Co-Immobilization of Thermostable Alpha-Amylase and Glucoamylase for Starch Hydrolysis

Co-immobilization of thermostable Alpha-amylase and Glucoamylase for starch hydrolysis

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Zifei Dai

Graduate Program in Food, Agricultural and Biological Engineering

The Ohio State University

2011

Dissertation Committee:

Professor Gonul Kaletunc, ―Advisor‖

Professor Sudhir K. Sastry

Professor Yebo Li

Professor Derek Hansford

Copyrighted by

Zifei Dai

2011

Abstract

Starch is one of the most important carbon and renewable energy sources. Starch hydrolysis products, such as glucose, high fructose syrups or maltose are widely used in food industries. Also, the produced sugars can be fermented for bioethanol production.

Starch hydrolysis involves large amount of enzymes consumption. The immobilization could recover enzymes and lower the production cost. A gel capsule system by dripping calcium chloride into a sodium alginate solution was developed for immobilization of recombinant glucoamylase (GA) by entrapment. Two types of starch hydrolysis enzymes, glucoamylase and alpha-amylase (AA), were co-immobilized inside and on the outside surface of calcium alginate gel capsules.

A His6-tagged thermostable recombinant GA from Sulfolobus Solfataricus P2 was expressed, purified and characterized. The recombinant thermostable GA had an optimum pH and temperature range of 5-5.5 and 90-95°C respectively. The effects of cations and chemicals on the recombinant GA activity were also studied. Thermal stability and inactivation energy of the recombinant thermostable GA were determined with a half-time of 5.3 minutes at 95°C and 218 kJ/mol.

GA from fungus (Aspergillus niger ) and AA from bacterial (Bacillus licheniformis) with low application temperature (50-65°C ) were immobilized on the gel

ii capsules by entrapment and adsorption. Then the optimum pH, temperature, reaction kinetics and reusability of the co-immobilized enzymes system were tested.

The recombinant thermostable GA and a commercially available thermostable

AA (Liquozyme X) were immobilized on the developed gel capsule. A co- immobilization method was developed by entrapping GA inside and covalently bounding

AA on the surface of capsules. The reaction kinetics, thermostability and reusability of the thermostable co-immobilized AA and GA system were tested both in a batch reactor and with a continuous stirred tank reactor.

iii

This work dedicated to truth and knowledge.

―Research under a paradigm must be a particularly effective way of inducing paradigm change.‖

— Thomas S. Kuhn

The Structure of Scientific Revolutions (1962), 52.

Acknowledgments

I would like to express my gratitude to my advisor Dr. Gonul Kaletunc. Her knowledge, research experience, patience, and dedication to science strongly motivated my research. She has not only mentored me for a better understanding of science and engineering by providing the logical research methodology, but, more valuably, encourage me to become an independent researcher with persistence.

I am very grateful to my committee members, Dr. Sudhir K. Sastry, Dr. Yebo Li, and Dr. Derek Hansford. Thanks for their time to review my dissertation and their help by providing insight suggestion for my research.

I must thanks to Dr. Eric Plum for introducing the idea of recombinant DNA technology and also tremendous help with the spectrophotometer. I feel deeply thankful to Professor John Reeve, Professor Tom Santangelo, Lubka Cubonova and Kirk Gaston from microbiology department. Without your help I could not complete this research.

I would like to express my gratitude to Jake Elmer in chemical engineering department for the discussion and helps with the enzyme purification process and gel

v electrophoresis. Thanks for Professor Andre F. Palmer letting me use the facilities in their lab.

I must acknowledge the support of teaching assistantship from the FABE department. Also I am grateful to thank the Ohio State University for providing me the opportunity to study in this great campus. I would like to express my appreciation to all of the coursework professors, thanks for opening the door of knowledge to me.

I am grateful to all my friends in Ohio State and my colleagues in FABE department.

Finally, I would like to thank my family, from the bottom of my heart, for your love and support.

Vita

2002 ...... B.E. Food Science and Technology,

Huazhong Agricultural University, China

2006 ...... M.S. Food Science and Technology,

...... Ghent University, Belgium

2006 to present ...... Graduate Teaching Associate, Department

of Food Agricultural and Biological

Engineering Department, The Ohio State

University

Fields of Study

Major Field: Food, Agricultural, and Biological Engineering

vii

Table of Contents

Abstract ...... ii

Acknowledgments ...... v

Vita ...... vii

Table of Contents ...... viii

List of Tables...... xv

List of Figures ...... xvi

Nomenclature ...... xx

Introduction ...... 1

Chapter 1 Background and Literature Review ...... 5

1.1 Starch ...... 5

1.2 Starch hydrolysis and enzymes ...... 6

1.2.1 Alpha-amylase (EC 3.2.1.1) ...... 8

1.2.2 Industrial glucoamylase (EC 3.2.1.2) ...... 9

1.2.3 Reducing starch hydrolysis steps ...... 11

1.3 Production of a thermostable GA...... 12

1.3.1 Recombinant DNA technology ...... 13 viii

1.3.2 Improvement of thermal stability by natural evolution, directed

evolution, or protein engineering ...... 14

1.3.3 Thermostable GA research ...... 14

1.4 Enzyme purification using immobilized metal-affinity chromatography

(IMAC) ...... 21

1.5 Enzyme reaction kinetics ...... 23

1.6 Thermal stability and enzyme inactivation ...... 24

1.7 Enzyme immobilization...... 25

1.7.1 Immobilization methods and materials ...... 26

1.7.2 Immobilization of GA ...... 33

1.7.3 Co-immobilization of enzymes ...... 35

Chapter 2: Hypothesis and Aims ...... 37

Chapter 3: Co-immobilization of AA and GA for starch hydrolysis ...... 43

3.1 Introduction ...... 43

3.2 Materials and Methods ...... 46

3.2.1 Materials ...... 46

3.2.2 Co-immobilization of GA and AA ...... 47

3.2.3 Starch hydrolysis reaction ...... 49

3.2.4 Protein test by Bradford method ...... 50

3.2.5 Loading efficiency of the immobilized enzyme ...... 51

3.2.6 Optimum pH and temperature for mixed-free and co-immobilized

enzyme system ...... 51

3.2.7 Determination of enzyme kinetics parameters, Km and Vmax ...... 52

3.2.8 Reusability of the co-immobilized enzyme ...... 52

3.2.9 Thermal stability of mixed free and co-immobilized enzymes system

...... 52

3.3 Results and Discussion ...... 53

3.3.1 Co-immobilization of GA and AA on calcium alginate beads and

capsules ...... 53

3.3.2 Effect of immobilization on optimum pH and temperature of enzyme

system ...... 58

3.3.3 Effect of immobilization on kinetic parameters ...... 63

3.3.4 Reusability of co-immobilized system...... 65

3.3.5 Comparison of thermal inactivation of free-mixed and co-

immobilized enzyme system ...... 70

3.4 Conclusion ...... 72

Chapter 4: Expression, purification and characterization a His6-tagged thermostable glucoamylase from Sulfolobus solfataricus P2 ...... 79

4.1 Introduction ...... 79 x

4.2 Materials and Methods ...... 82

4.2.1 Materials ...... 82

4.2.2 Cloning, nucleotide sequence analysis and construction of

expression vectors ...... 83

4.2.3 Growth conditions and Expression of recombinant GA ...... 85

4.2.4 Purification of recombinant GA ...... 87

4.2.5 Gel electrophoresis and protein content ...... 89

4.2.6 Enzyme activity tests ...... 90

4.2.7 Thermal stability of GA ...... 93

4.2.8 Michaelis-Menten kinetics parameters ...... 94

4.3 Results and Discussion ...... 94

4.3.1 The effects of E. coli growth factors on thermostable enzyme

production ...... 94

4.3.2 Effects of IPTG concentration ...... 99

4.3.3 Effects of osmotic pressure (sorbitol) and increased growth

temperature ...... 100

4.3.4 Results of purification and activity yield at optimum growth

conditions ...... 101

4.3.5 Effects of pH and temperature on enzyme activity ...... 107

4.3.6 Substrate specific activity ...... 110 xi

4.3.7 Metal effects on enzyme activity ...... 114

4.3.8 Effects of chemical reagents on enzyme activity ...... 115

4.3.9 Thermal stability of recombinant GA ...... 116

4.3.10 Michalies-Menten kinetics of recombinant GA ...... 119

4.4 Conclusion ...... 122

Chapter 5 Co-immobilization of thermostable alpha-amylase and glucoamylase for starch hydrolysis ...... 132

5.1 Introduction ...... 132

5.2 Materials and Methods ...... 135

5.2.1 Materials ...... 135

5.2.2 Co-immobilization of AA and GA ...... 136

5.2.3 Mixed enzyme capsules preparation ...... 138

5.2.4 Evaluation of the effect of covalent binding procedure on GA

activity ...... 138

5.2.5 Encapsulation efficiency of co-immobilized biocatalyst system ... 141

5.2.6 Effects of pH and temperature on immobilized GA ...... 141

5.2.7 Thermal inactivation of entrapped GA ...... 141

5.2.8 Optimum pH and temperature of co-immobilized GA and AA

system ...... 142

xii

5.2.9 Michaelis-Menten parameters for immobilized GA and AA system

...... 142

5.2.10 Reusability and thermal stability of GA and AA system ...... 142

5.2.11 CSTR setup and operation ...... 143

5.3 Results and Discussion ...... 146

5.3.1 Effects of covalent binding procedure to entrapped GA activity ... 146

5.3.2 Effects of pH on entrapped thermostable GA ...... 147

5.3.3 Influence of temperature on entrapped thermostable GA ...... 149

5.3.4 Thermal stability and inactivation kinetics of entrapped GA ...... 151

5.3.5 Effects of pH on the co-immobilized biocatalyst system ...... 154

5.3.6 Effects of temperature on the co-immobilized biocatalyst system . 156

5.3.7 Michaelis-Menten kinetics of co-immobilized thermostable GA and

AA ...... 158

5.3.8 Comparison of reusability of mixed immobilized and co-

immobilized GA and AA ...... 161

5.3.9 Starch hydrolysis in a continuous stirred tank...... 164

5.3.10 Comparison of CSTR with batch reactor ...... 168

5.3.11 Comparison of thermostable GA performance with industrial GA

...... 170

5.4 Conclusion ...... 172 xiii

Chapter 6 Future work and Investigations ...... 179

References ...... 181

Appendix A: Starch iodine test calibration curve ...... 210

Appendix B: Glucose test calibration curve ...... 211

Appendix C: Bradford test calibration curve ...... 212

Appendix D: Glucose production rate versus GA concentration ...... 213

Appendix E: Glucose production rate versus thermostable GA concentration ...... 214

Appendix F: Modified Lowery protein test calibration curve ...... 215

Appendix G: Amino acid sequence of thermostable GA from S. solfataricus P2 ...... 216

Appendix H: Protein purification with Zn column in FPLC ...... 217

Appendix I: Thermostable GA cost level ...... 218

xiv

List of Tables

Table 1. 1 Summary of thermostable GA from different sources ...... 16

Table 1. 2 GA immobilization methods and supports ...... 33

Table 3. 1 Gel capsule and bead comparison ...... 55

Table 3. 2 Optimum reaction temperature and pH for different systems ...... 59

Table 3. 3 Kinetic parameters of free and co-immobilized enzymes ...... 64

Table 4. 1 Variables for fermenter OTR calculation ...... 98

Table 4. 2 Effects of sorbitol and temperature for protein expression ...... 101

Table 4. 3 Purification steps and yield of recombinant GA ...... 105

Table 4. 4 Activity yields comparison in recent publications ...... 107

Table 4. 5 Specific activity of GA for various substrates ...... 110

Table 4. 6 GA substrate percent relative activity comparison...... 112

Table 4. 8 Influence of chemical reagents on GA activity ...... 116

Table 4. 9 GA inactivation at different temperatures ...... 117

Table 4. 10 Substrates effects on GA kinetics parameters ...... 121

Table 5. 1 Inactivation parameters of entrapped GA at different temperatures ...... 151

Table 5. 2 Michaelis-Menten kinetics parameters in the substrate range of 0 -75m/ml.. 160

Table 5. 3 Summary of immobilized GA and AA performance in CSTR...... 166

Table 5. 4 Enzyme/starch ratio effects on glucose yields ...... 171

List of Figures

Figure 1. 1 Amylose and amylopectin chemical structures...... 6

Figure 1. 2 Starch hydrolysis and enzymes ...... 8

Figure 1. 3 Structure model of A. awamori GA ...... 10

Figure 1. 4 Principles of enzyme immobilization ...... 27

Figure 2. 1 One step starch hydrolysis at high temperature ...... 38

Figure 3. 1 Co-immobilization of GA and AA in calcium alginate beads and capsules. .. 48

Figure 3. 2 Co-immobilized calcium alginate gel capsule system...... 49

Figure 3. 3 Calcium alginate gel capsules and beads...... 56

Figure 3. 4 Comparison of glucose production versus time of entrapped GA in beads and capsules ...... 56

Figure 3. 5 Comparison of the activities of co-immobilized system and entrapped GA capsules. In 10mg/ml maltose, pH 5.5, 55°C ...... 56

Figure 3. 6 Calibration curves of Bradford test with and without alginate (0.05% w/v). . 58

Figure 3. 7 Optimum pH of free-mixed and co-immobilized enzyme in capsule systems.

...... 61

Figure 3. 8 Optimum temperature of free-mixed and co-immobilized enzyme in calcium alginate capsules...... 62

Figure 3. 9 Michaelis-Menten kinetics of free-mixed and co-immobilized enzyme calcium alginate capsules. At pH 5.5 and 65°C...... 64

xvi

Figure 3. 10 Relative activities in terms of glucose production and AA remaining percentage on the capsules for the starch hydrolysis with co-immobilized GA and AA system...... 65

Figure 3. 11 Effect of GA/AA ratios on glucose production rate...... 66

Figure 3. 12 Reusability comparisons of co-immobilized and mixed-immobilized calcium alginate gel capsules systems for GA and AA immobilization...... 67

Figure 3. 13 Co-immobilized enzyme (GA and AA) and mixed individual immobilized enzyme in calcium alginate beads. pH 5.5, 65°C, 5mg/ml starch...... 68

Figure 3. 14 Comparision of co-immobilized capsules and beads for GA and AA immobilization...... 69

Figure 3. 15 Co-immobilized GA and AA in capsules for starch degradation and glucose production of 1st usage...... 70

Figure 3. 16 Thermostability of different systems up to 5 usages, free mix enzymes, co- immobilized enzymes, and modified co-immobilized system by considering the AA lose.

