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UNIVERSITI PUTRA MALAYSIA
IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM HOT SPRINGS IN MALAYSIA
AZURA LIANA BINTI KAMARUZAMAN
FSTM 2008 7
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IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA
ISOLATED FROM HOT SPRINGS IN MALAYSIA
By
AZURA LIANA BINTI KAMARUZAMAN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfilment of the Requirements for the Degree of Master of Science
January 2008
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Specially for my loving parents, Kamaruzaman Mohd Yatim and Rashidah Daud,
and siblings, Azila Asfar and Azrul Azlan
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM HOT SPRINGS IN MALAYSIA
By
AZURA LIANA BINTI KAMARUZAMAN
January 2008
Chairman: Associate Professor Nazamid Saari, PhD Faculty: Food Science and Technology
Lipase-producing thermophilic microorganisms were successfully isolated from water
samples collected from six hot springs in the west coast of Peninsular Malaysia and were
identified up to species level. Five hundred and forty-four microbial colonies were
qualitatively screened using Rhodamine B-olive oil agar plate method and sixty-six were
found to be positive as bacterial lipase producers after they formed orange fluorescent
colour around the colonies when irradiated with UV light. Lipolytic activities were
assayed using titrimetric method with olive oil emulsion as substrate. Comparative
analysis among the positive bacterial lipase producers indicated that isolate ST 7 had the
highest lipolytic activity of 4.58 U/ml, followed by isolate ST 6 with an activity of 3.51
U/ml. Eighteen isolates that showed high lipolytic activity were further examined for
thermostability. Thermostability was determined by incubation of the crude lipase at
temperature ranging from 40 to 80°C for 30 minutes. It was found that isolates ST 7 and
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ST 8 produced the most thermostable lipase, which retained 86.70% and 87.52% of the
original activity after incubation at temperature 80°C for 30 minutes, respectively.
Eighteen lipase-producing thermophilic bacteria were further characterized and
identified using morphological characteristics, Biolog Microlog® Bacterial
Identification System and conventional biochemical tests. Strains KW 6, KW 7, ST 1,
ST 6 and ST 10 were identified as CDC Group IVc-2 (Alcaligenes-like) or Ralstonia
paucula. Strains KW 8, KW 10 and DT 17 were identified as Burkholderia
cocovenenans, B. glumae and Photobacterium logei, respectively. Biolog identified
strains ST 7, ST 8, SY 7, KW 1, KW 9, KW 12 and TBN 3 were belonging to genus
Bacilli and strains SY 9, DT 9 and DT 12 as Staphylococcus species. Four strains of the
Bacillus genus with the highest thermostability, ST 7, ST 8, SY 7 and KW 12 were
chosen for further identification using 16S rRNA gene sequence analysis.
The protocol of DNA extraction method applied in this study was Qiagen DNeasy
Tissue Kit. Based on the quantification of extracted DNA and estimation of the purity by
UV spectrophotometer, the Qiagen DNeasy Tissue Kit produced a high DNA yield of all
samples. The primer pair used for specific-PCR generated the same expected PCR
product for all the strains with molecular weight of 1500 bp. Sequences were determined
using an automated sequencer; which was aligned with reference sequences of the
closest related organisms in NCBI database. Based on 16S rRNA gene sequencing, the
highest sequence similarity was found between strain ST 8 and Bacillus subtilis gene for
