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UNIVERSITI PUTRA MALAYSIA

NOR AZIZAH BINTI PARMIN

FBSB 2013 19

EXPRESSION OF TRUNCATED CHALCONE SYNTHASE FROM Boesenbergia rotunda L. MANSF. IN Escherichia coli

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EXPRESSION OF TRUNCATED CHALCONE

SYNTHASE FROM Boesenbergia rotunda L. MANSF.

IN Escherichia coli


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2013

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EXPRESSION OF TRUNCATED CHALCONE SYNTHASE FROM Boesenbergia

rotunda L. MANSF. IN Escherichia coli

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfillment of the Requirement for the Degree of Master of Science

March 2013

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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 Science

EXPRESSION OF TRUNCATED CHALCONE SYNTHASE FROM Boesenbergia

rotunda L. MANSF. IN Escherichia coli

By


March 2013

Chairman: Prof. Raja Noor Zaliha Raja Abd. Rahman, PhD

Faculty: Biotechnology and Biomolecular Sciences

Chalcone synthase (CHS) is an entrance enzyme in the biosynthetic pathway of different

flavonoids compound and become a critical regulatory enzyme. Truncated CHS was

isolated from Boesenbergia rotunda (B. rotunda), also known as Chinese ginger, which

is a medicinal and culinary herb from China and Southeast Asia. The popularity of its

medicinal usage has drawn the attention of scientists to investigate on its medicinal

properties. This research is a fundamentals works to study on the mechanism of

truncated CHS from B. rotunda in E. coli. This study is new for this plant but CHS has

been studied by many researchers in other plants. Optimization of B. rotunda specific

CTAB RNA extraction method developed in this study was able to produce high yield of

RNA with minimum polysaccharide contamination. The value of RNA yield for

optimization of B. rotunda specific CTAB RNA extraction method was 2.0 µg/g. A

reverse transcriptase-polymerase chain reaction (RT-PCR) based approach was used to

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identify and isolate a partial-length cDNA coding for CHS gene. The gene encoding

truncated CHS from B. rotunda was cloned into pTrcHis2 expression vector and placed

under the control of the trc promoter for high level expression in E. coli. The effect of

inducer concentration and induction time on CHS production from inducible system was

evaluated. The recombinant truncated CHS protein was characterized and optimized for

higher expression. The optimization of the recombinant truncated CHS production of

inducible system in 50 ml culture was done for the best clone truncated CHS from E.

coli TOP10. The effect of IPTG concentration and induction time on truncated CHS

production from inducible system was evaluated. A time course profile of recombinant

truncated CHS production with the optimized condition was performed and produced

after 16 h of induction time from recombinant truncated CHS5, while optimal IPTG

concentration was 0.5 mM.

The recombinant truncated CHS was purified by using affinity chromatography and Ion-

Exchange Chromatography (IEX). The expressed CHS protein had a molecular weight

of about 21 kDa, a size that matched with the prediction by bioinformatic analysis.

Truncated CHS was highly active at 40oC, pH 8.0 with a half-life of 1 h 10 min at 30

oC.

3D structure of truncated CHS from B. rotunda was predicted using CHS from alfalfa

(Medicago sativa) as a template with sequence identity 74.87%. Tyr-42, His-130 and

Asp-163 were assigned as catalytic triad residues. Truncated CHS was evaluated with

Ramachandran plot, Verify 3D and ERRAT to validate the structure. This study may

provide useful information on this enzyme and its function in the flavonoids biosynthetic

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pathway in B. rotunda that offers significant pharmaceutical properties for human

health.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

PENGEKSPRESSAN SEPARA CHALCONE SYNTHASE DARIPADA

Boesenbergia rotunda L. MANSF. DI DALAM Escherichia coli

Oleh


Mac 2013

Pengerusi: Prof. Raja Noor Zaliha Raja Abd. Rahman, PhD

Fakulti: Bioteknologi dan Sains Biomolekular

Chalcone synthase (CHS) merupakan enzim pintu masuk ke dalam tapak jalan

biosintesis sebatian flavonoid berbeza menjadi enzim pengawalatur yang kritikal. CHS

separa diasingkan daripada Boesenbergia rotunda (B. rotunda), juga dikenali sebagai

halia Cina yang merupakan herba perubatan dan masakan dari China dan Asia Tenggara.