...... 71

Figure 4. 1 Construction of expression vector pGKGA...... 84

Figure 4. 2 Production of thermostable GA by recombinant DNA technology...... 86

Figure 4. 3 Purification of recombinant thermostable glucoamylase...... 88

Figure 4. 4 Purification setup...... 89

Figure 4. 5 A pressurized stirring tank reactor for enzyme activity test...... 91

Figure 4. 6 Growth at different conditions...... 95

Figure 4. 7 Growth comparison with and without adding IPTG...... 96

xvii

Figure 4. 8 SDS-PAGE gel of 0.5mM IPTG expression...... 99

Figure 4. 9 Effects of different IPTG concentration for protein expression of cell pellets.

...... 100

Figure 4. 10 SDS-PAGE of purification process...... 102

Figure 4. 11 Two bands after purification...... 104

Figure 4. 12 pH effects on free GA activity at 95°C (relative activities are normalized by the activity at pH 5), use 10mg/ml maltose as substrate...... 108

Figure 4. 13 Effects of temperature on free GA activity at pH 5, use 10mg/ml maltotriose as substrate...... 1099

Figure 4. 14 Proposed explanation of maltotriose with best substrate activity...... 113

Figure 4. 15 Inactivation of GA at different temperatures...... 117

Figure 4. 16 Arrhenius plot of the thermal inactivation rates for GA in free form. .... 11919

Figure 4. 17 Michalies-Menten model fitting with respect to substrates, maltotriose and maltose...... 120

Figure 5. 1 A covalent binding procedure for the co-immobilization of GA and AA in sodium alginate capsules...... 137

Figure 5. 2 Evaluation of chemical effects on entrapped GA...... 140

Figure 5. 3 CSTR setup...... 145

Figure 5. 4 pH influences on free and entrapped GA at 95 oC ...... 147

Figure 5. 5 Temperature influence on free and entrapped GA...... 149

Figure 5. 6 Inactivation of entrapped GA at various temperatures...... 151

xviii

Figure 5. 7 Arrhenius plots of the thermal inactivation rates for recombinant GA in free nd immobilized form...... 153

Figure 5. 8 Influence of pH on the free mix and co-immobilized system...... 156

Figure 5. 9 Influence of temperatures on the co-immobilized and free mix enzymes

(thermostable GA and AA) system...... 158

Figure 5. 10 Michaelis-Menten kinetics of free mixed and co-immobilized GA and AA.

...... 159

Figure 5. 11 Reusability and inactivation rates of free, co-immobilized and mixed individually immobilized enzymes...... 163

Figure 5. 12 CSTR glucose concentration (a) change by using co-immobilized and mixed individually immobilized enzymes. Starch concentration (b) change by using co- immobilized, mixed individually immobilized biocatalysts (GA and AA) and blank capsules...... 165

Figure 5. 13 CSTR starch and glucose concentration change by using co-immobilized enzyme in 100mg/ml starch with a flow rate of 20mg/ml...... 167

Figure 5. 14 The cumulative glucose production in batch reactor ...... 169

xix

Nomenclature

A340: net absorbance at 340 nm

AA: alpha-amylase bp: base pair

BSA: bovine serum albumin

CSTR: continuous stirred tank reactor

DP: degree of polymerization

EDAC: N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide

EDTA: Ethylenediaminetetraacetic acid

GA: glucoamylase

GRAS: Generally Recognized As Safe

His6: hexahistidine tag

IMAC: immobilized metal affinity chromatography

IPTG: isopropyl--D-1-thiogalactopyranoside kb: kilobase pairs kd: enzyme denaturation rate constant kDa: kilodaltons

Km: Michaelis-Menten constant

LB: Luria-Bertani growth medium

M: moles/liter

MES: 4-morpholineethanesulfonic acid

MW: molecular weight xx

OD600: optical density as measured at 600 nm

OTR: oxygen transfer rate

PCR: polymerase chain reaction

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

Sulfo-NHS N-hydroxysulfosuccinimide t1/2 : half-life of enzyme inactivation

U: unit of enzyme activity

Vmax: maximum achieved velocity of an enzymatic reaction w/v: weight to volume ratio w/w: weight to weight ratio

xxi

Introduction

Starch hydrolysis products have wide applications in the food industry. For the past four decades, the starch industry has shifted from acid hydrolysis to enzymatic hydrolysis (Leisola et al., 2002; van der Maarel et al., 2002) due to the high efficiency and specificity of biocatalysts.

The industrial hydrolysis of starch involves several steps and several enzymes, including alpha-amylase (AA), glucoamylase (GA), and pullulanase. First in the liquefaction step, the starch is gelatinized (105°C , 5-10 min) and degraded to oligosaccharides at pH 6-6.5 and 95°C for 2 hours by a bacterial thermostable AA. Then the pH and temperature are adjusted to the optimum temperature (55-60°C ) and pH (4-

4.5) for the glucoamylase in the saccharification step. The pH and temperature adjustments are adapted by enzyme specification rather than ideal process conditions

(Crabb and Shetty, 1999).

Possible improvements for the current industrial practice of starch hydrolysis could be recovering enzymes, developing thermostable GA, minimizing the pH difference between the liquefaction and saccharification steps, and lowering the calcium requirement of AA.

AA and GA are major enzymes for starch hydrolysis. These hydrolysis enzymes represent about 30% of the worldwide industrial enzyme production (van der Maarel et

1 al., 2002; Janeček, 2009). It is projected that by 2012, approximately $320 million of $2.7 billion annual spending in industrial enzymes will be starch hydrolysis enzymes with an average annual growth rate of 4% (Pandey, 2006; BCC, 2008; Illanes, 2008). Enzymes are biocatalysts, which catalyze the reaction by lowering the activation energy. It could be cost effective and efficient to recover and reuse them. Recovery methods include membrane filtration and immobilization. The immobilization of AA and GA has received attention due to its potentially easy recovery from starch hydrolysis products (Kucera et al., 1982; Arica et al., 1998; Ida et al., 2000; Tanriseven et al., 2002; Roy and Gupta,

2004; Silva et al., 2005; Konsoula and Liakopoulou-Kyriakides, 2006; Sanjay and

Sugunan, 2007). The goal behind immobilization is attachment of enzymes to a support that can be recovered easily and that may offer a potential improvement in thermal stability.

Regardless of methods of immobilization, the immobilized system has two functions. One is non-catalytic (support material) for the purpose of separation, and the other is catalytic (enzymes). Cao (2005) pointed out that high specific activity (U/gram of total immobilized enzyme) is beneficial when designing an immobilized enzyme system rational. Co-immobilization could be used to increase the specific activity of the immobilized enzyme system and reduce the cost of support materials. Immobilizing two or more enzymes on one system has generated increasing interest in recent years and in many areas, such as biosensor and bioproducts (Erhardt et al., 2008; Minakshi and

Pundir, 2008; Cortina-Puig et al., 2009; Odaci et al., 2010; Olcer and Tanriseven, 2010;

Quan and Shin, 2010; Yang et al., 2010; Bankar et al., 2011; Shim et al., 2011; Xu et al.,

2011). Only a few studies of co-immobilization were reported as applied in starch hydrolysis. Yang et al. (2010) and Park et al. (2005) co-immobilized AA and GA for starch hydrolysis at 50-60°C . Atia et al. (2003) co-immobilized β-amylase and pullulanase to reduce the saccharification time with an increased yield of maltose.

Chakrabarti and Storey (1990) investigated a co-immobilized system of GA and pullulanase to enhance starch hydrolysis. There is no literature about co-immobilization of thermostable GA and AA for starch hydrolysis at high temperature. Development of a system with both immobilized and thermostable features could be beneficial for starch hydrolysis.

There are several studies focusing on improving the thermal stability of GA to the level of thermostable AA as well as matching the optimum pH of both enzymes

(Ganghofner et al., 1998; Uotsu-Tomita et al., 2001; Nguyen et al., 2002; Nielsen et al.,

2002; Serour and Antranikian, 2002; Kim et al., 2004; Gomes et al., 2005; Chen et al.,

2007; Dock et al., 2008; Michelin et al., 2008; da Silva et al., 2009; Zheng et al., 2010;

Pavezzi et al., 2011). It was found that fungal GAs have high yields but low thermal stability, with an optimum temperature less than 70°C (Gomes et al., 2005; Chen et al.,

2007). Bacterial GAs have a lower yield and increased optimum temperature up to 80°C

(Ganghofner et al., 1998; Zheng et al., 2010;). Archaeal GAs have been reported as the most thermostable enzymes with optimum temperature up to 90°C (Serour and

Antranikian, 2002; Kim et al., 2004). However, some archaeal GAs are from thermoacidophilic archaea, such as Thermoplasma acidophilum, with a low optimum pH

3 of 2 (Serour and Antranikian, 2002). GAs from archaea Sulfolobus, which have a high optimum temperature (95°C ) and desired range of optimum pH (5-6), are very promising.

In terms of calcium requirements of GA and AA, the lower calcium requirement for thermostable AA will decrease the production cost and downstream ion exchange cost. Low or no calcium dependent GAs are also favorable.

Considering food safety issues, the genetically engineered thermostable AA or

GA should not appear in final products. Immobilization can help to bind and/or confine enzymes on inert material so that they can be separated from the process stream, to assure product safety.

This study has developed a novel immobilized thermostable AA and GA biocatalyst system that can be used for starch hydrolysis in one step for glucose production, thereby reducing the number of steps for the current starch hydrolysis process. Also co-immobilization of thermostable enzymes to a support makes it possible to use in a continuous process, leading to savings in enzyme, labor, and downstream costs and hence increases the efficiency of the process.

Chapter 1 Background and Literature Review

1.1 Starch

Starch is a polysaccharide consisting of hundreds of glucose monomers linked by glycosidic bonds. It is the second most abundant polysaccharide in plant cells used for storing glucose by plants. Starch is one of nature’s most complex materials due to its heterogeneity of structure (Gidley, 2001). It is composed of two types of glucose polymers, amylose and amylopectin, which are arranged in a granule (partially crystalline) (Lindeboom et al., 2004).

Amylose is a linear polymer joined by α, 1-4 glycosidic bonds and its chains show spiral-shaped single or double helixes with six glucose units per rotation (Cunha and

Gandini, 2010). In potato or tapioca starch, it has a degree of polymerization (DP) of

1,000-6,000 with an amylose content of 10-20% (van der Maarel et al., 2002; Talja et al.,

2008). Amylopectin is a branched polymer with an average DP of 2,000,000.

Amylopectin has a cluster structure model consisting of α, 1-4 linked backbone chain and

α, 1-6 linked side chains with 15-45 glucose units (van der Maarel et al., 2002;). The branching locations are every 22-70 glucose units (Cunha and Gandini, 2010). About

95% of glycosidic bonds of amylopectin are α, 1-4 glycosidic bonds. The other 5% are α,

1-6 glycosidic bonds. The structures of amylose and amylopectin are shown in Figure

1.1.

4 1 4 (a) 1

α, 1-4 glycosidic bond

4 (b) 1 α, 1-6 glycosidic bond 4 6 1 4 1

Figure 1. 1 Amylose (a) and amylopectin (b) chemical structures.

(modified from Buchholz et al., 2005).

1.2 Starch hydrolysis and enzymes

Starch hydrolysis can be induced by enzyme, acid, or alkaline treatment. Heating, microwaving, or extrusion sometimes can facilitate hydrolysis when combined with enzymes, acids, or alkaline materials (Hui, 2006).

Under these conditions, the glycosidic bonds of starch are susceptible to hydrolysis, leading to bond cleavage. Resulting hydrolysis products are oligosaccharides or simple reducing sugars such as malotriose, maltose, or glucose.

Several enzymes are involved in starch hydrolysis process (Figure 1.2). The AA, an endo-amylase, cuts the α, 1-4 glycosidic bonds randomly and produces oligosaccharides. The GA, an exo-amylase, produces the glucose sequentially from the non-reducing ends. Often, a debranching enzyme, pullulanase, will also be added to cut the α, 1-6 glycosidic bonds to increase the glucose production efficiency. The glucose is fermented to produce ethanol, In the food industry, glucose is converted to fructose by glucose isomerase because fructose has the highest sweetness among sugars. This enzyme is used in immobilized form and magnesium is added as co-factor (Crabb and

Mitchinson, 1997). However, Ca2+ has inhibitory effects on the glucose isomerase. Thus, ion exchanging was applied to remove the Ca2+, which was added in the liquefaction step to stabilize the AA (Hashida and Bisgaard-Frantzen, 2000). If intending to produce maltose, β-amylase will be used instead of GA. The β-amylase cuts the amylose every two glucose units to produce maltose. Maltose can be used for brewing.

α-1,6 Randomly α-1,4 α-1,6

Non- Reducing reducing α-1,6 end end

α-1,6 α-1,6 Reducing end

Every two units

Maltose AA (Endos)

GA (Exo-NR)

Pulluanases (debranching) F F β-amylase (Exo) Isomerase Frucose Glucose

Figure 1. 2 Starch hydrolysis and enzymes

1.2.1 Alpha-amylase (EC 3.2.1.1)

AA exists widely in plants, microorganisms, and animals. AA is produced commercially through microbial fermentation. The microbial enzyme production of AA generally can meet industrial demands (Gupta et al., 2003). AA internally hydrolyzes 1,4- alpha-D-glucosidic linkages in polysaccharides in which DP is equal to or larger than 3.

The optimum reaction pH and temperature varies with microbial source. It normally requires calcium (1-20 ppm) as a cofactor for activity and stability (Brenda database).

Lowering the calcium dependence of AA can decrease the starch hydrolysis cost (Santa-

Maria et al., 2009). Some other metal ions, such as Mg2+ and Mn2+, are also reported as stimulating cofactors (Krishnan and Chandra, 1983).

