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UNIVERSITI PUTRA MALAYSIA

NORHAYATI BINTI RAMLI

FBSB 2012 9

DEVELOPMENT OF A LOCAL BACTERIAL ISOLATE EXPRESSING CYCLODEXTRIN GLYCOSYLTRANSFERASE THROUGH MOLECULAR

CLONING APPROACHES

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DEVELOPMENT OF A LOCAL BACTERIAL ISOLATE EXPRESSING

CYCLODEXTRIN GLYCOSYLTRANSFERASE THROUGH MOLECULAR

CLONING APPROACHES

By


Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in fulfillment of the requirements for the Degree of Doctor of Philosophy

July 2012

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DEDICATED TO MY LATE MOTHER, FATHER AND FAMILY

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the Degree of Doctor of Philosophy

DEVELOPMENT OF A LOCAL BACTERIAL ISOLATE EXPRESSING

CYCLODEXTRIN GLYCOSYLTRANSFERASE THROUGH MOLECULAR

CLONING APPROACHES

By


July 2012

Chairman : Professor Suraini Abd. Aziz, PhD

Faculty : Biotechnology and Biomolecular Sciences

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) exhibited as an important

member of the amylolytic glucosylase family that catalyzed the formation of

cyclodextrins (CDs) through cyclization reaction. The ability of CGTase to convert

starch into CD brings a great interest to the researchers. The formation of CD-

inclusion complex with variety of guest molecules was advantageous as the

enhancement of the physical and chemical properties of the inclusion complex

formed were beneficial in biotechnology, pharmaceutical, food, cosmetic, chemical

and agricultural field. However, the obstacle in producing the CD in industrial scale

was due to the production of different ratios of α-, β- and γ-CD catalyzed by

CGTases. The above phenomenon contributed to the complicated and tedious

purification strategy of the final targeted product. Due to the high viscosity of

starches, pre-treatment stage was needed in order to prevent the blockage in the

membrane system in filtration stage. Furthermore, the low concentration of CGTase

produced by wild type strain at longer incubation time caused the major problem for

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CGTase production in large scale. Hence, the objectives of this study were to isolate

and screen the potential CGTase producer. The isolated strain was then subjected to

the isolation of CGTase gene using primer screening technique and used for

construction of CGTase expression system in Escherichia coli. The enzyme

produced from recombinant strains was evaluated.

In this study, the CGTase-producing bacteria were successfully isolated from the soil

in Malaysia. Out of 65 strains, eleven CGTase producers has been further screened

using modified Horikoshi agar type II with specific indicator. The size of halo zones

formation on the plate indicates a good qualitative measurement of CGTase producer

as the biggest diameter formed is indicating the highest CGTase activity obtained.

All eleven isolates showed the characteristics of Gram positive and identified as

Bacillus sp. Furthermore, the selection for the best CGTase producer was carried put

with the highest CGTase activity (11.709 U/mL) and CD concentration (0.011, 2.504

and 0.188 mg/mL for α-, β- and γ-CD, respectively) were produced by Bacillus sp.

NR5 UPM using raw soluble starch as a substrate at 48 hours of fermentation. This

isolate also showed the highest CGTase activity (15.514 U/mL) at 32 hours of

fermentation with the used of sago starch as a substrate. The isolate Bacillus sp. NR5

UPM has the capability in predominantly producing β-CD as a main product and has

a potential in degrading the raw starch, thus known as a raw-starch degrading

enzyme producer.

Further study on the isolation of CGTase gene from Bacillus sp. NR5 UPM DNA

was successfully carried out using DNA walking strategy. Analysis of the nucleotide

sequences revealed the presence of an open reading frame of 2112 bp which encoded

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a protein containing 704 amino acids with a putative molecular weight of 78.6 kDa.

The deduced amino acids sequence showed about 98% homology with the CGTase

from Bacillus sp. KC201. The recognition of TTG as a start codon was assisted by

the presence of Shine-Dalgarno sequence, which located at 6 bp upstream from the

initiation codon. Meanwhile, the presence of 29 amino acids residues of the isolated

CGTase was functioned as signal peptide which facilitated in the secretion of protein.

