Post on 31-Aug-2019
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
NORHAYATI BINTI RAMLI
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
NORHAYATI BINTI RAMLI
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
NORHAYATI BINTI RAMLI
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Norhafizah binti Abdullah, PhD
Associate Professor
Faculty of Engineering
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Mohd Ali Hassan, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Noorjahan Banu Alitheen, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
______________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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.
_________________________
NORHAYATI BINTI RAMLI
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 Materials and Methods
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 Results and Discussion
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
Bacillus sp. NR5 UPM
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 Materials and Methods
5.2.1 Recombinant bacterial strains and storage of
cultures
5.2.2 Media
5.2.3 Expression of CGTase gene from recombinant
Bacillus sp. NR5 UPM
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 Results and Discussion
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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