Among thermostable AAs, a well-known one is Liquozyme X, which was commercialized by Novozyme in 2001. It is a genetically engineered thermostable AA from a thermophilic bacterium Bacillus licheniformis, which has an optimum growth temperature around 50°C and the highest secretion yield at 37°C . Liquozyme X has a high optimum temperature (95°C ), relatively low pH value (5.2-5.6) and low calcium requirement (0.25-5 ppm). Also this thermostable AA can be inactivated in 10 minutes by lowering the pH to 3.8 and holding at 95°C , which can terminate the reaction easily. The food industry has used this enzyme for starch liquefaction for years.

1.2.2 Industrial glucoamylase (EC 3.2.1.2)

GA is also available widely in many species of plants, animals, and microorganisms. Industrial GA has been produced by filamentous fungi by submerged fermentation with large amounts of extracellular secretion (Kumar and Satyanarayana,

2009). The major source microorganisms for GA are Aspergillus and Rhizopus spp.. The optimum reaction pH and temperature of GA from Aspergillus are pH 4- 4.5 and 55-

60°C . Unlike AA, activity and stability of GA has little dependence on calcium (James and Lee, 1997). GA could have multiple forms from a single gene. It was reported that there are two forms, GAI and GAII, in fungal GA (A. awamori and A. oryzae). GAI, the larger MW form, has a separate starch binding domain (Goto et al., 1997; Hata et al.,

1997 ). Also, even one GA could have two subunits (Koc and Metin, 2010).

The most commonly accepted 3-D structure of GA from fugal or bacterial sources includes three domains: catalytic domain (CD), starch binding domain (SBD) and a linker. The most extensively studied GA from A. awamori, as an example, is shown in

Figure 1.3. The CD starts from the N-terminal linked by a linker region with C-terminal

SBD. The linker region of fugal GA has the tendency to undergo glycosylation with carbohydrates (Nielsen et al., 2002).

Tertiary CD Linker SBD structure

Secondary (α-helix/α-helix)6 β - sheets structrue barrel

Primary Amino acid Amino acid Amino acid structure 1-470 471-508 509-612 55kD 13kD 12kD

Figure 1. 3 Structure model of A. awamori GA (Coutinho and Reilly, 1994; Kumar and Satyanarayana, 2009) SBD has the function of binding starch, and normally there are two binding sites.

Some bacterial GAs may not have SBD and hence cannot hydrolyze raw starch directly

(Zheng et al., 2010). This may distinguish them from fungal GAs. 10

The catalysis mechanism of GA was also generally explained by a two-catalytic carboxylates model (James and Lee, 1997; Sauer et al., 2000; Kumar and Satyanarayana

2009). One carboxylate is a base and the other is an acid. The nucleophilic attack of water causes the transfer of proton from ring oxygen to glycoside oxygen leading to anomeric carbon inversion. The glycoside is cleaved and the hydroxyl ion is added to the anomeric carbon. The glucose was finally released from substrates.

1.2.3 Reducing starch hydrolysis steps

The industrial starch enzymatic hydrolysis is currently performed in several steps.

The first three steps are: gelatinization at 105-110°C, liquefaction by thermostable AA at

95°C to oligosaccharides, and saccharification with GA at 60°C to glucose. The starch hydrolysis process conditions are limited by the enzymes properties. Liquefaction and saccharification steps could potentially be combined most effectively by developing a thermostable GA.

Some researchers investigated a one-step process to produce glucose from starch.

Kim et al. (1988) genetically engineered a Saccharomyces strain to secrete the GA and

AA at the same time. The resulting enzymes had converted 92.8% of available starch into reducing sugars in 2 days. However, the highest reaction temperature was 55°C and the primary reducing sugars were maltose and maltotriose. Janse and Pretorius (1995) used a recombinant strain of yeast, Saccharomyces cerevisiae, to express AA, GA, and pullulanase at the same time. The yeast strain was utilized to convert starch to glucose directly. In their experiments, the strain was grown in starch for 5 days and the optimum

11 temperature was only 50-60°C . Paolucci-Jeanjean et al. (2000) mixed starch with thermostable AA in a continuous recycle membrane reactor at 80°C. The final reactions products were DP1-7 with 70% starch conversion in two hours. The system had limitations due to membrane fouling and enzyme inactivation.

The advantages of using thermostable GA to achieve a one-step starch hydrolysis have been discussed by many authors (Crabb and Mitchinson, 1997; Crabb and

Shetty,1999; Synowiecki et al., 2006). Legin et al. (1998) pointed out that the greatest improvement for current industrial starch hydrolysis would be degrading the starch in one step. They also suggested that using thermostable (>90°C ) GA from archaea, with an optimum pH 5.5-6.5, could be the solution for the improvement. Leveque et al. (2000) also agreed that hydrolysis enzymes from thermophilic or hyperthermophilic archaea could be the top candidates for a one-step bioconversion of starch to glucose, which can decrease the glucose production cost.

1.3 Production of a thermostable GA

The advantages and demands for producing a thermostable GA are apparent.

During the last three decades, many researchers have investigated extensively a variety of microorganisms and utilized different methods to produce thermostable GA.

In order to develop an enzyme with desirable properties, the first step is to find a proper source. The natural source could be animal cells, plant cells, or microorganisms.

By screening microorganisms, the target enzyme that can convert or degrade certain compounds can be identified and classified. However, the yield of enzymes from wild microorganisms could be low or the growth environments of microorganisms could 12 require extreme conditions or the microorganisms may not be available in the Class I microorganisms range (GRAS). The enzyme gene can be transferred into Class I microorganisms to grow and express. Currently, most of the enzymes used in industrial processes are from recombinant microorganisms (Buchholz et al., 2005).

1.3.1 Recombinant DNA technology

Gene cloning is also called recombinant DNA technology. The DNA from the donor organism (such as a wild microorganism: archaea) is extracted, digested, and fused into a vector (plasmid) and hence creates a new combined DNA molecule (DNA construct). This construct is transferred and stays within a host cell (such as E. coli

DH5α). The host cells with the construct are then selected and picked up.

This process involved several steps. Plasmids are independent extrachromosomal entities (circular DNA molecules) in bacterial cells. Most of the plasmid cloning vectors for protein expression are genetically engineered with certain properties such as small size, unique restriction enzyme recognition sites, and antibiotics resistance.

Restriction endo enzymes were used to cut the circular DNA vector into linear DNA molecules and to create the sticky ends of the inserted gene. The sticky ends joined the inserted gene to the vector and formed a recombinant vector. The vector then was inserted into the host cell. The host cells grew in a proper medium and at an optimum temperature, and then were induced by the isopropyl-beta-D-thiogalactopyranoside

(IPTG). IPTG is a highly stable synthetic analog of lactose. It inactivates the lac repressor and induces synthesis of beta-galactosidase, an enzyme that promotes lactose utilization.

The IPTG is used to induce the expression of cloned genes, which are under control of the lac operon. The pQE-80L series of expression vectors, which encodes a lacIq repression module, allows use of any E. coli host strain.

1.3.2 Improvement of thermal stability by natural evolution, directed

evolution, or protein engineering

To achieve an enzyme with desirable properties, there are several methods, such as natural evolution, directed evolution, or protein engineering. Many enzymes have been screened, isolated, and characterized from nature. Methods for modifying and improving enzyme properties have also been developed. Researchers have developed many enzymes into commercially available products with high efficiency and stability using directed evolution. Knowledge of the enzyme evolutionary history can provide useful information in designing and engineering proteins. Most protein engineering research was conducted with rational design, based on knowledge of the protein structure, catalytic properties of the active site residues, and structural stability.

Some authors, such as Antranikian et al. (2005), used natural screening. Others use directed evolution for thermostable GA (Chen et al., 2007; Pavezzi, et al., 2011).

Many studies used protein engineering technology for thermostable GA production (Kim et al., 2004; Egorova and Antranikian, 2005; Dock et al., 2008; Zheng et al., 2010).

1.3.3 Thermostable GA research

Many studies of thermostable GA have been cited in the literature. Efforts in the past three decades are summarized in Table 1.1. 14

Table 1.1 shows that thermostable GAs were produced in three ways: grown directly form thermophilic fungi and bacteria, or mutant forms grown by the directed evolution from wild type, or using protein engineering to improve thermal stability. The optimum reaction temperature of most fungal GAs is lower than 70°C . GA from bacteria can reach optimum temperature of 75-80°C . The archaeal GAs have the highest optimum temperature of 90-95°C . In terms of pH, the fungus GA is in pH range of 3-5. The optimum pH of bacterial GA is around 4-5 whereas the optimum pH of archeal GA can be around 5.5-6. Liquozyme X has an optimum pH of 5.5-6. The development of a GA with an optimum pH value that is close to the pH of thermostable AA could lead to a one- step starch hydrolysis at high temperature.

Table 1. 1 Summary of thermostable GA from different sources

Source Organism Optimum Optimum Specific Thermal Growth Remarks Authors type pH Temp. Activity stability conditions °C U/mg# Fungus Thermomyces 5.0 70 80 Ea=61.5KJ/mol 50°C, shaking 39.6% yield, Rao et al., lanuginosus culture, 48hr MW=57kDa 1981 extracellular Talaromyces flavus 4.0-4.8 50 20 min at 60°C 30°C , 240rpm MW=42kDa Huang Lost 90% of its 96 hr and activity Woodams, 1993 Thermomyces 5.0 70 122.2 t1/2=6 min 14 days 2.6% yield, Li et al., lanuginosus A236 at 80°C MW=72kDa 1998 Thermomyces 4.4-5.6 70 60 half-life times 47°C , Km=0.68mg/ml Nguyen et

1 lanuginosus longer than 1 220 rpm MW=75kDa al., 2002

ATCC 34626 day at 60 ◦C 2 days Talaromyces emersonii 4-4.5 70 3.7 t1/2=48hr, 65 °C Vector Nielsen et in 30% (w/v) pCaHj483 al., 2002 glucose. MW=70kDa Thermomucor indicae- 7.0 60 134.28 t1/2=420 min 40°C, 250 rpm Mold Kumar seudaticae at pH 7, 80°C MW=42kDa and Ea=43KJ/mol Satyanara yana, 2003 Aspergillus flavus A1.1 4.0 65 t1/2=300 min 45°C, 2hr 3.2U/ml Gomes et at 60°C enzyme al., 2005 production (continued)

Table 1.1 continued

Source Organism Optimum Optimum Specific Thermal Growth Remarks Authors type pH Temp. Activity stability conditions °C U/mg# Fungus Thermomyces 4.5 70 t1/2=480 min 45°C 2.8U/ml Gomes et lanuginosus at 60°C al., 2005 A 13.37 Chaetomium 4.0 65 71.2 t1/2=20 min at 50°C, 7days, 3.57% yield, Chen et thermophilum 70°C 120 rpm MW=64kDa al., 2005 Scytalidium 5.5 55 35 t1/2=3.8 min 45ºC, 10% yield, Cereia et thermophilum 15.8 at pH 5, 60°C static,7days MW=83kDa al., 2006 Chaetomium 4.5-5.0 65 t1/2=40 min at Express in Chen et thermophilum 80°C, t1/2=10 vector pPID9K al., 2007

17 min at 90°C Paecilomyces variotii 5.0 55 t1/2=45 min at 30°C, 6days, MW=86.5kDa Michelin 60°C No agitation extracellular et al., 2008 Fusarium solani 4.5 40 t1/2=26 min at 150 rpm at MW=40kDa Bhatti et 60°C 30°C for 48 h Km=1.9mg/ml al., 2007 Aspergillus niveus 5.0-5.5 65 100% activity 40°C, 72h, pH MW=76kDa da Silva et remain 4h at 6.5, static al., 2009 60°C Aspergillus awamori 4.5 65 1250 Ea=100KJ/mol Submerged Vector Pavezzi et fermentation YepPM18 al., 2011 Expressed by MW=100kDa Saccharomyces cerevisiae (continued)

Table 1.1 continued

Source Organism Optimum Optimum Specific Thermal stability Growth Remarks Authors type pH Temp. Activity conditions °C U/mg# Bacterial Clostridium 5-6 75 75°C, 1hr 60°C Km=0.41 Hyun and thermohydrosulfuricum 39 activity 100% anaerobic mg/ml Zeikus, loss 4days 1985 Clostridium 5.0 70 54.4 at 75°C,1hr, 60°C, 30hr MW=75kDa Specka thermosaccharolyticum pH5, 8% of et activity remained al.,1991 4.5 The Express in Ohnishi Clostridium sp. G0005 reactions E. Coli. et al., were run MV1184 1992 at 25 vector pHI901 0.5mM 18 IPTG

Km=3.7mM (maltose) Thermoanaerobacterium 4.0-5.5 40-65 51 <25% maximum 63°C, 42hr, 6% yield, Ganghof thermosaccharolyticum activity remain Anaerobic, MW=75kDa ner et al., DSM 571 at 75°C pH 7.2 1998

Thermoanaerobacterium 4.5 65 Ea=38.8kJ MW=77kDa Feng et thermosaccharolyticum al., 2002 DSM 571 (continued)

Table 1.1 continued

Source Organism Optimum Optimum Specific Thermal Growth Remarks Authors type pH Temp. Activity stability conditions °C U/mg# Bacterial Bacillus 4.5 65 65°C,150 rpm Culture hot Natalia et acidocaldarius RP1 LB spring, al., 2002 medium,12hr Km=11.7 mg/ml Bacillus sp. Strain 5.0 70 34* t1/2=220 min pH 7, 65°C, Gill and J38 at pH 7, Luria Kaur, 2004 70°C broth,17-20hr Thermoanaerobacter 5.0 75 175 t1/2=2.6hr at 37°C, 16hr MW=77kDa Zheng et al., tengcongensis MB4 80°C pET42b vector 2010 auto induction

19 medium

Archaea Methanocaldococcus 6.5 80 37 °C LB E. coli BL21 Uotsu- jannaschii MJ1610 medium Plasmids Tomita et 0.4mM IPTG AMJBX50 and al., 2001 AMJKW69 Thermoactinomyces 6.5 80 overnight E. coli MV1184 Uotsu- vulgaris R-47 at 37 °C LB plasmid Tomita et medium pTGA6060 al., 2001 0.5mM IPTG Picrophilus oshimae 2 90 t1/2=1200 min 60 °C and pH extracellular Serour and at 90°C 0.7 MW=140 and Antranikian, 85 kDa 2002 (continued)

Table 1.1 continued

Source Organism Optimum Optimum Specific Thermal Growth Remarks Authors type pH Temp. Activity stability conditions °C U/mg# Archaea Picrophilus 2 90 t1/2=1440 min 60 °C and pH extracellular Serour and torridus at 90°C 0.7 MW=133 and Antranikian, 90 kDa 2002 Thermoplasma 2 90 t1/2=1440 min 60 °C and pH extracellular Serour and acidophilum at 90°C 1-2 MW=141 and Antranikian, 95 kDa 2002 Sulfolobus 5.5-6 90 56.3 at LB medium MW=65kDa Kim et al., solfataricus P2 80°C 37°C Tetramer, 2004 expressed in 20 E.coli. pGNX4

Sulfolobus 4.5 80 D-value of 17 Cloned and Njoroge et tokodaii hr at 80°C expressed in al., 2005 E. coli. Dimer Picrophilus 5 50 5.6 t1/2=60 min 60°C pH 0.9 MW=73kDa Schepers et torridus at 60°C al., 2006

Thermoplasma 5 75 4.2 t1/2=120 min 60°C pH 1.5 MW=66kDa Dock et al., acidophilum at 80°C with 2008 DSM 1728 presence of 5mM Ca2+ # One U is defined as the amount of enzyme that produced 1 µmol glucose per minute under assay conditions.