The pUC19CGT-SS expression system has been constructed by cloning the full

length of CGTase gene under the transcriptional control of lac promoter of pUC19

into an E. coli. Compared to wild type, the CGTase that was produced in

recombinant strain only required one-fourth of culture time and neutral pH to

produce CGTase. After 12 hours of cultivation, the CGTase activity in the culture

medium reached 29.6 U/mL, which was approximately 2.5-fold higher than the

CGTase produced by the parental strain. Interestingly, 94% of the CGTase activity

was detected in an extracellular space, indicating the signal peptide was functional in

E. coli. In addition, the isolation of the promoter and transcriptional terminator of

CGTase from Bacillus sp. NR5 UPM was carried out. The functionality of an

insertion of putative promoter regions upstream of CGTase gene was verified by the

construction of E. coli strain harbouring pTZCGT-BS, which showed approximately

3.2-fold increment of CGTase activity compared to the parent strain. These findings

support the important function of the strong promoter in regulating the expression

level of enzyme. In conclusion, the study on the strain development from Bacillus sp.

NR5 UPM by expressing CGTase gene into E. coli expression system was

successfully carried out with the improved enzyme activity from the recombinant

strain.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PEMBANGUNAN BAGI EKSPRESI SIKLODEKSTRIN

GLIKOSILTRANSFERASE OLEH BAKTERIA PENCILAN TEMPATAN

MELALUI KAEDAH PENGKLONAN MOLEKUL

Oleh


Julai 2012

Pengerusi : Profesor Suraini Abd. Aziz, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Siklodekstrin glikosiltransferase (CGTase, EC 2.4.1.19) merupakan enzim penting

dalam keluarga amilolitik glukosilase yang memangkinkan penghasilan

siklodekstrins (CDs) melalui tindak balas penghasilan gelung. Kebolehan enzim

CGTase untuk menukarkan kanji kepada CD menarik minat penyelidik.

Pembentukan pencantuman kompleks CD dengan pelbagai molekul asing memberi

kelebihan di mana peningkatan sifat fizikal dan kimia bagi kompleks yang terbentuk

berkepentingan dalam bidang bioteknologi, farmaseutikal, makanan, kosmetik, bahan

kimia dan pertanian. Namun, penghasilan CD dalam skala besar adalah sukar kerana

penghasilan α-, β- dan γ-CD dalam nisbah yang berbeza yang dimangkinkan oleh

CGTase. Fenomena di atas menyumbangkan strategi penulenan yang sulit dan sukar

kepada produk yang dikehendaki. Oleh kerana kelikatan kanji yang tinggi, langkah

pra-rawatan diperlukan untuk mengelakkan sistem membran tersumbat pada langkah

penapisan. Tambahan pula, masalah utama penghasilan CGTase dalam skala besar

adalah kerana kepekatan enzim CGTase yang rendah yang dihasilkan oleh strain liar.

Oleh itu, objektif kajian ini adalah untuk memencil dan menyaring penghasil

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CGTase yang berpotensi. Strain yang dipencilkan akan digunakan untuk pemencilan

gen CGTase menggunakan teknik penyaringan pemula bagi pembentukan sistem

ekspresi CGTase dengan Escherichia coli. Penghasilan enzim dijalankan oleh strain

rekombinan yang diperoleh ditentukan.

Dalam kajian ini, bakteria penghasil CGTase telah berjaya dipencilkan daripada

sumber tanah di Malaysia. Daripada 65 strain yang dipencilkan, sebelas penghasil

CGTase disaring dengan lebih lanjut menggunakan agar Horikoshi jenis II yang

dimodifikasi dengan penunjuk spesifik. Saiz zon lingkaran yang terbentuk pada plat

menandakan pengukuran kualitatif yang baik bagi penghasil CGTase. Kesemua

sebelas bakteria yang dipencilkan menunjukkan ciri Gram positif dan dikenalpasti

sebagai spesies Bacillus. Tambahan lagi, pemilihan untuk penghasil CGTase terbaik

telah dijalankan dengan aktiviti CGTase yang terbaik (11.709 U/mL) and kepekatan

CD (0.011, 2.504 and 0.188 mg/mL untuk setiap satu α-, β- dan γ-CD) telah

dihasilkan oleh Bacillus sp. NR5 UPM menggunakan kanji mentah terlarut pada jam

ke-48 proses fermentasi. Bakteria yang dipencil ini juga menunjukkan penghasilan

CGTase tertinggi (15.514 U/mL) pada jam ke-32 proses fermentasi dengan

menggunakan kanji sagu sebagai substrat. Bakteria pencilan Bacillus sp. NR5 UPM

mempunyai kebolehan dalam menghasilkan lebih banyak β-CD sebagai produk

utama dan berpotensi dalam mengurai kanji mentah, yang juga dikenali sebagai

penghasil enzim-pengurai kanji mentah.