16S rRNA partial sequence (AB110598 with 100% similarity between nucleotides 1-
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1532). Strains KW 12 and ST 7 were also showed a high sequence similarity at 99.0%;
comparable to those of Anoxybacillus kamchatkensis 16S rRNA gene partial (accession
number: AF510985) and Anoxybacillus flavithermus isolate AB05 16S rRNA gene
partial sequence (accession number: AF001964), respectively. Strain SY 7 displayed
98.0% sequence similarity to Bacillus cereus strain J-1 16S rRNA gene partial sequence
(accession number: AY305275). The 16S rRNA gene sequences for ST 8, ST 7, KW 12
and SY 7 have been deposited into Genbank Data Library and assigned the accession
number DQ401073, DQ193516, DQ401072 and DQ401074, respectively.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENGENALPASTIAN BAKTERIA TAHAN HABA PENGHASIL ENZIM LIPASE DIPENCILKAN DARI KOLAM AIR PANAS DI MALAYSIA
Oleh
AZURA LIANA BINTI KAMARUZAMAN
Januari 2008
Pengerusi: Profesor Madya Nazamid Saari, PhD Fakulti: Sains dan Teknologi Makanan
Mikroorganisma tahan haba penghasil enzim lipase telah berjaya diasingkan dari
sampel-sampel air yang diambil dari enam buah kolam air panas yang terletak di pantai
barat Semenanjung Malaysia dan dikenalpasti hingga ke paras spesies. Lima ratus empat
puluh empat koloni mikroorganisma telah disaring secara kualitatif menggunakan
kaedah piring agar Rhodamine B-minyak zaitun dan enam puluh enam didapati positif
sebagai penghasil enzim lipase setelah membentuk lingkaran cahaya berwarna jingga
mengelilingi koloni apabila disinari cahaya UV. Aktiviti lipolitik dicerakin
menggunakan kaedah titrimetrik dengan emulsi minyak zaitun sebagai substrat. Analisis
perbandingan di antara semua bakteria penghasil lipase yang positif menunjukkan
pencilan ST 7 mempunyai aktiviti lipolitik yang tertinggi iaitu 4.58 U/ml, diikuti oleh
pencilan ST 6 dengan aktiviti 3.51 U/ml. Lapan belas pencilan yang menunjukkan
aktiviti lipolitik yang tinggi kemudiannya diperiksa untuk ujian thermostabiliti.
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Thermostabiliti ditentukan dengan menginkubasi lipase mentah pada suhu 40 hingga
80°C selama 30 minit. Pencilan ST 7 dan ST 8 didapati menghasilkan enzim lipase
yang paling tahan haba, iaitu mengekalkan 86.70% dan 87.52% daripada aktiviti asal
setelah diinkubasi pada suhu 80°C selama 30 minit.
Pencirian dan pengenalpastian lapan belas bakteria tahan haba penghasil enzim lipase
telah dilakukan menggunakan kaedah pencirian morfologi, sistem ‘Biolog Microlog®’
pengenalpastian bakteria dan ujian-ujian biokimia. Strain KW 6, KW 7, ST 1, ST 6 dan
ST 10 telah dikenalpasti sebagai CDC Group IVc-2 (Alcaligenes-like) atau Ralstonia
paucula. Bagi strain KW 8, KW 10 dan DT 17 pula, mereka telah dikenalpasti sebagai
Burkholderia cocovenenans, B. glumae, dan Photobacterium logei. Hasil ujikaji Biolog
mendapati strain ST 7, ST 8, SY 7, KW 1, KW 9, KW 12 dan TBN 3 tergolong sebagai
genus Bacilli dan strain SY 9, DT 9 dan DT 12 sebagai spesies Staphylococcus. Empat
strain daripada genus Bacillus dengan thermostabiliti yang tertinggi, ST 7, ST 8, SY 7
dan KW 12 telah dipilih untuk pengenalpastian selanjutnya menggunakan kaedah ‘16S
rRNA Gene Sequence Analysis’.
Kaedah untuk pengekstrakan DNA yang digunakan di dalam kajian ini ialah Qiagen
DNeasy Tissue Kit. Berdasarkan kepada keputusan kuantifikasi DNA ekstrak dan
penganggaran ketulenannya menggunakan spektrofotometer UV, kaedah Qiagen
DNeasy Tissue Kit memberikan kualiti DNA tertinggi untuk untuk semua strain.
