EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF

MALTOGENIC AMYLASE FROM Bacillus lehensis G1

SAMSON PACHELLES

A thesis submitted in fulfilment of the

requirements for the award of the degree of

Master of Engineering (Bioprocess)

Faculty of Chemical Engineering

Universiti Teknologi Malaysia

MAY 2013

iv

ACKNOWLEDGEMENT

First and foremost, I would like to extend my sincere appreciation to my

supervisor, Prof. Dr. Rosli Md. Illias for his stern yet valuable advicse as well as his

guidance throughout the duration of this research. I would also like to thank and

acknowledge the assistance of the laboratory staff of the Department of Bioprocess

Engineering, UTM and my laboratory colleagues.

I would like to express my gratitude to Malaysia Genome Institute (MGI) for

the financial aid since the research was part of a project under Genomics and

Molecular Biology Initiatives Programmes (07-05-16-MGI-GMB12).

Last but not least, I would like to convey my absolute appreciations to my

parents and families for their continual support, love and patience.

v

ABSTRACT

This study was carried out for the expression and characterization of

maltogenic amylase (MAG1) from Bacillus lehensis G1. Amplification of 1741 base

pair gene fragments encoding MAG1 and expression in Escherichia coli have been

successful. The expression of MAG1 was optimized at 30°C and 0.5 mM isopropyl

β-D-1-thiogalactopyranoside (IPTG) for a period of 12 hour post-induction time.

Purification of the crude enzyme was done using the ACTAprime System which uses

the concept of affinity chromatography. The optimum temperature and pH of the

purified MAG1 were 40 °C and pH 7.0 respectively. The enzyme did not show

potent thermostability and was stable at pH ranging from 7.0 to 9.0. The purified

MAG1 has a preference towards cyclodextrin (CD) specifically β-CD. Starches were

least favored by MAG1 followed by pullulan. Only the metal ion Mn2+

increased the

activity of MAG1 while K2+

, Li2+

and Mg2+

slightly affect its activity. The metal ion

Ca2+

significantly reduced MAG1 activity while Fe2+

, Co2+

, Zn2+

, Cu2+

, Pb2+

and Ni2+

drastically reduced the activity of MAG1. In terms of additives, only 2-

mercaptoethanol (2-ME) managed to enhance the activity of MAG1 while

ethylenediaminetetraacetic acid (EDTA) and Tween20 did not affect its activity.

Drastic reduction of MAG1 activity was caused by phenylmethylsulfonyl fluoride

(PMSF), sodium dodecyl sulfate (SDS), methanol and ethanol. The hydrolysis

pattern of MAG1 was studied using CDs and maltooligosaccharides. The hydrolysis

of CDs resulted in the formation of maltose but no glucose was detected. MAG1 was

also able to linearize the CDs. MAG1 did not react with glucose, maltose and

maltotriose but did react with maltotetraose, maltopentaose, maltohexaose and

maltoheptaose to produce mainly maltose and maltotriose. The Km and Vmax of

MAG1 towards β-CD were 6.358 mg/mL and 91.63 µmol/min respectively.

vi

ABSTRAK

Penyelidikan ini telah dijalankan untuk ekspresi dan mencirikan enzim

maltogenic amylase (MAG1) yang diperolehi daripada Bacillus lehensis G1.

Amplifikasi sebanyak 1741 pasang bes fragmen yang mengkodkan MAG1 dan

ekspresi dalam Escherichia coli telah berjaya. Ekspresi MAG1 adalah optimum pada

suhu 30°C dengan induksi 0.5 mM isopropil β-D-1-tiogalaktopiranosid (IPTG)

selama 12 jam. Penulenan enzim telah dijalankan menggunakan sistem ACTAprime

yang menggunakan konsep kromatografi afiniti. Suhu dan pH optimum bagi enzim

yang telah ditulenkan adalah pada 40 °C dan pH 7.0. Enzim ini tidak menunjukkan

kestabilan yang positif pada suhu tinggi dan stabil pada pH dari 7.0 ke 9.0. MAG1

yang telah ditulenkan lebih cenderung kepada siklodekstrin (CD) terutamanya β-CD.

