyalkanoate, oligoesters, superheated steam, hydrolysis

final-23rd biotech colloquium 2015 tentative programme · 2020-03-14 · 23rd biotech colloquium...

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