Pasangan primer yang digunakan untuk PCR-spesifik menghasilkan ‘produk PCR’ yang
diharapkan untuk semua strain dengan berat molekul 1500 bp. Rangkaian jujukan
dikenalpasti dengan menggunakan program rangkaian jujukan automatik; di mana
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rangkaian jujukan akan dibandingkan dengan rujukan yang terhampir dengannya di
dalam pangkalan maklumat NCBI. Keputusan dari analisis ‘jujukan gen 16S rRNA’
mendapati rangkaian jujukan ST 8 telah mendapat nilai persamaan tertinggi dengan
Bacillus subtilis gene for 16S rRNA partial sequence (AB110598 dengan 100%
persamaan antara nukleotida 1-1532). Strain KW 12 dan ST 7 juga menunjukkan nilai
persamaan yang tinggi pada 99.0%; dibandingkan dengan Anoxybacillus kamchatkensis
16S rRNA gene partial (nombor kemasukan: AF510985) dan Anoxybacillus flavithermus
isolate AB05 16S rRNA gene partial sequence (nombor kemasukan: AF001964). Strain
SY 7 mempamerkan 98.0% persamaan rangkaian jujukan dengan Bacillus cereus strain
J-1 16S rRNA gene partial sequence (nombor kemasukan: AY305275). Hasil dari ujikaji
jujukan gen 16S rRNA untuk ST 8, ST 7, KW 12 dan SY 7 telah disimpan dalam
‘Genbank Data Library’ dan ditetapkan di bawah nombor kemasukan DQ401073,
DQ193516, DQ401072 dan DQ401074.
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ACKNOWLEDGEMENT
In the name of Allah, The Most Gracious and The Most Merciful. First and foremost, I
would like to express my heartfelt thanks to Almighty for giving me the will, strength
and the perseverance to pursue and complete my Master of Science. It would not have
been possible to complete this study without His grace.
I would like to express my sincere gratitude and appreciation to the chairman of my
supervisory committee, Assoc. Prof. Dr. Nazamid Saari for his invaluable guidance,
dedicated efforts, his tremendous help in teaching me to write proper scientific papers
and continuous support throughout the study. Because of his guidance, I have been able
to make my dream come true of combining two of my favourite fields, microbiology and
biotechnology together to write this thesis.
My special thanks and appreciation goes to my co-supervisor, Dr. Nazimah Sheikh Abd.
Hamid and Professor Dr. Son Radu for their advises, suggestions and consistent
encouragement. Their strong enthusiasm for research helps me a lot in maintaining my
confidence to achieve such high-level results in scientific work.
I am indebted to my dear friend, Halifah Mat Yasin for her willingness and constant
assistance and help in time especially when I was having difficulties with my molecular
works during my early years of research. Words cannot express my gratitude for her
patience and support.
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My sincere gratitude also goes to all the staff in the biochemistry lab and microbiology
lab of the Faculty of Food Science and Technology, Universiti Putra Malaysia, who has
contributed one way or another throughout the course of study.
Needless to say, that I am grateful to all of my friends, Yuli Haryani, Marlina, Tunung,
Rozila, Lesley, Belinda, Rasyila, Kqueen, Jurin, Tuan, Suwaibah, Yin Tze, Sia Yen,
Daniel, Henie, Tung and Maslihana from Microbiology Lab. Sincere thanks are also
extended to my friends, Ermina Sari, Wan Zunairah, Azimah, Ching, Reza, Raja Rohaya
and Rosnizam from Nutraceutical and Enzymology Lab. Thank you for being around
and sharing so much fun and several good times together during my research in the lab.
All of you have made my student life an interesting and a delightful one. I am thankful
to all of you and wish you the best of luck.
All of my hard work would have never been done properly without continuous support
and encouragement especially from my dearest parent and my loving brother and sister.
Because of their financial and spiritual support, I have been able to study at an advanced
academic level.