Populariti nilai perubatan yang terkandung di dalam B. rotunda telah menarik minat para

saintis untuk mengkaji nilai perubatan yang tersembunyi di dalamnya. Penyelidikan ini

bertujuan untuk mengetahui mekanisme CHS separa daripada B. rotunda di dalam

sistem E. coli. Kaedah penyelidikan ke atas enzim separa CHS adalah baru untuk

tumbuhan B. rotunda tetapi CHS daripada tumbuh-tumbuhan yang lain telah banyak

dibuat kajian. Bagi mengeksploitasikan potensi B. rotunda sebagai herba perubatan ke

arah kajian saintifik, kaedah pengekstrakan CTAB RNA spesifik B. rotunda yang

dioptimumkan dilakukan dalam kajian ini telah dapat menghasilkan RNA yang tinggi

dengan polisakarida yang minima. Hasil RNA untuk pengoptimuman kaedah

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pengekstrakan CTAB RNA spesifik ialah 2.0 µg/g. Enzim penjujukan berbalik

tindakbalas rantai berpolimer (RT-PCR) yang telah digunakan untuk mengenal pasti dan

mengasingkan cDNA separa panjang bagi gen CHS. Gen yang membawa CHS separa

daripada B. rotunda diklonkan ke dalam vektor pengekspressan pTrcHis2 vektor dan

diletakkan di bawah kawalan promoter trc untuk pengekspressan yang tinggi dalam E.

coli. Kesan kepekatan pengaruh dan masa induksi bagi penghasilan CHS daripada

sistem perangsangan telah dinilai. Protein rekombinan CHS separa dicirikan dan

dioptimumkan untuk penghasilannya yag lebih tinggi. Pengoptimuman pengeluaran

rekombinan CHS separa daripada sistem aruhan dalam 50 ml kultur telah dilakukan

untuk klon separa CHS5D dari E. coli TOP10. Kesan kepekatan IPTG dan masa induksi

untuk penghasilan separa CHS dari sistem aruhan telah dinilai. Profil masa penghasilan

rekombinan CHS separa keadaan optimum telah dijalankan dan dihasilkan selepas 16

jam daripada masa induksi manakala kepekatan pengaruh terbaik IPTG adalah pada 0.5

mM.

Rekombinan CHS separa rekombinan telah ditulenkan dengan menggunakan

kromatografi afiniti dan IEX. Protein CHS separa yang mempunyai berat molekul lebih

kurang 21 kDa, saiz yang sepadan dengan ramalan oleh analisis bioinformatik. CHS

separa adalah sangat aktif pada 40oC dan pH 8.0 dengan separa hayatnya daripada 1 j 10

minit pada 30oC. Struktur 3D CHS separa daripada B. rotunda telah diramalkan dengan

menggunakan CHS daripada alfalfa (Medicago sativa) sebagai templat dengan urutan

identiti 74.87%. Tyr42, His130 dan Asp163 telah bertindak sebagai tapak pemangkin

enzim. CHS separa dinilai dengan plot Ramachandran dan 3D ERRAT untuk

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mengesahkan strukturnya. Kajian ini boleh memberikan maklumat berguna untuk enzim

ini dan fungsinya dalam tapak jalan biosintesis flavonoid dalam B. rotunda yang

mempunyai banyak ciri-ciri farmaseutikal untuk kesihatan manusia.

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ACKNOWLEDGEMENTS

All praises are due to Allah, Lord of the universe for strength, wisdom and grace in

times when human abilities fail. Only by His grace and mercy this thesis can be

completed.

I would like to express my gratitude to individuals who played an important roles in

completion of this study. First and foremost, thank you to my supervisor Professor Dr.

Raja Noor Zaliha Raja Abd. Rahman for giving me the opportunity to enter a post-

graduate career, having faith in my progress and supporting, advising and inspiring me

throughout. Also, most sincere thanks to my cosupervisor, Professor Dato’ Dr. Abu

Bakar Salleh and Professor Dr. Mahiran Basri for their valuable advice and teachings

concerning both moral and scientific issues.

A special word of thanks to the following people for their generous contributions and

technical assistance regarding the experimental procedures: Dr Adam Leow Thean Chor,

Dr Mohd Shukuri Mohamad Ali, Dr Wong Chee Fah and Dr. Fairolniza Mohd Shariff

for their unlimited guidance, advice, and suggestion.