Kanji kurang digemari oleh MAG1 diikuti oleh pullulan. Aktiviti MAG1 telah

dipertingkatkan oleh Mn2+

manakala K2+

, Li2+

dan Mg2+

tidak terlalu memberi kesan

kepada aktiviti MAG1. Aktiviti MAG1 telah direncat oleh Ca2+

dan direncat dengan

mendadak oleh Fe2+

, Co2+

, Zn2+

, Cu2+

, Pb2+

and Ni2+

. Selain daripada itu, 2-

merkaptoetanol (2-ME) juga meningkatkan aktiviti MAG1 tetapi aktivitinya tidak

dipengaruhi oleh asid atelindiamintetraasetik (EDTA) dan Tween20. Perencatan

yang mendadak pada aktiviti MAG1 adalah disebabkan oleh fenilmetilsulfonil

florida (PMSF), natrium dodecil sulfat (SDS), metanol dan etanol. Hasil hidrolisis

oleh MAG1 dikaji dengan menggunakan CD dan maltooligosakarida. Hidrolisis CD

menghasilkan maltosa sebagai produk utamanya tetapi tiada penghasilan glukosa

dapat dikesan. MAG1 juga mampu menukar CD kepada bentuk linearnya. MAG1

tidak bertindak balas dengan glukosa, maltosa dan maltotriosa tetapi bertindak balas

dengan maltotetraosa, maltopentosa, maltoheksosa dan maltoheptosa untuk

menghasilkan maltosa dan maltotriosa. Nilai Km dan Vmax bagi MAG1 terhadap β-

CD adalah 6.358 mg/mL dan 91.63 µmol/min.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xiii