Kajian lanjut dalam pemencilan gen CGTase daripada DNA Bacillus sp. NR5 UPM

telah berjaya dijalankan menggunakan strategi perjalanan DNA. Analisis ke atas

jujukan nukleotida menunjukkan kehadiran satu rangka kehadiran terbuka sepanjang

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2112 bp, dengan jujukan peptida CGTase sebanyak 704 asid amino, dengan berat

molekul putatif 78.6 kDa. Jujukan rantaian asid amino menunjukkan kira-kira 98%

homologi dengan CGTase daripada Bacillus sp. KC201. Pengenalan TTG sebagai

kodon permulaan dibantu oleh kehadiran jujukan Shine-Dalgarno yang terletak 6 bp

di sebelah hilir daripada kodon permulaan. Manakala, kehadiran 29 jujukan asid

amino daripada CGTase yang dipencilkan mungkin bertindak sebagai peptida isyarat

yang mana bertindak membantu dalam perembesan protein.

Sistem ekspresi pUC19CGT-SS telah dibina dengan mengklonkan gen CGTase

penuh di bawah kawalan transkripsi pemula lac pUC19 ke dalam E. coli. Berbanding

dengan strain liar, CGTase yang dihasilkan oleh strain rekombinan hanya

memerlukan satu perempat daripada masa kultur dan pH neutral untuk menghasilkan

CGTase. Selepas 12 jam pengkulturan, aktiviti CGTase dalam medium kultur

mencapai 29.6 U/mL, yang merupakan kira-kira 2.5 kali ganda lebih tinggi daripada

CGTase yang dihasilkan daripada strain liar. Menariknya, 94% daripada aktiviti

CGTase dikesan di luar sel, menunjukkan peptida isyarat berfungsi di dalam E. coli.

Di samping itu, pemencilan pemula dan penamat transkripsi CGTase daripada

Bacillus sp. NR5 UPM telah dijalankan. Fungsi pemula yang dipencilkan dengan

kehadiran pemula vektor-T7 dibuktikan dengan pembinaan strain E. coli yang

mengandungi pTZCGT-BS, dengan kenaikan kira-kira 3.2 kali ganda aktiviti

CGTase berbanding strain asal. Penemuan ini menyokong fungsi penting pemula

yang kukuh dalam mengawal selia ekspresi enzim. Kesimpulannya, kajian ke atas

pembangunan strain daripada Bacillus sp. NR5 UPM dengan mengekspresi gen

CGTase ke dalam sistem ekspresi E. coli berjaya dijalankan dengan peningkatan

aktiviti enzim daripada strain rekombinan.

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ACKNOWLEDGEMENTS

My deepest thanks and praise to Allah for His love and blessings throughout

the research and preparation of this thesis.

I would like to express my sincere gratitude and whole-hearted appreciation

to my supervisory committee chairman, Professor Dr. Suraini Abd. Aziz, whose

encourage and give guidance and support throughout my study. Appreciation is

extended to my co-supervisors, Assoc. Prof. Dr. Noorjahan Banu Alitheen and

Professor Dr. Mohd Ali Hassan for their support and assistance.

I gratefully acknowledge staffs at SIRIM Berhad for their advice and

assistance especially in molecular cloning works. I also wish to acknowledge all

lecturers, students and staffs of Kyushu Institute of Technology, Japan for their

guidance and support during the JENESYS programme. Special thanks to all

lecturers and members in Environmental Biotechnology Research Group and to all

my colleagues that supported me in one way or another during the completion of the

project.

My deepest gratitude and appreciation goes to my beloved family; my late

mother, father and siblings who have consistently motivated me, giving me spiritual

encouragement and support throughout these years. Lastly, I would like to thank

Universiti Putra Malaysia and Ministry of Science and Technology (MOSTI) for

providing funding for this study.

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I certify that a Thesis Examination Committee has met on 10 July 2012 to conduct

the final examination of Norhayati binti Ramli on her thesis entitled “Development

of a Local Bacterial Isolate Expressing Cyclodextrin Glycosyltransferase through

Molecular Cloning Approaches” in accordance with the Universities and University

Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U(A)

106] 15 March 1998. The Committee recommends that the student be awarded the

Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Norhani binti Abdullah, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Muhajir bin Hamid, PhD

Associate Professor



(Internal Examiner)

Norhafizah binti Abdullah, PhD

Associate Professor

Faculty of Engineering


(Internal Examiner)

Ya-Wen He, PhD

Professor

Shanghai Jiao Tong University

China

(External Examiner)

__________________________

ZULKARNAIN ZAINAL, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 27 August 2012

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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 Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Suraini Abd. Aziz, PhD

Professor



(Chairman)

Mohd Ali Hassan, PhD

Professor



(Member)

Noorjahan Banu Alitheen, PhD

Associate Professor



(Member)

______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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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 at

any other institution.