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I certify that an Examination Committee met on 22 January 2008 to conduct the final examination of Azura Liana Binti Kamaruzaman on her Master of Science thesis entitled “Identification of Lipase-Producing Thermophilic Bacteria Isolated from Hot Springs in Malaysia” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Master of Science. Members of the Examination Committee are as follows:
Yaakob Che Man, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Farinazleen Mohd Ghazali, PhD Lecturer Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Shuhaimi Mustafa, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Thong Kwai Lin, PhD Professor Faculty of Science University Malaya (External Examiner)
________________________________
HASANAH MOHD GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 10 July 2008
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Nazamid Saari, PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Nazimah Sheikh Abd. Hamid, PhD Faculty of Food Science and Technology Universiti Putra Malaysia (Member) Son Radu, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member)
AINI IDERIS, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 10 July 2008
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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently submitted for any other degree at Universiti Putra Malaysia or any other institution. AZURA LIANA BINTI KAMARUZAMAN Date:
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TABLE OF CONTENTS
Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xix CHAPTER 1 INTRODUCTION 1 1.1 General Objectives of the Study 4 2 LITERATURE REVIEW 5
2.1 Extremophilic Microorganisms 5 2.1.1 Thermophiles 6 2.1.2 Resistance of Thermophiles 7 2.2 Sources of Thermophilic Microorganisms 8 2.3 Lipase-producing Bacteria 10
2.3.1 Pseudomonas 12 2.3.2 Bacillus 14 2.4 Screening and Isolation of Lipase- Producing Thermophilic Bacteria 15 2.4.1 Assay of Lipase Activity 18
2.5 Identification of Lipase-Producing Thermophilic Bacteria 21 2.5.1 Traditional Phenotypic Identification 22 2.5.2 Modern Genotypic Identification 23
3 SCREENING AND ISOLATION OF LIPASE- PRODUCING THERMOPHILIC BACTERIA FROM MALAYSIAN HOT SPRINGS 30 3.1 Introduction 30
3.2 Materials and Methods 32 3.2.1 Materials 32 3.2.2 Methods 33 3.3 Results and Discussion 36 3.3.1 Screening and isolation of lipase-
producing bacteria 36 3.3.2 Comparative lipolytic activity of
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positive bacterial lipase producer 39 3.3.3. Thermostability of lipases 43 3.4 Conclusion 49 4 IDENTIFICATION OF LIPASE PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM MALAYSIAN HOT SPRINGS 50 4.1 Introduction 50 4.2 Materials and Methods 52 4.2.1 Materials 52 4.2.2 Methods 53 4.3 Results and Discussion 57 4.3.1 Morphological characteristics 57 4.3.2 Identification of strains 59 4.4 Conclusion 87 5 CONFIRMATION OF BACILLUS GENUS LIPASE- PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM MALAYSIAN HOT SPRINGS BY 16S RIBOSOMAL RNA GENE SEQUENCING 88 5.1 Introduction 88 5.2 Materials and Methods 90 5.2.1 Samples 90 5.2.2 DNA Extraction Method 90 5.2.3 Quantification Of DNA Quality 92 5.2.4 Polymerase Chain Reaction (PCR) 92 5.2.5 Sequencing of 16S rRNA Gene 94 5.3 Results and Discussion 96 5.3.1 DNA Extraction 96 5.3.1.1 DNA Yield and Purity 96 5.3.2 Specific PCR for Strains Identification 98 5.3.3 16s rRNA Sequence Analysis for Confirmation of PCR products 99 5.4 Conclusion 114 6 CONCLUSION 116 REFERENCES 118 APPENDICES 135 BIODATA OF STUDENT 158 LIST OF PUBLICATIONS 161
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LIST OF TABLES Table Page
2.1 Lipase-Producing Bacteria 11
3.1 The Occurrence of Lipase-Producing Bacteria from Six Hot Springs in West-Coast of Peninsular Malaysia 39 3.2 Lipases Thermostability of the 18 Lipase- Producing Bacteria Isolates 44 4.1 Morphological Characteristics of Lipase-
Producing Thermophilic Bacteria 58