Thanks to everyone in Enzyme and Microbial Technology Research group who have

either given me advice or showed me friendship. I will miss all of you when I look back

on my time in this group. I would like to thank my parents; Parmin bin Kiram and Siti

Aissah bt Haji Masturi and also my family for their unlimited love and constant support.

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I certify that an Examination Committee has met to date of viva to conduct the final

examination of Nor Azizah bt Parmin on her degree of Masters of Science thesis

entitled, “Expression of truncated chalcone synthase from Boesenbergia rotunda (L.)

Mansf. in Escherichia coli” in accordance with Universiti Pertanian Malaysia (Higher

Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981.

The Committee recommends that the candidate be awarded the Degree of Masters of

Science.

Members of the Examination Committee were as follows:

Chairman, Ph.D.

Dr. Mohd Puad bin Abdullah

Associate Professor

Faculty of Biotechnoogy and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Examiner 1, Ph.D.

Dr. Janna Ong binti Abdullah

Associate Professor



(Internal Examiner)

Examiner 2, Ph.D.

Dr. Nor’aini bt. Abd Rahman



(Internal Examiner)

External Examiner, Ph.D.

Dr. Ainon Hamzah

Associate Professor

Faculty of Sciences and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

__________________________________

NORITAH OMAR, PhD

Assoc. Professor and Deputy Dean

School of Graduate Studies


Date:

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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 Masters of Science. The

members of the Supervisory Committee were as follows:

Raja Noor Zaliha Raja Abd. Rahman, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences


(Chairman)

Abu Bakar Salleh, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences


(Member)

Mahiran Basri, PhD Professor

Faculty of Science


(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 institutions.