LIST OF SYMBOLS/ABBREVIATIONS xvii

LIST OF APPENDICES xx

1 INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives of Research 3

1.3 Scopes of Research 3

2 LITERATURE REVIEW 4

2.1 Starch 4

2.2 Starch Degrading Enzyme 7

2.3 Amylases 8

viii

2.4 Alpha-amylase 9

2.5 Maltogenic Amylase 13

2.6 Biochemical Characteristics of

Maltogenic Amylase 17

2.7 Reaction Patterns of Maltogenic Amylase 19

2.7.1 Hydrolysis Pattern of Maltogenic

Amylase 19

2.7.2 Transglycosylation Pattern of Maltogenic

Amylase 20

2.7.3 Coupled Reactions of Maltogenic Amylase 22

2.8 Maltogenic Amylase as an Anti-Staling Agent 24

3 METHODOLOGY 26

3.1 Bacterial Strains 27

3.2 Chemicals 27

3.3 Preparation of Bacterial Glycerol Stock 27

3.4 Medium and Agar Preparation 28

3.4.1 Horikoshi Broth and Agar 28

3.4.2 Luria-Bertani Broth and Agar 28

3.5 General DNA Technique 29

3.5.1 Agarose Gel Preparation 29

3.5.2 Genomic DNA Extraction 29

3.5.3 Quantification of DNA using

Spectrophotometer 30

3.6 Polymerase Chain Reaction (PCR) for the

Gene of Maltogenic Amylase 31

3.6.1 Primer Design 31

3.6.2 PCR Mixture and Protocol 31

3.6.3 Purification of PCR Product 32

3.7 Plasmid Extraction 33

3.8 Digestion of DNA and Plasmid 34

3.9 Ligation of DNA to Plasmid 34

3.10 Preparation of Competent Cell using TSS Method 34

ix

3.11 Transformation into E. coli Hosts 35

3.12 Screening for Positive Transformant 36

3.13 Expression of Recombinat Maltogenic Amylase 36

3.14 Cell Lysis using Sonicator 36

3.15 Purification of Recombinant Maltogenic Amylase 37

3.15.1 System Wash 37

3.15.2 Connecting Column 38

3.15.3 Selecting the Program for Purification 38

3.15.4 Collecting Samples 38

3.16 Analysis of the Purification Result using

SDS-PAGE 39

3.17 Western Blot 39

3.18 Protein Determination 40

3.19 Assay for Maltogenic Amylase 41

3.20 Biochemical Characterization of Recombinant

Maltogenic Amylase 41

3.20.1 Optimum Temperature and pH 41

3.20.2 Thermal and pH Stability 41

3.20.3 Susbstrate Specificity 42

3.20.4 Effect of Metal Ions and Additives 42

3.20.5 Hydrolysis Pattern of the Recombinant

Maltogenic Amylase 43

3.20.6 Analysis of Hydrolysis Pattern using TLC 43

3.20.7 Determination of Kinetic Parameters 43

4 RESULTS AND DISCUSSION 45

4.1 Cloning of Recombinant Maltogenic Amylase 45

4.1.1 Amplification of Maltogenic

Amylase Gene 45

4.1.2 Cloning of Maltogenic Amylase Gene 46

4.2 Bioinformatic Analysis of Maltogenic

Amylase Gene 47

4.2.1 Full Sequence Analysis of Maltogenci

x

Amylase Gene 47

4.2.2 Hydrophobicity of the Recombinant

Maltogenic Amylase 57

4.3 Expression of Maltogenic Amylase 59

4.3.1 Expression at Different Temperature and

Different Concentration of IPTG 60

4.3.2 Expression in Different Expression Hosts 64

4.4 Western Blot 66

4.5 Purification of the Crude Recombinant

Maltogenic Amylase 67

4.6 Biochemical Characterization 70

4.6.1 Effect of Temperature and Temperature

Stability 70

4.6.2 Effect of pH and pH Stability 72

4.6.3 Substrate Specificity 74

4.6.4 Effect of Metal Ion 76

4.6.5 Effect of Additives 77

4.6.6 Hydrolysis Pattern 78

4.6.6.1 Hydrolysis of Cyclodextrin (CD) 78

4.6.6.2 Hydrolysis of

Maltooligosaccharides 80

4.7 Kinetic Parameter of Recombinant

Maltogenic Amylase 82

5 CONCLUSION 83

5.1 Conclusions 83

5.2 Recommendations 84

REFERENCES 85

Appendices A - E 93 - 105

xi

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Differences of amylose and amylopectin 6

2.2 The members in the α-amylase family 10

2.3 Recently discovered maltogenic amylase from different

sources with its basic biochemical characteristic 18

2.4 Example of donor, acceptor and products that have been

studied for the transglycosylation reaction of

maltogenic amylase 21

3.1 PCR primers for the amplification of maltogenic amylase

from B. lehensis G1 31

3.2 PRC components used to obtain the gene of

maltogenic amylase 32

4.1 Nucleotides distribution table of MAG1 50

4.2 Amino acids distribution table of MAG1 51

4.3 Identity comparison of MAG1 with other maltogenic

amylase from different sources 55

xii

4.4 Hydrophobicity scores at the conserved regions

of MAG1 58

4.5 Activity of MAG1 expressed in E. coli BL21 at

different temperature and IPTG concentration 62

4.6 Activity of MAG1 expressed in different host 65

4.7 Purification table of recombinant maltogenic

amylase expressed at the optimized conditions 69

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 General structure of starch 5