*One U is defined as the amount of enzyme that produced 1 µmol of reducing sugar per minute under assay conditions.

1.4 Enzyme purification using immobilized metal-affinity chromatography (IMAC)

Although protein content in a living prokaryotic or eukaryotic cell is less than

20% (Buchholz et al., 2005), there are still hundreds of proteins present in cells. To recover the target enzyme, purification methods are designed based on the properties of protein such as size, charge, biological affinity, or hydrophobicity.

Chromatography is one of the most important technologies for separation.

Immobilized metal-affinity chromatography (IMAC) is used widely for protein purification, especially for recombinant proteins. IMAC was first introduced by Porath et al. (1975). The method is based on the chelating affinity between certain amino acid residues, specifically histidine and cysteine, and transition metal ions (Ni2+, Zn2+,

Co2+, and Cd2+). The metal ions were immobilized on resins to create a stationary phase with an affinity to ligands of target proteins. The ligands in protein are electron donors. IMAC can be a rapid and efficient purification method that can remove most contaminants and hence achieve a high purity. A homogenous target protein with more than 95% purity can be obtained by IMAC (Zachariou and Bailon, 2008). Therefore,

IMAC is used widely in modern recombinant protein production and purification.

IMAC has three main steps: adsorption, washing, and elution. The adsorption is usually in alkaline pH to assure that the electron donors are unprotonated. Normally a high concentration (1M) of sodium chloride is added to quench the non-specific electrostatic interactions (Sulkowski, 1985). During washing, a large amount of the non-competitive buffer is passed through the column to remove the contaminants.

Ligand exchange is a common method for elution. The ligands in the proteins are replaced by the elutant (other electron donors), such as imidazole, at neutral pH, and hence the target proteins are released. The advantage of using a neutral pH and imidazole is that the conditions used during this process can produce the soluble protein in a biologically native form either from the intracellular cytoplasm or the extracellular secretion (Bornhorst and Falke, 2000).

By the recombinant technology, proteins are designed with affinity tags (e.g.,

His6-tag), which are fused to the C or N terminal of the target gene. With the addition of affinity tags, the purification process can be simple and effective by using IMAC method. Among amino acids, histidine shows the strongest interaction with immobilized metal ions (Bornhorst and Falke, 2000). The relative molecular weight of

His6-tag is around 780 Da. The use of relatively small molecule has two advantages.

First, the small size enables the tag to be fused easily into many express vectors.

Second, the small size rarely affects the protein activity. In many cases, it is not necessary to remove the His6-tag (Zachariou and Bailon, 2008). If required, the tag can be removed by adding a cleavage site between the tag and the protein. For example, N- terminal His6-tag can be cleaved by using thrombin and be removed by a Ni-NTA column (Kim et al., 2001). Commercially available cloning vectors for the expression of the His6-tagged recombinant are often used, such as the pQE from Qiagen.

1.5 Enzyme reaction kinetics

The kinetics of enzyme reactions can generally be described by Michaelis-

Menten equation as follows:

Eq. 1.1

where E is the enzyme (M), S is the substrate (M), ES is the enzyme-substrate

-1 -1 -1 -1 complex in M. k1 (M s ), k-1(s ) and kcat(s ) are rate constants.

The reaction rate is:

Eq. 1.2

where ,

is the initial enzyme concentration. The Vmax and Km was calculated from the initial reaction rate versus substrate concentration curve. The initial reaction rate for each substrate concentration was determined from a progress curve developed by measuring the product or substrate concentration versus time during the enzyme

reaction. Km represents the affinity of the substrate for the enzyme. Rate constant, kcat, provides information for maximum number of substrate molecules converted to produce per enzyme molecule and per second. The ratio of kcat to Km is also an

23 important property as it describes how efficiently a substrate is converted into product by the enzyme.

1.6 Thermal stability and enzyme inactivation

Enzymes provide many advantages compared to traditional inorganic catalysts.

Enzymes can catalyze reactions under mild conditions of temperature and pH with high efficiency, specificity, and selectivity. However, enzymes can be inactivated during storage or reaction when conditions such as pH, temperature, ionic strength, solvent, pressure, and mechanical force are not favorable for their activity.

In order to develop a thermostable enzyme, the temperature effects on the structure and functionality of enzymes need to be considered. Enzymes, as proteins, may go through denaturation, and furthermore agglomeration.

The thermal inactivation of enzymes is caused by the weakening of the intermolecular forces that maintain the 3D structure of enzymes (Illanes, 2008). The inactivation of proteins may involve the disruption of covalent or non-covalent bonds.

When the proteins can not recover their initial conformation (improper folding) or start to aggregate, the catalytic activity is lost. The enzyme thermal inactivation can be investigated by plotting the activities versus time at a certain temperature. Most enzymes follow a first-order inactivation when induced by heat as described in

Equation 1.3.

Eq. 1.3

where kd is the inactivation rate constant and E(t) is the remaining enzyme activity at time t. In many situations there are actually two stages. 24

Eq. 1.4

where En is the native form of an enzyme, Er is the reversible unfolding form of an enzyme, and Ei is the irreversible unfolding form. If an enzyme is exposed to a high temperature only for a short time, the enzyme can fold spontaneously to native form when the temperature recovers to initial conditions. One-step simple inactivation assumes irreversible conformational change and unfolding of the enzyme. The temperature effects on the kd can be described by the Arrhenius equation.

1.7 Enzyme immobilization

Immobilization of enzymes can improve thermal stability by decreasing the conformation flexibility through the forced interactions between supports and enzymes

(Unsworth et al., 2007). Enzyme immobilization is utilized to physically or chemically confine or localize enzymes to or within a certain region of an inert material with retention of catalytic activities for repeated and continuous usage (Bickerstaff, 1997;

Beatriz and Francisco, 2006).

Michaelis and Ehrenreich (1908) were the first to adsorb the enzyme invertase on charcoal (Dunnill, 1980). Since then, many immobilization methods, such as covalent binding, entrapment, adsorption, and crosslinking, have been developed. The successful application of enzyme immobilization for industrial use included glucose isomerase, lactase, penicillinamidase, aminoacylase, aspartase, fumarase hydantoinase, and β-galactosidase (Gerbsch and Buchholz, 1995; Beatriz and Francisco, 2006). 25

Enzyme immobilization also has been applied in medical and pharmaceutical areas, using glucose oxidase biosensor, drug delivery systems, and tumor identification

(Spahn and Minteer, 2008).

There are several reasons for using an enzyme in an immobilized form.

Immobilization is preferred to reduce enzyme costs, stabilize enzymes, and to use in continuous process. In addition to more convenient handling of the enzyme, immobilization allows easy separation from the products, thereby minimizing or eliminating protein contamination by the products. This characteristic is especially important for recombinant enzymes. A further benefit of immobilization is often enhanced thermal stability under both storage and processing conditions. Enhanced stability and repeated use improve catalyst productivities.

1.7.1 Immobilization methods and materials

Various methods of enzyme immobilization are shown in Figure 1.4. Enzyme immobilization methods can be classified into three major categories: binding to supports, entrapment in supports, and cross-linking (Bickerstaff, 1997; Beatriz and

Francisco, 2006).

Enzyme immobilization in supports

Entrapping Binding to Cross linking into supports supports

Complex Covalent Adsorption Ionic binding (binding to binding metals)

Figure 1. 4 Principles of enzyme immobilization (modified from Buchholz et al., 2005)

Many materials were used as enzyme support. The support materials include inorganic materials (such as bentonite, silica, metal, and glass) and organic materials

(alginate, chitsan, carrageenan, cellulose, sepharose, agarose, xanthan gum, gelatin, polyacrylamide, polyamides, and polystyrene) (Cao, 2005; Beatriz and Francisco,

2006)

1.7.1.1 Adsorption

Adsorption of enzymes onto the surface of support materials under appropriate pH and ionic strength is the simplest method for the enzyme immobilization (Chaplin and Bucke, 1990). This method involves reversible interactions between enzymes and

27 the surface of the support materials. The involved electrostatic forces are hydrogen bonding, hydrophobic bonding, ionic bonding, and van der Waals force (Bickerstaff,

1997). This method has several advantages: First, the adsorption is simple and there is no need to add other chemical reagents leading to cost reductions. Second, because adsorption method does not involve the use of chemicals, the enzyme catalytic activity is retained, which is particularly important for labile enzymes (Beatriz and Francisco,

2006). Last, because the weak non-covalent interaction is reversible, the reloading of supports with fresh enzymes is possible. However, this weak interaction may also reduce the effectiveness of immobilized enzyme system due to the desorption of enzymes if changes of pH, temperature, ionic interaction, or solvent polarity in the environment occur (Beatriz and Francisco, 2006). After adsorption, the immobilized enzyme system needs to be washed extensively because the adsorption tends to be non-specific and other contaminants could be present on the support. There are also overloading problems, which can reduce enzyme activity due to the steric hindrance between two catalytic sites on the support (Bickerstaff, 1997). Many materials have been used for adsorption, such as calcium alginate, metal oxide, and silicone gel. An appropriate support should have high affinity and binding capacity for enzyme binding with little effect on enzyme catalytic activity (Brodelius, 1970).

1.7.1.2 Entrapment

The entrapment method is based on the physical inclusion of enzymes within a polymeric network, which allows the free diffusion of low molecular weight substrates and products to pass through but restricts the relatively larger molecular weight of enzymes (Kilara and Shahani, 1979; Beatriz and Francisco, 2006).

Entrapped enzymes in a gel matrix are different from adsorped enzymes, which can be similar to a free enzyme in solution. The porosity of the gel matrix or membrane is controlled to prevent enzyme leakage and to allow passage of the substrates and products freely (Brodelius, 1970). The advantage of entrapment is that the immobilization process only involves physical occlusion, which can minimize immobilization effects on enzyme activity. Methods of enzyme immobilization by entrapment are relatively easy and enzymes are entrapped in a gel matrix microenvironment that can protect the enzymes from harmful chemicals, proteins, and solvents (Bickerstaff, 1997). Furthermore, the gel matrix or membrane permeability can be designed to meet different application purposes. Finally, the distinct advantage of entrapment is that it allows co-immobilization by using the combination of two or more immobilized enzymes (Bickerstaff, 1997).

One drawback is that the support may hinder the mass transfer of substrates and products through the gel matrix or membrane leading to reduced enzyme reaction rate. Also, the entrapment method may not allow the big molecular weight substrate to approach the catalytic sites within the gel matrix (Chaplin and Bucke, 1990). Many

29 materials have been used for enzymes entrapment, such as cellulose triacetate, calcium alginate, diacetyl cellulose, chitosan, and polyacrylamide (Cao, 2005).

1.7.1.3 Covalent binding

Covalent binding is used widely for enzyme immobilization. It usually provides the strongest linkage between enzymes and supports (Cao, 2005). The covalent bond is formed between the amino acid residues of enzymes and active functional groups on or attached to the support surface. The most involved amino acid residues are amino group, carboxyl group, hydroxyl group, and sulfydryl group

(Bickerstaff, 1997). The functional groups attached to the support surface normally need to be activated by specific reagents (Brodelius, 1970). Then the enzymes are added and covalent bonds between enzymes and support are formed by a chemical reaction. The activated functional groups attached to the support normally are designed to be electrophilic, reacting with strong electron donor group such as amino group on the enzyme surface (Bickerstaff, 1997). The amino groups, which are responsible for the enzyme catalytic activity, should not be involved in the covalent binding (Beatriz and Francisco, 2006).

The advantage of the covalent binding is that the enzyme is retained on the support surface with minimal leakage due to the strong interactions provided by covalent binding. This is important for the application of catalytic reaction at a high temperature or in a high ionic strength solution. On the other hand, the reaction of covalent bond formation could block some active sites of the enzyme. Also the cost of

30 covalent binding is relative high compared to other immobilization methods

(Brodelius, 1970 ).

Tee and Kaletunc (2009) developed a procedure for covalent binding of AA on calcium alginate beads. The carboxyl groups on calcium alginate (support) were activated by a dehydrating agent, EDAC ( N-(3-Dimethylaminopropyl)-N’- ethylcarbodiimide). Sulfo-NHS (N-hydroxysulfosuccinimide ),which can improve the coupling efficiency by forming semi-stable amine-reactive NHS-ester with EDAC, was also used. EDAC itself is not efficient for the crosslinking with amines because

EDAC reacts with a carboxyl group on the support, forming an intermediate. The formed amine active O-acylisourea intermediate is very unstable, with a half life of seconds under acid or neutral pH (Hoare and Kshland, 1967; Tee and Kaletunc, 2009).

The intermediate may react with an amine on the AA, which will form a stable amide bond. Other intermediates failed to react with amines, resulting in hydrolysis and the regeneration of carboxyl groups. The addition of Sulfo-NHS can stabilize the amine- reactive intermediate by reacting with EDAC to form an amine-reactive Sulfo-NHS ester (Hermanson, 2008). This amine-active Sulfo-NHS ester intermediate is stable from minutes at pH 8-9 to hours at pH 4-5 (Schasfoort and Tudos, 2008). The 4- morpholineethanesulfonic acid (MES) is normally used as buffer environment for the amine-active Sulfo-NHS ester intermediate to react with amines. Either by the addition of 2-mercaptoethanol or by the buffer exchange of activation buffer through desalting column, the EDAC reaction can be quenched (Hermanson, 2008). Finally, the amine

31 groups in the AA will form amide bonds with carboxyl groups on the supports and hence the proteins are bound covalently to the supports.