_______________________________


Date: 14 March 2013

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

APPROVAL vii

DECLARATION x

LIST OF TABLES xv

LIST OF FIGURES xvii

LIST OF APPENDICES xx

LIST OF ABBREVIATIONS xxi

CHAPTER

1.0 INTRODUCTION 1

2.0 LITERATURE REVIEW 4

2.1 Chalcone synthase 4

2.1.1 Application of CHS 6

2.1.3 Hexanoyl-CoA as aliphatic substrate 7

2.2 Flavonoid 8

2.3 Prokaryotic cell expression 12

2.4 Expression of truncated gene from eukaryote in prokaryotic

system 13

2.5 Analysis of secretory expression with SDS-PAGE 14

2.6 Detection of recombinant protein in E. coli 15

2.7 Purification strategy of recombinant protein in E. coli 16

2.7.1 Polyhistidine tag 18

2.7.2 Ion Exchange Chromatography (IEX) 19

2.8 Characterization study of CHS 19

2.8.1 Byproducts from the reaction of CHS 21

2.8.2 Substrate Promiscuity 22

2.9 Protein structure modeling 23

2.10 Homology modeling 24

2.10.1 Fold assignment and template selection 26

2.10.2 Template-target alignment 26

2.10.3 Model building 27

2.10.4 Model evaluation 28

2.11 Circular dichroism (CD) of protein 31

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3.0 MATERIALS AND METHODS

3.1 Materials 32

3.2 General method 32

3.3 Sample collection and preparation 33

3.4 Modified CTAB method 33

3.5 Quantification of total RNA 34

3.6 Formaldehyde-agarose gel electrophoresis 35

3.7 The first strand cDNA synthesis 36

3.8 Amplification of cDNA encode truncated CHS gene with degenerate

primer 37

3.9 Purification of PCR product 38

3.10 Cloning of RT-PCR products into cloning vector 39

3.10.1 Preparation of E. coli competent cell 39

3.10.2 Cloning of RT-PCR products into pGEMT cloning vector 40

3.10.3 Heat shock transformation of E. coli 40

3.10.4 Analysis of recombinant plasmids 41

3.10.5 Plasmid extraction 41

3.10.6 Sequencing of recombinant truncated CHS gene. 42

3.11 Phylogenetic analysis 43

3.12 Transformation of the expression vector pTrcHis-truncated CHS in

E. coli 44

3.12.1 Amplification of truncated CHS gene with specific primer 44

3.12.2 Ligation reaction into expression vector 45

3.13 Heterologous expression in E. coli 46

3.13.1 Small scale expression 46

3.13.2 Scale-up of expression 47

3.14 Optimization studies of inducible truncated CHS expression 46

3.14.1 Effect of IPTG concentration on truncated CHS

expression 48

3.14.2 Effect of induction time on truncated CHS expression 48

3.14.3 Effect of induction Abs600nm on protein expression 49

3.15 Analysis of recombinant truncated CHS expression 49

3.15.1 Quantitative determination of truncated CHS

activity 49

3.15.1.1 Truncated CHS assay 50

3.15.1.2 Standard curve 4-hydroxy-6-methyl-2-

pyrone 50

3.15.2 High performance liquid chromatography (HPLC)

analysis 51

3.15.3 Determination of protein concentration 51

3.15.4 Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) analysis 52

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3.15.5 Western Blot analysis 53

3.16 Purification of recombinant truncated CHS 54

3.16.1 Affinity Chromatography using Ni Sepharose 6 Fast

Flow 54


3.16.3 Native polyacrylamide gel electrophoresis

(Native-PAGE) 55

3.17 Characterization of purified truncated CHS 56

3.17.1 Effect of pH on truncated CHS activity 56

3.17.2 pH stability of enzyme activity 56

3.17.3 Effect of temperature truncated CHS activity 56

3.17.4 Thermostability of enzyme activity 57

3.17.5 Substrate specificity of truncated CHS from B. rotunda 57

3.18 Sequence analysis of truncated CHS of B. rotunda 58

3.19 Comparative protein modeling of truncated CHS 58

3.20 Circular Dichroism (CD) spectral analysis of truncated CHS 59

3.20.1 Denatured protein analysis 59

3.20.2 Weight of truncated CHS of various temperature 60

4.0 RESULTS AND DISCUSSION 61

4.1 Total RNA extraction method 61

4.2 Sequence aligment of CHS genes 63

4.3 Analysis result of degenerate primer design 66

4.4 RNase H Digestion before PCR 68

4.5 Analysis of recombinant plasmid contain insert gene

encoding truncated CHS from B. rotunda 71

4.6 Sequencing analysis of recombinant truncated CHS from

B. rotunda 75

4.7 Phylogenetic tree of truncated CHS 78

4.8 Secretory expression of truncated CHS from B. rotunda in prokaryotic

system 81

4.8.1 Effect of inducer concentration on truncated CHS

secretion 81

4.8.2 Effect of induction time on truncated CHS secretion 83

4.8.3 Effect of induction Abs600nm on truncated CHS secretion 85

4.9 Standard curve of 4-hydroxy-6-methyl-2-pyrone 86

4.10 Identification of truncated CHS end product 87

4.11 Relation between incubation period and enzyme

concentration 90

4.12 Western blot 91

4.13 Purification of recombinant truncated CHS 92

4.13.1 Purification of recombinant truncated CHS by using

Ni-Sepharose 6 Fast Flows 92


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4.13.3 Native polyacrylamide gel electrophoresis

(Native-PAGE) 108

4.14 Characterization of purified CHS 109

4.14.1 Effect of pH on truncated CHS activity 109

4.14.2 pH stability of enzyme activity 111

4.14.3 Effect of temperature for truncated CHS activity 112

4.14.4 Thermostability of enzyme activity 113

4.14.5 Substrate specificity of truncated CHS from B. rotunda 116

4.15 Sequence analysis of truncated CHS from B. rotunda 118

4.16 Comparative modeling, structure analysis of truncated CHS and

activity of recombinant CHS protein 119

4.17 Catalytic triads of truncated CHS from B. rotunda 124

4.17.1 Tyrosine 124

4.17.2 Histidine 124

4.17.3 Asparagine 126

4.18 Structure validation of predicted truncated CHS of B. rotunda 127

4.19 Circular Dichroism (CD) spectra analysis of truncated CHS 134

5.0 CONCLUSION AND RECOMMENDATIONS 136

5.1 Conclusion 136

5.2 Recommendations 138

REFERENCES 140

APPENDICES 147

BIODATA OF STUDENT 159

EXPRESSION OF TRUNCATED CHALCONESYNTHASE FROM Boesenbergia rotunda L. MANSF.IN Escherichia coliABSTRACTTABLE OF CONTENTSCHAPTERSREFERENCES

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