_________________________


Date: 10 July 2012

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TABLE OF CONTENTS

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER

1 INTRODUCTION

2 LITERATURE REVIEW

2.1 Starch

2.1.1 Structure and properties of starch

2.1.2 The uses of starch as carbon sources

2.1.3 Pre-treatment of starch

2.2 Cyclodextrin glycosyltransferase (CGTase)

2.2.1 CGTase catalyzed reaction

2.2.2 CGTase-producing microorganisms

2.2.3 Properties of bacterial CGTase

2.3 Recombinant cyclodextrin glycosyltransferase (CGTase)

2.3.1 Cloning of CGTase gene

2.3.2 Escherichia coli expression system

2.3.3 Secretion of recombinant CGTase

2.3.4 Promoter and transcriptional terminator

2.4 Cyclodextrin

2.4.1 History of cyclodextrin

2.4.2 Structure and properties of cyclodextrin

2.4.3 Applications of cyclodextrin

2.5 Concluding Remarks

3 ISOLATION, SCREENING AND CHARACTERIZATION

OF CYCLODEXTRIN GLYCOSYLTRANSFERASE

(CGTASE)-PRODUCING BACTERIA

3.1 Introduction

3.2 Materials and Methods

3.2.1 Chemicals

3.2.2 Bacterial strains and storage of cultures

3.2.3 Media

3.2.4 Isolation and screening of microorganism

3.2.5 Morphological characterization

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3.2.6 Bacterial 16S ribosomal RNA (rRNA)

identification

3.2.7 Production of crude CGTase from Bacillus sp.

NR5 UPM

3.2.8 Analytical methods

3.3 Results and Discussion

3.3.1 Isolation of microorganisms

3.3.2 Morphological characterization

3.3.3 Bacterial 16s rRNA identification

3.3.4 Selection for the best CGTase-producing bacteria

3.3.5 Effect of starch pre-treatment on CGTase

production

3.3.6 Cyclodextrin production

3.4 Conclusion

4 ISOLATION OF CYCLODEXTRIN

GLYCOSYLTRANSFERASE (CGTASE) GENE USING

PRIMER SCREENING TECHNIQUE FOR

CONSTRUCTION OF CGTASE EXPRESSION SYSTEM

WITH Escherichia coli

4.1 Introduction


4.2.1 Bacterial strains and plasmids

4.2.2 Chemicals and biological enzymes

4.2.3 Media

4.2.4 General DNA techniques

4.2.5 Cyclodextrin glycosyltransferase (CGTase) gene

isolation

4.2.6 Construction of CGTase expression system with E.

coli

4.2.7 Ligation of DNA

4.2.8 Transformation into E. coli JM109 cells

4.2.9 Confirmation of insert


4.3.1 Isolation of known sequence of CGTase gene from

Bacillus sp. NR5 UPM

4.3.2 Isolation of full length of CGTase gene from


4.3.3 Isolation of promoter and transcriptional

terminator of Bacillus sp. NR5 UPM CGTase

4.3.4 Construction of CGTase expression system in E.

coli under the control of lac promoter

4.3.5 Construction of CGTase expression system in E.

coli under the control of isolated promoter and

transcriptional terminator

4.3.6 Transformation and screening of the transformants

4.3.7 Amino acid sequence analysis with other CGTases

and amylase

4.4 Conclusion

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5 PRODUCTION OF CYCLODEXTRIN

GLYCOSYLTRANSFERASE (CGTASE) FROM

RECOMBINANT Bacillus sp. NR5 UPM

5.1 Introduction


5.2.1 Recombinant bacterial strains and storage of

cultures

5.2.2 Media

5.2.3 Expression of CGTase gene from recombinant


5.2.4 Protein precipitation

5.2.5 Characterization of crude recombinant CGTase

5.2.6 Cell fractionation

5.2.7 Assay of β-CGTase


5.3.1 Cyclodextrin glycosyltransferase (CGTase)

productions from E. coli strain harbouring

pUC19CGT-SS

5.3.2 Effect of culture conditions on CGTase synthesis

by recombinant construct

5.3.3 Effect of insertion of putative promoter regions

and transcriptional terminator on enzyme

expression

5.3.4 Characterization of crude recombinant CGTase

5.4 Conclusion

6 CONCLUSION AND RECOMMENDATIONS FOR

FUTURE WORKS

6.1 Summary

6.2 Conclusion

6.3 Recommendations for future works

REFERENCES

APPENDICES

BIODATA OF STUDENT

LIST OF PUBLICATIONS

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