4.2 Physiological Characteristics of Lipase- Producing Thermophilic Bacteria 63
4.3 Results for Biochemical Tests of the Gram-Negative Short Rods Bacteria at 45°C 64
4.4 Results for Biochemical Tests of the Gram-Negative Short Rods Bacteria at 35, 37 or 42°C 66
4.5 Results for Biochemical Tests of the Gram-Positive Rods Bacteria at 45°C 72
4.6 Results for Biochemical Tests of the Gram-Positive Rods Bacteria at 35, 37 or 42°C 74
4.7 Results for Biochemical Tests of the Gram-Positive Cocci Bacteria at 45°C 81
4.8 Results for Biochemical Tests of the Gram-Positive Cocci Bacteria at 35, 37 or 42°C 83
5.1 Oligonucleotide Primers for PCR Amplification and Sequencing 96
5.2 Comparison of Identification Results of Bacillus Genus Lipase-Producing Thermophilic Bacteria Against Three Identification Methods 113
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LIST OF FIGURES
Figure Page
2.1 The Different Steps in PCR 25
2.2 The Exponential Amplification of the Gene in the PCR 26
3.1 Six Hot Springs Sampling Locations in West-Coast of Peninsular Malaysia 37
3.2 The Difference between a Positive Lipase Producer and a Negative Lipase Producer 38 3.3 Lipolytic Activities of Positive Lipase-Producing Thermophilic Bacteria Isolated from Malaysian Hot Springs (A-F) 40
3.4 High Thermostable Lipases Secreted by ST 6, ST 7, ST 8, ST 10, SY 7 and KW 6 46
3.5 Moderate Thermostable Lipases Secreted by KW 7, KW 10, KW 12 and TBN 3 47
3.6 Low Thermostable Lipases Secreted by SY 9, KW 1, KW 8, KW 9, ST 1, DT 9, DT 12 and DT 17 48
5.1 Agarose Gel Electrophoresis Shows the Quality of DNA
Isolated from Thermophilic Bacteria by Qiagen Kit 98
5.2 Agarose Gel Electrophoresis of PCR Amplification Products of Thermophilic Bacteria 99
5.3 The Alignment of Sequencing Results from Each Primer to Provide a Partial Sequence for Genus Bacillus Strains 101 5.4 16S rRNA Gene Sequence Alignment Comparison of ST 8 as Subject Compared to the Published Sequence of Bacillus subtilis Gene for 16S rRNA Partial Sequence (AB110598) with 1532 Nucleotide 102
5.5 16S rRNA Gene Sequence Alignment Comparison of ST 7 as Subject Compared to the Published Sequence of Anoxybacillus flavithermus isolate AB05
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16S rRNA Gene Partial Sequence (AF001964) with 1411 Nucleotide 104 5.6 16S rRNA Gene Sequence Alignment Comparison of KW 12 as Subject Compared to the Published Sequence of Anoxybacillus kamchatkensis 16S rRNA Gene Partial Sequence (AF510985) with 1548 Nucleotide 107 5.7 16S rRNA Gene Sequence Alignment Comparison of SY 7 as Subject Compared to the Published Sequence of Bacillus cereus Strain J-1 16S rRNA Gene Partial Sequence (AY305275) with 1479 Nucleotide 110
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LIST OF ABBREVIATIONS
rpm revolution per minute
v/v volume per volume
min minute
h hour
g gram
ml millileter
mg milligram
μm micrometer
nm nano meter
mM milliMolar
µmol micromole
ng nanogram
μl microliter
M Molar
N Normality
Gly Glycine
Ser Serine
kDa kilo Dalton
ATCC American Type Culture Collection
pI isoelectric point
U/ml unit per millileter
U/g unit per gram
xix
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xx
w/v weight per volume
UV ultraviolet
DNA deoxyribonucleic acid
rRNA ribosomal ribonucleic acid
bp base pair
kb kilo basepair
C18 stearate/ stearic acid
C18:1 oleate/ oleic acid
BUG Biolog Universal Growth medium
GN Gram-negative
GP Gram-positive
IF Inoculation fluid
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CHAPTER 1
INTRODUCTION
Nowadays, lipases arise amongst the most important biocatalysts carrying out novel
reactions in both aqueous and non-aqueous media. This is mainly due to their ability
to utilize a wide spectrum of substrates, high stability towards extremes of
temperature, pH and organic solvents, and show chemo-, regio- and
enantioselectivity. In recent times, the determination of their three-dimensional
structures has thrown light into their unique structure–function relationships.