1.7.1.4 Crosslinking

This method is based on the aggregation of whole cells (microorganisms) but uses only a single enzyme catalytic activity of the cells. The cells serve as the matrix and normally are crosslinked by glutaraldehyde or by physical methods (Buchholz et al., 2005). Therefore, this type of immobilization of whole cells or enzymes, by forming a 3D structure, is support free. This makes the crosslinking method a simple and natural form of immobilization. However, the toxicity of the crosslinking reagents limits its application to many enzymes (Bickerstaff, 1997). Crosslinking of enzymes also can combine with other immobilization methods, such as crosslinking the absorbed enzymes or crosslinking the entrapped enzymes. These combination methods can improve the stability of the enzymes (Brodelius, 1970).

1.7.2 Immobilization of GA

GA has been immobilized by using various support materials and methods as outlined in Table 1.2.

Table 1. 2 GA immobilization methods and supports

Immobilization Support material Enzyme Reaction References method source Glyoxyl-agarose GA from 45-55°C, pH Tardioli et Covalent Aspergillus 7.0 al., 2011 binding niger 1mg/ml starch epoxy groups GA from pH 5.5, 55°C Bai et al., carriers Aspergillus 10mg/ml starch 2009 niger polyglutaraldehyde- pH 4.3, 65°C, Tanriseven activated gelatin 30mg/ml, and Olcer, maltodextrin 2008 cellulose-based carrier Liquid GA pH 4.5, 60°C, Bryjak et (Amigase) 12.5mg/ml al.,2007 starch polyaniline after Aspergillus pH 4-5, 50°C, Silva et al., activation with niger GA 10mg/ml starch 2005 glutaraldehyde dialdehyde cellulose GA from pH 3.5-5, 60- Varavinit Aspergillus 65°C et al., 2001 niger 2%(w/v) starch Magnetic poly- GA from pH 5-6, 50- Arica et al., methylmethacrylate Aspergillus 55°C 2000 microspheres niger soluble dextrin 20mg/ml Porous glass GA pH 4.65, 55- Marsh et 60°C al., 1973 starch Adsorption magnetic metal-chelate GA from 65°C, pH 4.5 Kok et al., beads, Aspergillus 15mg/ml starch 2011 m-poly(ethylene glycol niger dimethacrylate-vinyl imidazole) [m- poly(EGDMA-VIM)] carbon supports Glucoawamor pH 4.6, 60°C, Kovalenko in® 30-35%, and dextrin Perminova, 2008 (continued)

Table 1.2 continued

Immobilization Support material Enzyme Reaction References method source Adsorption Fibrous Polyelectrolytes GA from 25°C and pH 4.7, Shkutina (aminocarboxylic ion- Aspergillus starch et al., 2005 exchangers) awatori Sepabeads EC-EP3 GA from pH 4.5, 60°C, Torres et supports coated with Aspergillus 100 mM maltose, al., 2004 polyethyleneimine niger 10mg/ml starch Magnetic poly(styrene) GA from pH 4-4.5, 50°C Bahar and Aspergillus 10% w/v Maltose Celebi, niger 2002 Amberlite CG-50 GA pH 4.5, 40°C Miyamoto 3.3mM Maltose et al.,1973 Adsorption gelatinized corn starch GA from pH 4.3, 60°C Tanriseven and subsequent and alginate fiber Aspergillus Maltodextrin, 5% et al., 2002 entrapment niger (w/v) Adsorption Chitin GA starch Sanroman Covalent et al., 1992 binding Entrapment Polyacrylamide-Gel GA from pH 4.2,62°C, 4% Yankov et Aspergillus (w/v) soluble al., 1992 niger starch Calcium alginate gel GA from pH 3-5,30- 50°C Kobayashi Rhizopus soluble starch et al., 1987 defemur poly(viny1 alcohol) GA from pH 4.5, 40°C Imai et al., Aspergillus 2% (w/v)maltose 1986 niger and 0.2%(w/v)soluble starch Ca-Alginate Gel coated GA pH 5, 30°C Tanaka et with (Boehringerl 0.5%(w/v)Maltose al., 1984 Partially Qua ternized Mannheim- Poly(ethy1eneimine) Y amanouchi Co. Ltd)

Most of the research focused on the immobilization of fungal GAs. The investigated pH and temperature ranges are pH 3-7 and 25-65°C. Covalent binding was studied more extensively than the other two methods due to its strong binding ability; however, it is also the most costly method (Bickerstaff, 1997). The entrapment 34 method for GA also has been investigated by several researchers, and the limitation of this method is that relatively small molecular weight substrates are preferred.

Although the entrapment method has been used for AA immobilization by using calcium alginate beads (Kumar et al., 2006; Ertan et al., 2007), the direct use of starch as a substrate by using entrapment fungal GA in catalysis was not preferred. Starch is a large molecule to diffuse through the support to reach the enzyme. However, use of a multi-enzyme system makes it possible to entrap the GA and use starch as a substrate.

1.7.3 Co-immobilization of enzymes

Brena and Batista-Viera (2006) reviewed the development of enzyme immobilization and summarized it into three periods. The first period was from 1815 to the 1960s and involved the application of immobilized enzymes in a commercial process such as in the production of acetic acid without the knowledge of the exact mechanism,. The second period was from the 1960s to 1985. Single enzymes were immobilized for industrial use, such as the isomerization of glucose. The third period, from 1985 to the present, involved the development of multiple-enzyme immobilization, including enzyme/enzyme, enzyme/cells, enzyme/cofactors. Mosbach

(1977) presented the idea of ―Togetherness‖ through immobilization by pointing out the multistep enzyme systems and enzyme-cofactor systems could have wide applications. Co-immobilization has been studied in the biosensor and bio-products area since the 1970s, including the co-immobilization of two cells (Nedovic et al.,

2000; Gardin and Pauss, 2001), one cell (or component of cell) and one enzyme

(Cocquempot et al., 1982; Champluvier et al., 1989; Chithra and Baradarajan, 1989), or one enzyme and cofactors (Ukeda et al., 1989; Chen et al., 2010). For the co- immobilization of enzymes, the purpose could be that the process involved multiple substrates (Carrin et al., 2001), or that one enzyme can help to stabilize the other enzyme (Bankar et al., 2011), or that creating the final products requires multi-step reactions (Chaubey et al., 2000; Atia et al., 2003, 2005; Erhardt et al., 2008).

The production of glucose from starch is a multi-step reaction during which the substrate changes. AA degrades the starch into oligosaccharides and then the GA uses oligosaccharides as substrates. The two enzymes can potentially be used as co- immobilized enzymes system.

Chapter 2: Hypothesis and Aims

This study is based on the hypothesis that using both thermostable AA and

GA as a co-immobilized biocatalyst system will increase the efficiency of the starch hydrolysis process. First, AA degrades starch by cutting the α-(1-4)-D-glucosidic bonds. Then the lower molecular weight oligosaccharides can diffuse inside the calcium alginate gel capsules, and gain access to GA, leading to the production of glucose. The glucose diffuses out of the capsules and releases into the solution. The use of thermostable GA and AA has the potential for developing a one-step starch hydrolysis at high temperature. Also the immobilization of GA and AA could improve the thermal stability at high temperature.

The goal of this study is to develop a calcium alginate gel capsule system to co-immobilize the thermostable AA and GA and to use the multi-enzyme system in a continuous starch hydrolysis process. The primary goal of the study is also described schematically in Figure 2.1. In the current industrial starch hydrolysis, the saccharification step is performed at a relatively low temperature (55-60°C), so the processing temperature needs to be reduced after the liquefaction step. Starch hydrolysis at elevated temperature could have faster kinetics. The co-immobilization of thermostable AA and GA can decrease the number of processing steps and hence reduce the cost.

Figure 2.1 describes the concept of using co-immobilized thermostable AA and

GA to improve the current starch hydrolysis process.

Starch slurry Starch slurry 35% pH 4.5 35% pH 4.5

NaOH Adjustment of pH to NaOH Adjustment of pH to solution value of 5-6 solution value of 5-6

Starch gelatinization Starch gelatinization 105ºC 105ºC

Starch liquefaction Thermostable Thermostable Starch liquefaction and Saccharification α-amylase and α-amylase 90-95ºC 90-95ºC by Co- thermostable immobilized enzyme Glucoamylase system

HCL Adjustment of pH to solution value of 4.5

Glucoamylase Saccharification 55-65ºC

Figure 2. 1 Conventional starch hydrolysis (left) and one step starch hydrolysis at high temperature (right).

This study pursued three major aims.

Aim 1: Develop a co-immobilized GA and AA biocatalyst system for one-step starch hydrolysis processing and verify the usability of the biocatalyst system at 60-65°C

This step is to test the hypothesis that the co-immobilized GA and AA can be utilized together to break down the starch directly to glucose. The development of a modified gel capsule method may reduce the rate of diffusion of substrates into and products out of the capsules. The gel capsules have a liquid-core with a semipermeable shell. Immobilized AA on the surface of the gel capsules can degrade the starch into oligosaccharides. Then the oligosaccharides will diffuse inside the gel capsules and reach the active sites of GA. After conversion of oligosaccharides to glucose, the product will diffuse out of the capsules thereby combining the liquefaction and saccharifications steps into one.

Aim 2: Express, purify, and characterize a thermostable GA from archeon Sulfolobus solfataricus P2 by adding His6-tag

There is no commercially available thermostable GA. According to the literature, GA from Sulfolobus solfataricus P2 is very promising. The His6-tag in the vector is fused on the target gene SSo0990. IMAC was used for the purification

2+ process by utilizing the affinity of transition metal ion such as Ni to the His6-tag on the GA. The application of the thermostable GA from archeon S. solfataricus in the starch hydrolysis process at a high temperature (95°C ) eliminates the demand of cooling after the liquefaction step.

Aim 3: Develop an immobilization process for co-immobilizing thermostable GA (gel entrapment) and AA (covalent binding) and test the thermostability, reusability, and kinetics of the co-immobilized enzyme system for starch hydrolysis at a high temperature

The co-immobilized enzyme system including entrapped GA and adsorbed AA was evaluated for immobilization efficiency, performance of the gel capsules for starch hydrolysis, and reusability in a batch reactor. This research demonstrated that the co-immobilized enzyme system can be used for glucose production from starch in one step. Covalent binding by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) as a crosslinker to calcium alginate was applied successfully to thermostable AA in our group (Tee and Kaletunc, 2009). It was demonstrated that covalently bound thermostable AA can be used at 95°C for seven consecutive starch hydrolysis cycles. Enzyme leakage was also reduced by the covalent binding. The co- immobilized thermostable GA (by entrapment method) and AA (by covalent binding method) biocatalyst system developed by using calcium alginate as a support was shown to hydrolyze starch into glucose in a single step. To this end, to the co- immobilized thermostable AA and GA system has a high potential to improve the efficiency of the starch hydrolysis process by reducing capital investment as well as operational costs.

Rationale and Significance:

Enzymes such as AA, GA, and pullulanase play very important roles in starch hydrolysis. Amylolytic enzymes (including AA, β-amylase, GA, α-glucosidase, and pullulanase) account for $225 million of $2 billion annual worldwide sales of industrial enzymes (Tee and Kaletunc, 2009). Immobilized enzymes have extensive applications in the food and biotechnology industries. A variety of immobilization methods have been developed, including adsorption, entrapment, and covalent binding. Immobilization allows the reuse of enzymes, and easy separation of the enzymes from the starch hydrolysis products, thereby saving enzymes, labor, and downstream costs (Gerhartz, 1990). Immobilization can also improve the thermal stability of enzymes because of the restriction of conformational flexibility in the immobilized form (Tumturk et al., 2000). Due to the industrial importance of AA and

GA, there are ongoing interests in producing thermostable AA and GA suitable for new industrial applications, especially in sugar and ethanol production. For example, the saccharification step costs $0.1 per gallon of ethanol by turning corn starch to ethanol. In the starch model, enzymes (including C6 fermentation at $0.01 per gallon of fuel ethanol) contribute approximately $0.045 to the cost of a gallon of fuel ethanol

(NREL, 2000). The total savings for a capacity of 110 MMgy (million gallons per year) is about $3,850,000 if it is assumed that there is no loss between each reuse of immobilized enzymes. About 5 billion gallons of ethanol were produced from corn in

2009 in the US (USDA, 2009). In industrial starch hydrolysis, the saccharification step is performed at a relatively low temperature (around 60°C), so the processing

41 temperature needs to be reduced after the liquefaction step. Cooling water (1$/1000 lb) can also be saved to reduce costs. In terms of pH adjustment, acid and alkaline material costs can also be reduced. The thermostable enzyme proposed can be used with a low calcium requirement. Further, GA doesn’t need the calcium to promote the enzyme reaction. The use of enzymes with low calcium requirement can decrease the cost of the ion exchange step.

There are several studies investigating immobilization of starch hydrolysis enzymes individually, however, only a few studies exist on the immobilization of a multi-enzyme system. The co-immobilization of GA and AA opens up a way for a one-step starch conversion. There is no research about the co-immobilization of thermostable AA and GA enzymes. This co-immobilization method may reduce the processing steps such as pH, temperature adjustment, and ion exchange. It offers the potential to decrease the cost of starch hydrolysis.

There are few studies in the literature investigating the cost savings by using a one-step starch hydrolysis process. The savings can come from the chemicals, by adjusting the pH; the enzyme, by immobilization; and the labor, by shortening the reaction time. Moreover, this knowledge can also be applied to the starch-to-ethanol process, and may improve the efficiency of the starch-to–glucose process.

Chapter 3: Co-immobilization of AA and GA for starch hydrolysis

3.1 Introduction

Alpha-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) are major enzymes in starch hydrolysis. Starch hydrolysis enzymes account for 30% of the worldwide industrial enzyme production. The ease of recovery, reusability, and improved thermal stability can be accomplished by immobilization of enzymes. Furthermore, the use of immobilized enzymes reduces enzymes, labor, and downstream costs for enzyme- catalyzed reactions (Gerhartz 1990). Immobilization of enzymes involved in the starch hydrolysis process has been investigated to improve the process and reduce cost

(Tanriseven et al., 2002; Bryjak, 2003; Roy and Gupta, 2004; Milosavic et al., 2005;

Sanjay and Sugunan, 2007; Gangadharan et al., 2009).