2.2 Schematic representation of the action pattern of

amylolytic enzymes on starch (amylopectin) polymers 11

2.3 Conserved sequence regions in the α-amylase family 12

2.4 The first three dimensional structure of the α-amylase 13

2.5 Structure of cyclodextrin 14

2.6 Schematic diagram on substrate preference of dimeric

maltogenic amylase 15

2.7 Ribbon diagram for a monomer of maltogenic amylase

from Thermus sp. 16

2.8 The ribbon diagram representation of dimeric maltogenic

amylase from Thermus sp. 16

2.9 Differences of amino acids in conserve regions for

TpMA and ThMA 20

xiv

2.10 Proposed models of coupled hydrolysis and

transglycosylation by maltogenic amylase from

Bacillus stearothermophilus ET1 22

2.11 Schematic drawings of products from reaction of acarbose

with maltogenic amylase from Thermus sp. 23

3.1 Overview of methodology 26

4.1 Gel electrophoresis of genomic DNA of

B. lehensis G1 extraction 46

4.2 Gel electrophoresis of amplified PCR product of the

maltogenic amylase gene 46

4.3 Gel electrophoresis of double-digestion

(Bam HI and Xho I) of the cloning vector pET21a 47

4.4 Nucleotide and deduced amino acid sequence of MAG1 50

4.5 Multiple alignment MAG1 with other amylolytic enzymes 54

4.6 Phylogenetic analysis of MAG1 based on amino

acid sequences 56

4.7 Hydrophobicity analysis of MAG1 57

4.8 SDS-PAGE electrophoresis of MAG1 expressed in

E. coli BL21 at 37 °C with 1 mM IPTG 60

4.9 SDS-PAGE of MAG1 expressed at 30°C with

different concentration of IPTG 62

4.10 SDS-PAGE of MAG1 expressed at 25°C with different

xv

concentration of IPTG 63

4.11 SDS-PAGE of MAG1 expressed at 20°C with

different concentration of IPTG 63

4.12 SDS-PAGE of MAG1 expressed in Origami 2 at 30°C

with different concentration of IPTG 65

4.13 SDS-PAGE of MAG1 expressed in Origami B at 30°C

with different concentration of IPTG 66

4.14 Western blot of MAG1 expressed at the 30°C and

0.5 mM IPTG 67

4.15 SDS-PAGE of the purified MAG1 68

4.16 Effect of temperature on the activity of MAG1 70

4.17 Temperature stability of MAG1 72

4.18 Effect of pH on the activity of MAG1 73

4.19 pH stability of MAG1 74

4.20 Relative activity of MAG1 on different substrate 76

4.21 Effect of metal ions on the activity of MAG1 77

4.22 Effect of additives on the activity of MAG1 78

4.23 TLC analysis of MAG1 hydrolytic activity on CD 79

4.24 TLC analysis of MAG1 hydrolytic activity on

xvi

Maltooligosaccharides 81

4.25 Partial multiple alignment of MAG1 with other

amylolytic enzymes showing the conserve region IV 81

4.26 Lineweaver-Burk plot for MAG1 using β-CD as

a substrate 82

xvii

LIST OF SYMBOLS/ABBREVIATION

Asn - Asparagine

Asp - Aspartic acid

BCIP - 5-Bromo-4-Chloro-3-Indolyl Phosphate

Blast - Basic Local Alignment Search Tool

Bp - base pair

DNA - Deoxyribonucleic acid

EC - Enzyme Commision

EDTA - ethylene diamine tetraacetic acid

eg. - example

et al. - and frinds

EtOH - Ethanol

g/L - gram per litter

Glu - Glutamate

His - Histidine

HPAEC - High Performance Anion Exchange

Chromatography

HPLC - High Performance Liquid

Chromatography

IPTG - IsoPropyl β-D-1-ThioGalactopyranoside

kb - kilo base

KCl - potassium chloride

KI - potassium iodide

LMW - Low Molecular Weight

MeOH - Methanol

mg/mL - milligram per milliliter

xviii

mM - millimolar

NaCl - sodium chloride

NaOH - sodium hydroxide

NBT - Nitro Blue Tetrazolium chloride

nm - nano meter

PAGE - polyacarylamide gel electrophoresis

PMSF - phenylmethanesulfonyl fluoride

rpm - revolutions per minute

SDS - sodium dedocyl sulphate

Ser - serine

TBE - Tris/Borate/EDTA

TBS - Tris Buffered Saline

TBSTT - Tris Buffered Saline Tween-20 Triton

TE - Tris/EDTA

TLC - Thin Layer Chromatography

TSS - Transformation and Storage Solution

U/mL - unit per milliliter

UV - ultraviolet

UV-Vis - ultraviolet visible

v/v - volume per volume

w/v - weight per volume

α - alpha

β - beta

γ - gamma

µL - micro litter

% - percentage

°C - degree Celsius

Ca2+

- calcium ion

Co2+

- cobalt ion

Cu2+

- copper ion

Fe2+

- iron ion

K+

- potassium ion

Li2+

- lithium ion

Mg2+

- magnesium ion

xix

Mn2+

- manganese ion

Ni2+

- nickel ion

Pb2+

- lead ion

Zn2+

- zinc ion

H2SO4 - sulfuric acid

KH2PO4 - monopotassium phosphate

MgSO4 - magnesium sulfate

Tm - melting temperature

xx

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Medium and Buffer 92

B Standard Curve for Maltogenic Amylase Assay 102

C Standard Curve for Bradford Assay 103

D pET-21a(+) VECTOR (PROMEGA) 104

E Publication 105

CHAPTER 1

INTRODUCTION

1.1 Introduction

Alpha-amylase (EC 3.2.1.1.) is one of the enzymes that have been

investigated most thoroughly and used most widely in the starch hydrolytic industry.