Lipase or glycerol ester hydrolases (EC 3.1.1.3) is defined by the Enzyme
Commission of International Union of Biochemistry and Molecular Biology as the
enzyme that catalyzes hydrolysis of ester bonds at the interface between an insoluble
substrate phase and the aqueous phase in which the enzyme is dissolved, under a
natural conditions. Under certain experimental conditions, such as in the absence of
water, they are capable of reversing the reaction. The reverse reaction leads to
esterification and formation of glycerides from fatty acids and glycerol. The catalytic
action that occurs is as follows:
TRIACYLGLYCERIDE + H2O GLYCEROL + FREE FATTY ACIDS
(Ester) (Alcohol) (Acid)
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In nature, lipases are ubiquitous and produced from various sources including
animals, plants and microorganisms. However, lipases of microbial origin are the
most versatile enzymes and are known to bring about a range of bioconversion
reaction including alcoholysis, aminolysis, hydrolysis, esterification and
interesterification (Pandey et al., 1999). Besides, microbes can be easily cultivated
and the lipases produced are mostly extracellular. Several Bacillus sp. have been
reported to be the main source of lipolytic enzymes (Schmidt et al., 1994).
The isolation of a large number of beneficial thermophilic microorganisms from
different exotic ecological zones of the earth and followed by the extraction of
thermostable lipases shows that the advances of this area have been possible
(Gowland et al., 1987; Dong-Woo et al., 1999; Kambourova et al., 2003).
Thermostable lipases, which are commercially available, are mostly produced from
mesophilic bacteria and fungi. Although many lipases from mesophilic group are
stable at elevated temperatures, lipases from thermophilic have become the subject of
special interest for structural investigation and biotechnological applications
(Herbert, 1992). Thermostable lipases, which have been isolated from thermophilic
organisms, play an important role in commercial applications because of their overall
inherent stability (Demirjian et al., 2001). They are also stable and active at
temperatures which are higher than optimum temperatures for the growth of the
microorganisms. This reduces the risk of contamination by common mesophiles.
Allowing a higher operation temperature has also a significant influence on the
bioavailability and solubility of organic compounds and thereby provides efficient
bioremediation (Becker et al., 1997).
2
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With the rapid development of enzyme technology, many novel biotechnology
applications for lipases have been identified. The esters produced play a relevant role
in the food industry as flavour and aroma constituents (Gandhi, 1997). Other
applications include the removal of the pitch from pulp in the paper manufacturing
industry, drug formulations in the pharmaceuticals industry and the removal of
subcutaneous fat in the leather industry (Pandey et al., 1999). Lipases are extensively
used in dairy industry for flavour development in cheese, butter and margarine and
hydrolysis of milk fat (Paiva et al., 2000). Other applications are in the fats and oil
industries including production of modified triacylglycerides and upgrading of low-
value oils and fats.
As industrial application increase, the requirements for large amounts of lipases
become a limiting factor. Therefore, several workers have tried to obtain
thermostable lipases from thermophilic or extreme thermophilic strains such as
Pseudomonas fluorescens SIK W1 (minimal activity at 60ºC) (Andersson et al.,
1979) or Bacillus sp. (maximal activity at 60ºC) (Emanuilova et al., 1993). The
alkalophilic and thermophilic Bacillus sp. has been the focus of a number of
investigations as the source of lipases that are stable and function optimally at
alkaline pH values and high temperature
This research was done with the ultimate goal of isolating lipase-producing
thermophilic bacteria from hot springs in Malaysia. The strain that produced a high
thermostable lipase was chosen for further studies. Identification and characterization
of the thermophilic bacteria producing lipase was carried out by using three methods;
3
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4
Biolog Microlog® Bacterial Identification System, conventional biochemical test and
16S rRNA gene sequencing.
1.1 General Objectives of the Study
The study was undertaken with the following general objectives:
a) To isolate lipase-producing thermophilic bacteria from hot springs in
Malaysia
b) To identify and characterize the thermophilic bacteria producing
lipase using three different methods
i) Biolog Microlog® Bacterial Identification System
ii) Conventional biochemical tests
iii) 16S rRNA gene sequencing