Immobilization method and choice of support are two major considerations in enzyme immobilization. Enzymes are immobilized in or on the support by entrapment, adsorption, or covalent binding. Entrapment is a suitable approach for the enzyme reactions involving small molecular weight substrate and products, while adsorption and covalent binding are used for large molecular weight substrates. Calcium alginates are used widely as a support for enzyme immobilization in the food industry due to the mild gel formation condition and the absence of risk to human health (Chaplin and

Bucke, 1990; Busto et al., 2006; Leick et al., 2010). Calcium alginate gel were used

43 for entrapment of enzymes in the form of beads (Dey et al., 2003; Roy and Gupta,

2004), or as gel capsules (Tanriseven and Dogan, 2001; Blandino et al., 2000, 2003).

Calcium alginate capsules were prepared for invertase entrapment by dripping 4%

(w/v) calcium chloride and 57% (w/v) maltodextrin into 1% (w/v) alginate solution

(Tanriseven and Dogan, 2001). Maltodextrin was used as a non-gelling polymer to adjust the viscosity in order to achieve a spherical capsule shape. The maltodextrin were expected to diffuse out of capsules after the completion of gel crosslinking

(Nigam et al., 1988). Blandino et al. (2000) used the calcium alginate gel entrapment method to encapsulate glucose oxidase to create liquid-filled capsules with thin semipermeable membrane. They found that the entrapped glucose oxidase leaked 8 ± 3% after 22 hr under 600 rpm agitation in acetate buffer at pH 5.1 from capsules prepared by using 1% (w/v) sodium alginate and 4% (w/v) calcium chloride.

A co-immobilization method that immobilizes two enzymes in one system has been investigated for use in the starch hydrolysis process. Yang et al. (2010) co- immobilized AA and GA on magnetic chitosan beads by using glutaraldehyde as a crosslinker (a dialdehyde connects the carboxylic group of the chitosan and amine group of protein) and used the enzyme system for starch hydrolysis up to 15 cycles.

However, the starch hydrolysis reaction was performed at 50°C (40 ml in a stirred tank) or 25°C (1.5 ml in a centrifuge tube under magnetic field). Park et al. (2005) applied the covalent binding method to immobilize AA and GA onto diethylaminoethyl (DEAE) cellulose and then entrapped the resulting system inside calcium alginate beads. The thermal stability of the immobilized enzyme system was

44 improved, with the optimum reaction temperature shift from 50°C (free AA and GA) to 60°C (co-immobilized system) concurrent with the optimum pH shift from 5 to 5.5.

Their findings demonstrate that the co-immobilization of GA and AA can produce glucose from starch in one step. However, the enzyme system included glutaraldehyde as a crosslinker for covalent binding of GA and AA to DEAE cellulose. Furthermore, in addition to covalent binding of GA and AA on DEAE cellulose, the entrapment inside the calcium alginate bead are expected to create a diffusion barrier for both the substrates and the products (Park et al., 2005).

The co-immobilization of enzymes may reduce the processing steps, such as pH adjustment and ion exchange, leading to the potential decrease of the cost for starch hydrolysis. GA has been shown to have affinity to alginate (Teotia et al., 2001; Roy and Gupta, 2004), which makes alginate an ideal support for immobilization of GA.

Calcium alginate gel prepared by using sodium alginate in the concentration range of

0.5-4% (w/v) has a small pore size, which can prevent the leakage of enzymes with

MW of 60 kDa to 100 kDa (Nigam et al., 1988). GA used for this study (from

Aspergillus niger ) has a MW of 68 kDa-94 kDa, and can also form a dimer in solution

(Jorgensen et al., 2008). At a concentration of 1-2% sodium alginate, a low level GA leakage was expected. Furthermore, AA also shows affinity to alginate, and can be immobilized on the alginate beads or capsules by adsorption method (Sardar and

Gupta, 1998). Konsoula and Liakopoulou-Kyriakides (2007) used a gel capsule method for entrapment of a thermostable AA to be used for starch hydrolysis. The results of studies by Konsoula and Liakopoulou-Kyriakides (2007) and Blandino et al.

(2000), who applied the gel capsule method for glucose oxidase immobilization, showed that it was possible to change the capsule properties (such as the thickness of membrane or permeability of different substrate) by adjusting the gelation conditions.

The hypothesis of co-immobilized GA and AA enzyme system is that the AA will first degrade the starch by cutting the α-(1-4)-D-glucosidic bonds, then the oligosaccharides can diffuse inside the alginate beads/capsules, and react with GA to produce glucose. Calcium alginate beads made with 2% (w/v) solution have an average pore diameter of 80 to 100 Å (Stewart and Swaisgood, 1993). The glucose and several oligosaccharides (DP<10) are smaller than this, if we consider the diameter of glucose as approximately 1 nm (Netrabukkana et al., 1996). Calcium alginate gel (1-

2% w/v) has no restriction of passage of neutral products or substrates up to MW of

5,000 (Kierstan and Buck, 1977). The aim of this study was to apply the modified gel capsule method to co-immobilize the AA (by adsorption) and GA (by entrapment) and verify the application of a multi-enzyme system in a batch reactor for one-step starch hydrolysis processing.

3.2 Materials and Methods

3.2.1 Materials

Glucoamylase (A7095) from A. niger, α-amylase (A4551) from B. licheniformis, sodium alginate (A2158 low viscosity), soluble potato starch (S2630), Bradford reagent (B6916), glucose assay reagent (G3293), maltose (M5885), and bovine serum albumin (P0834) were purchased from Sigma (St. Louis, MO). Calcium chloride, 46 sodium acetate, acetic acid, hydrochloric acid, and sodium hydroxide were purchased from Fisher Scientific (Pittsburg, PA, USA).

3.2.2 Co-immobilization of GA and AA

GA was entrapped inside beads or capsules. Beads were produced by dripping a mixture of sodium alginate (2% w/v) and GA into 100mM calcium chloride solution, while capsules were produced by dripping a mixture of calcium chloride and GA into alginate solution. Then, AA was immobilized on the surface of beads or capsules by adsorption.

3.2.2.1 Immobilization of GA in beads or in capsules

Alginate beads were produced by dripping a mixture of GA (1.67 mg/ml) and alginate (2% w/v) into 100 mM CaCl2, solution in acetate buffer at pH 5.5 through

26G (ID = 0.26mm) using a peristaltic pump (0.6 ml/min) (Variable-Flow Peristaltic

Pump Low Flow, Fisher Scientific, Pittsburg, PA). The beads were incubated in 0.1 M

CaCl2 solution for 2 hours before use.

Alginate capsules were formed by dripping a mixture of CaCl2 (4% w/w) solution and GA (1.67 ± 0.1 mg/ml) through a 26G (ID = 0.26mm) needle (Becton

Dickinson & Co, Rutherford, NJ) into 1% (w/v) 40 ml of alginate solution at 0.15 ml/min by a peristaltic pump. Sodium alginate solution was kept at 4°C with a stirring speed of 400 rpm. The distance from needle to solution level was maintained at 27cm.

The alginate capsules were washed with de-ionized water, and were collected using a ceramic filter. Gel capsules were cured in 50 mM sodium acetate buffer containing

100 mM calcium chloride at pH 5.5, for 2 hours.

3.2.2.2 Adsorption of AA on beads or capsules

For adsorption of AA on bead/capsule surfaces, 2 grams of capsules or beads containing entrapped GA were immersed inside a 30 ml AA solution (22 ± 1 µg/ml) and stirred at 60 rpm for 2 hours. The co-immobilized capsules or beads were stored in

100 ml 50 mM acetate buffer at pH 5.5 containing 5 mM calcium chloride at 4°C .

The flow charts of co-immobilization procedure for AA and GA as beads and capsules are shown in Figure 3.1.

GA+ sodium alginate GA+ CaCl2

Dripping Dripping

CaCl2 Sodium alginate

Calcium alginate beads Calcium alginate capsules with entrapped GA with entrapped GA

AA AA solution solution Mixing Mixing

Co-immobilized AA and GA Co-immobilized AA and GA beads capsules

Figure 3. 1 Co-immobilization of GA and AA in calcium alginate beads (left) and capsules (right). 48

Figure 3. 2 Co-immobilized calcium alginate gel capsule system.

3.2.3 Starch hydrolysis reaction

A batch system was utilized for mixed-free enzyme and for co-immobilized enzyme systems. Starch hydrolysis enzyme kinetic studies were performed in a 150-ml beaker fitted with a silicone stopper with openings. A thermocouple probe was installed through one opening to monitor the temperature of reaction medium. A condenser unit was connected to the second opening to prevent the loss of water by evaporation. The third opening was used for sample removal. The reaction beaker was placed on a hot plate with temperature control (Model Isotemp, Fisher Scientific,

Pittsburg, PA). The temperature was controlled by the hot plate heating unit to ±1°C .

The solution was stirred at 260 rpm using a magnetic stir bar. 49

For the experiments, starch solution concentrations of 1.25, 3, 5, 10, 15, and 20 mg/ml were used. Starch solutions were prepared by dissolving soluble potato starch in 50 mM acetate buffer at pH 5.5 acetate buffer solution containing 5 mM CaCl2. In a batch reactor, 50 ml of starch solution at desired pH was heated to the selected reaction temperature and 1 ml free enzyme solution or 2 grams of alginate capsules or

2 grams of alginate beads with co-immobilized AA and GA were added to starch solution. Approximately 0.2-ml samples were removed every 15 s for free enzyme and every 1 minute for co-immobilized enzyme from reaction medium (up to 12 minutes) to monitor the starch and glucose concentrations. The reaction was stopped by addition of 0.8-1 ml 0.5N HCl prior to starch and glucose assays.

A glucose test kit (Sigma G3293) was utilized to determine glucose concentration. The pH was adjusted to neutral (6-7) by 0.5N sodium hydroxide. Then a 50-200-µL sample was added into 1 ml glucose test reagent. The absorbance was measured at 340 nm wavelength by using 1-cm path length quartz cuvette.

3.2.4 Protein test by Bradford method

The protein content of the enzyme solution before and after each step of immobilization procedure was determined by using the Bradford reagent. Bovine serum albumin (BSA) was used as a standard. Two standard calibration curves were used for the protein test. One of them was prepared with acetate buffer containing 100 mM CaCl2. The other one was prepared with acetate buffer containing 5 mM CaCl2 for detection of protein in wash solutions. For GA immobilization in calcium alginate

50 capsules, protein content in alginate solution subsequent to immobilization was determined from the calibration curve prepared as activity versus GA concentration using maltose (10 mg/ml) as substrate.

3.2.5 Loading efficiency of the immobilized enzyme

The amount of protein immobilized in or on calcium alginate capsules was calculated by applying mass balance. The total mass of protein in the alginate solution and washing buffers after immobilization (Pf) were determined and subtracted from the initial mass of protein (Pi) to calculate the protein content of gel capsules for both

AA and GA. The loading efficiency (LE) was defined by the following equation.

3.2.6 Optimum pH and temperature for mixed-free and co-immobilized

enzyme system

The activities of free GA and AA were determined individually at 55°C over a pH range of 4.5-7. Glucose test was used for GA activity analysis. The initial reaction rate was calculated from glucose concentration versus time data at each pH studied.

The optimum reaction temperature was investigated at pH 5.5 and at temperatures of

45, 55, 65, 75, and 85°C . Optimum pH study was conducted at 55°C (free mix AA and

GA) or 65°C (co-immobilized AA and GA) reaction temperature by varying the pH of the starch solution at pHs 4.5, 5, 5.5 (acetate buffer) and 6, 7 (phosphate buffer). 51

3.2.7 Determination of enzyme kinetics parameters, Km and Vmax

The starch hydrolysis reaction was performed at various initial starch concentrations of 1.25, 3, 5, 10, 15, and 20 mg/ml, at pH 5.5, and at 65°C. The initial rate of reaction in terms of glucose produced per min was determined and plotted versus initial starch concentration. The data were fitted to the Michaelis-Menten kinetics equation to calculate the kinetic parameters, Vmax and KM of the free-mixed and co-immobilized enzyme systems.

3.2.8 Reusability of the co-immobilized enzyme

The co-immobilized enzyme system was used for starch hydrolysis six times (six cycles) in a batch system. The enzyme reaction for each cycle was stopped after 12 minutes of reaction time. After each cycle, the gel capsules or beads were removed from the reaction medium using a ceramic filter and they were washed with acetate buffer. AA and GA leakages from gel capsules were determined by analyzing starch degradation and glucose production, respectively, using the samples from reaction medium.

A mix of gel capsules containing only entrapped GA and gel capsules containing only adsorbed AA was used as a control to investigate the effect of adsorbed AA on rate of glucose production for the co-immobilized enzyme system.

3.2.9 Thermal stability of mixed free and co-immobilized enzymes system

Thermal stability of mixed free enzyme at 65°C was studied by heating the enzyme solution for five consecutive cycles of 12 minutes each to simulate the 52 immobilized enzyme reusability tests as described by Tee and Kaletunc (2009). The amounts of total and of each free enzyme were kept identical to the amounts in co- immobilized enzyme system. The mixed free enzymes were incubated at 65°C , and every 12 minutes a mixed GA and AA (0.4mg and 0.43 mg) activity test was performed by using 5mg/ml starch.

After each use for 12 minutes, the capsules were collected by using a ceramic filter. The capsules were washed with 100 ml of acetate buffer at pH 5.5 to remove the starch and other reaction products.

3.3 Results and Discussion 3.3.1 Co-immobilization of GA and AA on calcium alginate beads and

capsules

The alginate beads or gel capsules with entrapped GA were produced. The formation of capsules required higher agitation speed (400 rpm) than alginate beads

(260 rpm) to avoid agglomeration of capsules formed. The optimum distance from needle to alginate solution to ensure proper shape was determined. Gel beads had higher internal diffusion resistance than capsules (Figure 3.3). The observation can be attributed to the hollow inside structure of capsules, which was confirmed by cutting a capsule in half (Figure 3.3a). The calcium alginate beads are shown in Figure 3.3b.