These amylase are endo-acting amylases which means the enzyme hydrolyze α-1,4-

glycosidic linkage of internal starch polymer randomly. This in turn resulted in

oligosaccharides with varying lengths and α-dextrins that retains its α-1,6-glycosidic

linkages. There are various amylolytic enzymes such as pullulanase (EC 3.2.1.41;

Kuriki et al., 1988), cyclodextrin glucanotransferase (EC 2.4.1.19; Sakai et al,.

1987), cyclodextrinase (EC 3.2.1.54; Kitahata et al, 1983), and maltogenic amylase

(EC 3.2.1.133; MAase; Kim et al,. 1992) in which these enzymes exhibits novel

enzymatic properties that clearly differentiate it from those of α-amylase, being

capable of hydrolyzing poly- and/or maltooligosaccarides such as pullulan and

cyclodextrins (CDs) as well as its extensive transglycosylation activity.

Maltogenic amylases which belong to the glycoside hydrolase family 13

(GH13) are very unique where these enzymes have multi-substrate specificity

towards starch, pullulan, and CDs, which is modulated by dimerization of the

2

enzyme. Other than its typical-α-amylase hydrolytic activity, maltogenic amylase is

also able to perform a transglycosylation activity via formation of various glycosidic

linkages such as α-1,3- and α-1,6-linkages to produce branched oligosaccharides

from substrate such as liquefied starch (Park et al., 2000). This enzyme is named

maltogenic amylase because in general it will hydrolyze CD into mainly maltose and

glucose.

The enzyme activity was found to be cell-bounded which means that the

enzyme is intracellular (Kolcuoğlu et al., 2010). It was proven by the predicted

amino acid sequence with no sign of signal sequence necessary for secretion of the

enzyme outside the cell. In order to exploit its benefits, maltogenic amylase has long

been discovered and isolated such as from B. licheniformis (Kim et al., 1992), B.

stearothermophilus (Cha et al., 1998), and a Thermus species (Kim et al., 1999).

From previous researches on maltogenic amylase, it has been proven that

maltogenic amylase is able to produce branched side chains when reacted with

amylopectin cluster (Kim et al., 2006). Moreover, this enzyme hydrolyzes acarbose,

a potent α-amylase inhibitor (Park et al., 1998). The combined activities of

maltogenic amylase which are hydrolysis and transglycosylation have been utilized

in the production of branched oligosaccharides and various modified sugars (Kim et

al., 1994; Lee et al., 1995). Maltogenic amylase is also a highly effective anti-staling

agent used for bread making in industrial bakeries.

Different source of enzymes exhibits their own unique characteristic even

though they have some similarities in terms of function, substrate they act upon or

their structure. Finding the enzymes of highly useful characteristic is very important

as it can contribute in variety of industries. This is where maltogenic amylase from

Bacillus lehensis G1 becomes an interest. Maltogenic amylase has very useful

industrial applications and exploring another source of this enzyme can give us more

option to consider.

3

Amount of this enzyme that can be obtained from the wild microorganism is

normally limited and their characteristics are unknown. Therefore, their applicability

to meet with the conditions of a reaction criteria such as high temperature is still

unclear. We intend to clone and over-expressed the maltogenic amylase in

expression system such as Escherichia coli (E. coli) and characterize the

recombinant enzyme. This will contribute in research to improve the characteristic of

the enzyme such as thermostability by protein engineering. It is an important

characteristic for industrial application where a thermostable enzyme is preferred.

Other than that, an efficient way to produce this enzyme is crucial in order to obtain a

low cost production.

1.2 Objectives of Research

The objectives of this research are to clone and express maltogenic amylase

from Bacillus lehensis G1 (MAG1) in E. coli and to characterize the purified

recombinant maltogenic amylase.

1.3 Scopes of Research

1.3.1 Amplification of maltogenic amylase gene using polymerase chain

reaction (PCR) method

1.3.2 Cloning and expression of maltogenic amylase gene in E. coli

expression system

1.3.3 Purification and characterization of recombinant maltogenic

amylase

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