Compared to capsules, beads had more solid structure and longer diffusion path.

Therefore, the internal diffusion resistance of beads may be higher than that of capsules.

(a)

(b)

Figure 3. 3 Calcium alginate gel capsules and beads. capsule (a) , solid bead (b) capsule/bead was cut into 2 halves as shown in the right .

The two systems were put into maltose (10 mg/ml) for the activity test (Figure

3.4), and the results showed that the two entrapment systems had very close protein encapsulation efficiency (52% for beads and 56% for capsules), but the specific activity (initial rate divided by entrapped GA amount) of the capsules was higher than the beads in terms of glucose production using maltose as a substrate (Table 3.1). The specific activity of GA in gel capsule was approximately 1.6-fold higher than GA entrapped in beads. The gel capsule method creates a hollow internal aqueous environment in contrast to the solid gel environment created by the bead method. The

54 free GA had a specific activity of 17.5 ± 0.1 (µmol glucose/min/mg enzyme). This indicated that the gel capsules had a diffusion limitation because the specific activity of free GA was 3.45 times that of the GA in gel capsules.

A control experiment for the comparison of the glucose production rate of the co-immobilized capsules and the entrapped GA capsules was also performed in 10 mg/ml maltose. With the adsorption of AA outside, the glucose production rate of capsules did not decrease (Figure 3.5). The results indicated that the adsorbed AA on calcium alginate gel capsules did not provide an additional diffusion barrier for maltose. The thickness of the capsule membrane was 0.39 ± 0.03 mm.

Table 3. 1 Gel capsule and bead comparison

Encapsulation efficiency Specific activity of entrapped of GA GA Size

[%] [µmol glucose/min/mg enzyme] [mm]

Beads 52.15% ± 2.90% 3.14 ± 0.07 1.97 ± 0.06

Capsules 56.11% ± 1.12% 5.07 ± 0.12 2.98 ± 0.21 50ml reaction medium, at pH 5.5 and 55°C .

160

140

120

100

Glucose production 60 [µg glucose/ml per GA] mg [µg 40

0 0 100 200 300 400 500 Time [s]

Figure 3. 4 Comparison of glucose production versus time of entrapped GA in beads (Δ) and capsules (ᵡ). 2 gram of beads or capsules in 50ml for systems, pH 5.5, 55°C , 10mg/ml maltose as substrate. 100

[µg glucose/(ml)] [µg 40 Glucose concentration

0 0 200 400 600 800 1000 Time [s] Figure 3. 5 Comparison of the activities of co-immobilized system(Δ) and entrapped GA capsules(ᵡ). In 10mg/ml maltose, pH 5.5, 55°C .

For the capsule method, the amount of GA inside the support was determined by subtracting the GA amount in alginate solution and wash solution from the initial amount of GA. The amount of alginate in wash solution at low concentration (0.05%) did not affect protein detection based on the standard curve with or without sodium alginate (Figure 3.6). However, the detection of enzyme in 1% sodium alginate required that the protein content had to be calculated based on the calibration curve of enzyme activity versus enzyme amount (Appendix D).

The adsorption efficiency of protein to gel capsules was found to be 67 ± 2.3%.

For AA, 0.43 mg was adsorbed on 2 grams of capsules and 0.4 mg of GA was entrapped inside the calcium alginate gel capsules. This leads to a GA/AA ratio of 1:1 on the co-immobilized gel capsule system. The adsorption efficiency of protein to calcium alginate beads was 54 ± 1.5%. For AA, 0.36 mg was adsorbed on 2 grams of beads and 2.82 mg of GA was entrapped inside the calcium alginate gel beads. This leads to a GA/AA ratio of 7.8:1 on the co-immobilized gel beads system. A 1.71-ml mixture of GA and CaCl2 solution produced approximate 7.45 gram of capsules. A

3.22-ml mixture of GA and alginate solution produced approximate 2 gram of beads.

1.2

0.8

0.6 Net Net Absat595nm 0.4

0.2

0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 BSA concentration[mg/ml]

Figure 3. 6 Calibration curves of Bradford test with (Δ) and without (ᵡ) alginate (0.05% w/v).

3.3.2 Effect of immobilization on optimum pH and temperature of enzyme

system

The optimum reaction temperatures and pH of free AA, GA, mixed AA and

GA, and co-immobilized beads or capsules were given in Table 3.2. For free AA, the optimum conditions were based on starch degradation rate using 5 mg/ml starch as substrate. For free GA, the optimum conditions were based on glucose production rate using 10 mg/ml maltose as substrate. For mixed free enzymes and the co-immobilized system, the optimum conditions were in terms of glucose production rate by using 5

58 mg/ml starch as substrate. The optimum pH and temperature of mixed free AA and

GA have the same optimum conditions as free GA.

Table 3. 2 Optimum reaction temperature and pH for different systems

Free Free Mixed Free Co-immobilized Co-immobilized GA AA# GA&AA (1:1) beads capsules

Optimum temperature(°C ) 55 60 55 60-65 65

5.5- Optimum pH 5.5 6 5-5.5 5.5 5.5 # the activity is in terms of starch (5mg/ml) degradation.

The optimum pH and temperature of the co-immobilized enzyme system, which had the same enzyme amount and ratio as the mixed free enzyme system, were

5.5 (Figure 3.7) and 65°C (Figure 3.8), respectively. The pH of the co-immobilized system shifted from 5 (mixed free GA and AA) to 5.5. Park et al. (2005) had a similar finding for covalently bound GA and AA on DEAE-cellulose and entrapped inside calcium alginate beads. The shift of pH could be caused by a secondary interaction, such as ionic and polar interactions or a hydrogen bond, between enzymes and support

(Arica et al., 1998). In this study the interaction could be an ionic interaction. The calcium alginate is negatively charged due to carboxylate groups (Tee and Kaletunc,

2009). The hydrogen ion concentration is higher in the alginate, which creates a microenvironment that shifts the displacement of bound or entrapped enzyme toward alkaline pH values. The optimum pH of many enzymes shifts to the alkaline region if

59 the carrier is anionic and to the acid region if the carrier is cationic because the pH will change the degree of ionization of amino acid residue of the active sites (Palmer and

Bonner, 2007).

The shifting of optimum pH to alkaline region of immobilized GA on poly(2- hydroxyethyl methacrylate) (pHEMA) hydrogels, diethylaminoethyl (DEAE) cellulose, and poly(ethylene glycol dimethacrylate 1-vinyl-1,2,4-triazole) beads was reported by other researchers (Arica et al., 1998; Park et al., 2005; Kok et al., 2011).

However, Tanriseven et al. (2002) and Milosavic et al. (2007) found that the free and immobilized GA had the same optimum pH. Tanriseven et al. (2002) mixed GA with gelatinized corn starch and alginate solution then added them into a mixture of CaCl2, glutaraldehyde, and maltodextrin to produce a calcium alginate fiber (2cm) and found that the optimum pH was 4.5 for both free and immobilized GA using maltodextrin as substrate. Milosavic et al. (2007) used copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate (poly(GMA-co-EGDMA)), applying glutaraldehyde or periodate as a crosslinker, to immobilize GA and found the optimum pH was 4.5 for both free and immobilized GA using soluble starch as substrate.

105

100

Relative rate [%]

65 3.5 4.0 4.5 5.0 5.5 6.0 6.5

Figure 3. 7 Optimum pH of free-mixed (○ normalized by activity at pH 5.0) and co- immobilized (●normalized by activity at pH 5.5) enzyme in capsule systems.

The optimum reaction temperature of the co-immobilized GA and AA capsule system increased from 55°C to 65°C . The increased optimum temperature by immobilization was reported in the literature (Arica et al., 1998; Park et al., 2005;

Milosavic et al., 2007; and Kok et al., 2011). The increase in optimum temperature could be caused by the physical or chemical change of GA. The immobilization could reduce the conformation flexibility of enzyme, leading to an increase in thermal stability (Arica et al., 2000; Bai et al., 2009). This result may indicate that the immobilization of enzyme can improve the thermal stability of enzyme by reducing

61 the mobility of the enzyme and/or creating a micro-environment in the sodium alginate gel capsules (Unsworth et al., 2007). It is expected that the beads would show greater improvement in optimum reaction temperature because the beads can provide more physical inclusion for entrapped GA. However, this was not observed, as shown in

Table 3.2. The relative activities (normalized by activity at 65°C ) of co-immobilized beads at 55°C , 60°C , and 65°C are 62.3%, 98.1%, and 100%, respectively.

120

100

Relative rate [%] Relative 40

0 40 50 60 70 80 90

o Temperature [ C ]

Figure 3. 8 Optimum temperature of free-mixed (○normalized by activity at 55°C ) and co-immobilized (● normalized by activity at 65°C ) enzyme in calcium alginate capsules.

3.3.3 Effect of immobilization on kinetic parameters

Kinetic parameters for co-immobilized GA and AA were determined in the stirred batch reactor using soluble potato starch as the substrate at 65°C and pH 5.5.

The Michaelis-Menten model was fitted to the collected data to determine the Km and

TM Vmax of the co- immobilized enzyme using KaleidaGraph software (Figure 3.9). The kinetic parameters of immobilized enzyme were determined using the data from first usage (due to thermal inactivation and possible enzyme leakage during reuse). Both

Vmax and Km were affected by the immobilization process. The immobilized enzyme exhibited a lower Vmax and a higher Km in comparison with the free enzyme (Table

3.3). The decreased Vmax and increased Km also were reported in literature for immobilized AA (Arica et al., 1995; Tumturk et al., 2000) and GA (Shkutina et al.,

2005; Kok et al., 2011). The Vmax of co-immobilized GA and AA was approximately six times lower than mixed free GA and AA. Park et al. (2005) also found the Vmax of the co-immobilized system was 5.2-fold lower than the mixed free GA and AA.

Compared to the mixed free enzymes, co-immobilized enzymes have a barrier against oligosaccharide diffusion in and the product diffusion out of the capsules, factors that could decrease the maximum reaction rate.

Km values imply the affinity of enzyme to substrate. The higher Km suggests a lower affinity for substrate by the co-immobilized enzyme. The change of affinity could be caused by structural change of the enzyme during immobilization, steric hindrance induced by support, or diffusion effects (Arica et al., 1998; Kok et al.,

2011). The active sites of the enzymes (in this case AA and GA) have to face the

63 substrate for reaction. When the enzymes were immobilized on a support surface, an orientation for access of the substrate was produced; the surface of support promoted steric hindrance, especially for the macromolecular substrates such as starch

(Bickerstaff, 1997). Diffusional limitations and steric effects of the co-immobilized system may contribute to the increased Km value for the substrate to access the enzyme active sites.

5 Glucoserate mediumin reaction [mg/min]

0 0 5 10 15 20 25

Starch concentration [mg/ml]

Figure 3. 9 Michaelis-Menten kinetics of free-mixed (□) and co-immobilized (●) enzyme calcium alginate capsules. At pH 5.5 and 65°C in 50ml reaction medium.

Table 3. 3 Kinetic parameters of free and co-immobilized enzymes

Km Vmax [mg/ml] [mg glucose/min] Free 1.25 ± 0.15 20.94 ± 0.51 Co-immobilized 1.98 ± 0.14 3.00 ± 0.41 enzyme

3.3.4 Reusability of co-immobilized system

Figure 3.10 shows the relative activities of the co-immobilized system for six consecutive use. The co-immobilized GA (entrapment) and AA (adsorption) lost 80% of their initial activity after four uses. Leakage of AA in the reaction medium was found. The losses of AA for each usage are 0.18, 0.13, 0.08, 0.03, and 0.005 mg respectively. GA leakage was not detected as verified by the glucose production activity test in the reaction medium using 10 mg/ml maltose as substrate at pH 5.5 and at 65°C .

120 120

100 100

80 80

60 60 Relative rate [%] Relativerate

40 40 AA remaining AA on the capsules[%]

20 20

0 0 0 1 2 3 4 5 6 7

Number of use

Figure 3. 10 Relative rates (∆) in terms of glucose production and AA remaining percentage on the capsules (x) for the starch hydrolysis with co-immobilized GA and AA system at pH 5.5, 65°C.

The AA leakage was taken into consideration to calculate, the GA/AA mass ratio during each use. Figure 3.11 shows that the relative change in activity with the

GA/AA ratios. Park et al. (2005) reported that the best GA/AA ratio is 3:1 within the range of 5:1 to 1:1 for free mixed enzymes. Yang et al.(2010) also reported that the best GA/AA ratio is 1:4-1:0.67 for the co-immobilized system based on glucose production. The ratio of GA/AA of 0.93:1 for the 1st use was between the optimum region of 1.5:1 and 0.67:1for the co-immobilized system reported by Yang et al.(2010). When the AA amount decreased due to leakage, the production of oligosaccharide reduced which was used by the GA as substrate for glucose production. Shortage of substrate may have been the reason for the observed decrease in the glucose production rate for the co-immobilized system during multiple reuse.

100 1st 90 80 70 2nd 60 50 3rd 40 30 4th Relative rate rate [%] Relative 20 5th 10 6th 0 0.93 1.60 4.21 20.00 40.00 80.00 GA/AA ratio

Figure 3. 11 Effect of GA/AA ratios on glucose production rate, data was normalized by GA/AA of 0.93/1 ratio. Numbers on the top of bar indicate different usages.

Compared to the co-immobilized enzyme in capsules, the reusability of mixed individual immobilized enzymes in capsules was not significantly different for the first usage (p = 0.1678 > 0.05). However, there were significant differences in the third and the fifth usage (p = 0.0256 < 0.05) and (p = 0.029 < 0.05) respectively. Both co- immobilized and individually immobilized mixed enzyme systems lost 80% of initial activity after four usages. The co-immobilized system had the advantage of using only half the amount of the support materials (calcium alginate) used for the mixed individually immobilized mixed enzyme system.

10 [µg glucose/(min*ml*mg protein)] glucose/(min*ml*mg [µg

Glucose production rateGlucose 0

0 1 2 3 4 5 6 Number of reuse

Figure 3. 12 Reusability comparisons of co-immobilized (●) and mixed-immobilized (○) calcium alginate gel capsules systems for GA and AA immobilization. pH 5.5, 65°C , 5mg/ml starch. The reusability of mixed individual immobilized enzymes in beads was not significantly different from co-immobilized enzymes in beads (Figure 3.13, all p- values > 0.05 for two-sample t-test). 67

10 Glucoseproduction rate

[µg glucose/(min*ml*mg protein)] [µg 5

0 0 1 2 3 4 5 6 7 8 Number of use

Figure 3. 13 Co-immobilized (∆) enzyme (GA and AA) and mixed individual (ᵡ) immobilized enzyme in calcium alginate beads. pH 5.5, 65°C, 5mg/ml starch.

The comparison of co-immobilized beads and capsules for starch hydrolysis is shown in Figure 3.14. For the first three usages of co-immobilized capsules and beads there were significant differences in glucose production rate per mg of enzyme.

However, for 4th through 6th usages, the glucose production rate appeared to be similar for bead and capsule systems.

Glucose production rateGlucose protein)] glucose/(min*ml*mg [µg

0 1 2 3 4 5 6 7

Number of use Figure 3. 14 Comparision of co-immobilized capsules (●) and beads (○) for GA and AA immobilization. pH 5.5, 65°C, 5mg/ml starch. It was also found that 82% of AA was lost after 3rd usage of co-immobilized beads. The leakage of AA after three usage explains why co-immobilized capsules and beads converged to a similar point, because there was not enough oligosaccharides supply for GA. For the first usage of co-immobilized capsules, the starch and glucose concentration versus time are described in Figure 3.15. The starch degradation rate was 1062 µg/(ml*min), and within 5 minutes all of starch was degraded to oligosaccharides. With the loss of AA, the starch degradation rate also decreased.

5 250

4 200

3 150

100 2

Starchconcentration [mg/ml] Glucose concentration [µg /ml] Glucose concentration[µg 50 1

0 0 0 200 400 600 800 1000 Time [s]

Figure 3. 15 Co-immobilized GA and AA in capsules for starch degradation (ᵡ) and glucose production(∆) of 1st usage. pH 5.5, 65°C , 5mg/ml starch, 0.22mg AA and 0.2 mg GA. Both the entrapped GA in capsules and co-immobilized GA and AA showed better glucose production rate than the beads system. This study focused more on the capsules system.

3.3.5 Comparison of thermal inactivation of free-mixed and co-

immobilized enzyme system

The activity of the co-immobilized system continued to decrease from the fourth usage to the sixth usage marginally (Figure 3.10). The thermal stability of free mixed enzymes was also investigated under the same conditions as the co-immobilized

70 system. The mixed free enzyme can only be used once while the co-immobilized enzyme can be used up to six times. Leakage was found during the reuse of the co- immobilized system. There are two factors that could contribute to the decrease of enzyme activity, thermal inactivation and GA/AA ratio change. The thermal inactivation of free and co-immobilized enzyme systems is shown in Figure 3.16. The

AA leakages were determined during the reuse of capsules. If we assume there was no leakage, by adding back the amount of leaked AA, the co-immobilized system was more stable than the free enzyme system. This indicates that the immobilized system is more thermally stable in terms of glucose production. If AA leakage did not occur, GA reaction appears to be the limiting step in overall starch hydrolysis processing.

120

100

60 Relative rate [%] Relativerate

0 0 10 20 30 40 50 60 70 Time [minute]

Figure 3. 16 Thermal stability of different systems up to 5 usages, free mix enzymes (◊), co-immobilized enzymes (ᵡ), and modified co-immobilized (∆) system by considering the AA lose. 71

AA was adsorbed onto the surface of gel capsules. The adsorption method involves reversible interaction between the enzyme and the surface of the support materials. The involved electrostatic forces are hydrogen bonding, hydrophobic bonding, ionic bonding and van der Waals force (Bickerstaff, 1997). This weak interaction may cause activity reduction of the immobilized enzyme system due to desorption of enzymes as a result of changes in pH, temperature, ionic interaction, or solvent polarity in the environment (Beatriz and Francisco, 2006). The loss of AA during reuse could be avoided by using a covalent binding method. Tee and Kaletunc

(2009) reported a covalent method for AA that can be used at high temperature (95

ºC ).

3.4 Conclusion A co-immobilized enzyme system using calcium alginate gel capsules was developed. A hollow aqueous environment created inside the calcium alginate gel capsules improved the availability of GA for the substrates similar to the free enzyme condition. Compared to solid beads, the capsules showed twice the specific activity in terms of glucose production. Gel capsules containing co-immobilized enzymes can be utilized at 65 ºC and pH 5.5 at least six times. Leakage of adsorbed AA was observed.

Thermal stability of the enzyme system was improved by immobilization when the results were corrected for AA leakage. The calcium alginate gel capsule demonstrated a model system for the co-immobilization of two enzymes. This system can be extended to multi-enzyme immobilization, and also is not limited to the combination of entrapment and adsorption. 72

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Chapter 4: Expression, purification and characterization a His6-tagged thermostable glucoamylase from Sulfolobus solfataricus P2

4.1 Introduction

In previous chapter, a co-immobilization system of AA and GA with an

AA/GA ratio of 0.43:0.4 has been developed and tested in a batch stirred reactor at

65 °C . To reduce the starch hydrolysis into one-step process, a co-immobilized thermostable GA and AA system which has an optimum reaction temperature above

85 °C need to be utilized.

Developing a thermostable GA has been attractive to many researchers due to its potential to improve the efficiency of starch hydrolysis process. Use of thermostable enzymes provides many advantages to starch hydrolysis process. First, the elevated temperature can lower the starch or reaction medium viscosity (Liu and

Wang, 2003). Second, the higher reaction temperature can lead to faster reaction kinetics (Liu and Wang, 2003; Wang et al., 2006). Third, hydrolysis at a high temperature can reduce the risk of microbial contamination (Liu and Wang, 2003;

Synowiecki et al., 2006; Fernandes, 2010). Furthermore, the thermostable enzyme also has the potential to decrease the ion dependence and have wider pH reaction range

(Synowiecki et al., 2006; Tee and Kaletunc, 2009). Production of a low cost and high yield thermostable GA can improve the current starch hydrolysis process by increasing 79 the saccharification temperature. The disadvantage of using thermostable enzyme is the additional cost of maintaining high temperature.

However, there is no commercially available GA which has an optimum temperature above 90 °C . Enzyme manufacturers such as AB Enzymes GmbH, Amano pharmaceutical Co., Biocatalysts Ltd, Christian Hansen, Danisco, Novozymes do not have the thermostable GA available for starch hydrolysis. The thermostable commercially available GA, Spirizyme® Fule, from Novozyme is for bioethanol processing with an optimum temperature of 65-70 °C . The optimum temperature range for G-ZYME® 480 Ethanol, from Danisco (Genencor enzyme), is 58-65°C. Also

OPTIDEX® L-400 from Danisco has the optimum temperature at 60-65°C for saccharification. The GA used for the starch hydrolysis in the food industry is reported to have an optimum temperature of 55-60 °C . Several studies have been dedicated towards improving optimum reaction temperature of fungus GAs by protein engineering (Nielsen et al, 2002; Liu and Wang, 2003) and directed evolution (Chen et al., 2007; Pavezzi, et al., 2011). However, the reported temperature improvement is small (5°C improvement) and most of the reported optimum reaction temperatures a still below 80 °C .

On the other hand, the GA purified from hyperthermophilic archaea can have an optimum temperature higher than 90 °C (Serour and Antranikian, 2002; Kim et al.,

2004; Egorova and Antranikian, 2005). Among thermostable GAs, GA from

Sulfolobus solfataricus P2 has an optimum pH range of 5-6 (Table 1.1), which is close to the commercially available thermostable AA. This makes it possible to use with

80 thermostable AA at the same time in order to develop a one step starch hydrolysis process at high temperature.

Sulfolobus solfataricus (S. solfataricus) is a hyperthermophilic archaea which can grow at 85 °C (Egorova and Antranikian, 2005). Thermophiles or hyperthermophiles often contain thermostable enzymes (Hyun and Zeikus, 1985,

Antranikian et al., 2005). The complete genome sequence of S. solfataricus P2 was studied by She et al. (2001). They found gene clusters (SSo0987–0991) encoding enzymes responsible for intracellular synthesis and degradation of glycogen. Kim et al.

(2004) inserted the chromosomal DNA from S. solfataricus P2 into an E. coli expression vector pGNX4 and determined the activity of the resultant thermostable glucoamylase using gene SSo0990.

However, Kim et al. (2004) did not provide information about the effects of metals, chemical reagents on activity of thermostable GA, or reaction kinetics parameters including the inactivation energy of the GA from S. solfataricus P2.

Furthermore, the purification method was complicated because it included several steps (hydrophobic column, size column) with relatively higher cost of purification.

With the development of recombinant enzyme technology and purification methods, it would be possible to add a tag to N or C terminal of the enzyme for the purpose of easy purification. This chapter provides information to produce a thermostable recombinant GA from the S. solfataricus P2 by adding His6-tag. It also presents the effects of pH, temperature, cations, chemical agents and substrates on the enzyme activity. The reaction kinetics and the thermal stability were also determined.

Producing and characterizing a thermostable GA will lead to the development of a co- immobilized AA and GA enzyme system in order to improve the efficiency of starch hydrolysis process.

4.2 Materials and Methods 4.2.1 Materials

In order to construct the gene inserted plasmid, the genomic DNA for S. solfataricus P2 was purchased from the ATCC (Product number: 35092D-5). The expression vector pQE80L was purchased from QIAGEN (Valencia

CA, USA). Oligonucleotides were obtained from Eurofins MWG Operon (Huntsville,

AL, USA). Two restriction enzymes, BamHI (R0136S) and PstI (R0140S), were purchased from New England Biolabs (Ipswich, MA, USA). Pfu DNA polymerase was purchased from Finnzymes (Thermo Fisher Scientific, Lafayette, CO, USA).

For transformation, the host, E. coli DH5α™ competent cells (order #18265-

017), was bought from Invitrogen (Carlsbad, CA, USA). For expression, purification, and characterization, Luria-Bertani (LB) medium (BP1427), EDTA, glacial acetic acid

( A38-500), sodium hydroxide, calcium chloride (BP510), hydrochloric acid solution

(SA48-4), magnesium chloride (M35-500), copper (II) sulfate, iron (III) chloride were purchased from Fisher-Scientific (Pittsburg, PA, USA); Carbenicillin (C2130) was purchased from TEKNOVA (Hollister, CA, USA). Isopropyl β-D-1- thiogalactopyranoside (IPTG, 17886-1gm) was purchased from USB (Cleveland, OH,

USA). Imidazole ( I202 ), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDAC,

E6383), sodium acetate (S8625), sodium chloride, 2-Mercaptoethanol (M3148), oligosaccharides kit (47265 Supelco), Bradford reagent (B6916), glucose assay reagent (G3293), maltose (M5885), bovine serum albumin (P0834) were obtained from Sigma (St. Louis, MO, USA). HisTrap HP (17-5247-01), packed with Ni

Sepharose, was purchased from GE Healthcare (Piscataway, NJ, USA). Modified

Lowry protein assay (PI-23240) and Sulfo-NHS (N-hydroxysulfosuccinimide) (24510) were purchased from Pierce (Thermo Fisher Scientific, Rockford, IL, USA). SDS-

PAGE (12% TGX) gel (456-1043), Laemmli sample buffer (161-0737), electrophoresis running buffer (161-0732) were purchased from BIO-RAD (Hercules,

CA, USA).

4.2.2 Cloning, nucleotide sequence analysis and construction of expression

vectors

The gene cloning was carried in Professor John Reever’s lab in the

Microbiology Department at the Ohio State University. The genomic DNA for S. solfataricus P2 was purchased from the ATCC (Product number: 35092D-5).

Oligonucleotide primers were designed based on the DNA sequence of annotated hypothetical glucoamylase gene (SSo0990). Two oligonucleotide primers, forward (5′-

CCGGTTGGATCCATGAGAGTTTCCTCCATAGGAAATG-3′) and reverse (5′-

GGAAGGCTGCAGTTATATATGGTTTAAGAGCTTA-3′) were used to introduce

BamHI and PstI restriction recognition sites (underlined). The DNA fragment of the hypothetical glucoamylase was amplified (1900 bp) from the genomic DNA by PCR

(mastercycler® gradient, Eppendorf) with Pfu DNA polymerase (Phusion®, 83

Finnzymes) at an annealing temperature of 72°C . After being digested with BamHI and PstI, the PCR fragment was inserted into E. coli expression vector pQE80L containing a N-terminal His6-tag. The PCR products (1900 bp) were cloned into pQE80L and in frame with sequences encoding His6 residues as a BamHI/PstI fragment to generate a plasmid. The vector, pQE80L, also contained antibiotics resistant sequence which would allow the growth of cells under added antibiotics. The resultant recombinant plasmid, pGKGA, was transformed into E. coli DH5α for cloning (as shown in Figure 4.1). DNA sequencing of the PCR gene was performed in

Plant-Microbe Genomics Facility at the Ohio State University.

Figure 4. 1 Construction of expression vector pGKGA. 84

4.2.3 Growth conditions and Expression of recombinant GA

Effects of LB medium concentration, agitation speed, liquid volume to air ratio, osmotic pressure (sorbitol) and growth temperature on the growth of recombinant cells were studied in order to achieve high yield of target soluble protein.

Recombinant E. coli cells were grown in LB medium (2% or 4% w/w) with carbenicillin (150 µg/ml). pQE80L was plasmid DNA containing a resistance gene for the antibiotic used for colony selection (i.e., an ampicillin- resistance gene). The cells were grown in a flask placed inside a shaking bath with 200 rpm rotation speed, or in a fermenter (BIOSTAT Aplus 2L, Sartorius, Goettingen, Germany). For growing in the flask, the effect of liquid to air volume ratios at 1:5, 2:5 or 1:10 were investigated. For the fermenter the dispersed air flow rate was 1.5L/min, and the motor rotation speed was 200 rpm. Sorbitol at 1% (w/v) final concentration was added in the culture medium to test its effect on the yield of thermostable protein.

After approximately 3hr of growing at 37°C , when OD at 600nm

(Spectrophotometer, Carry 5000, Varian, Inc.) reached 0.6-0.8, IPTG was added to reach a final concentration of 0.1, 0.2, 0.3, 0.5, or 1 mM for the induction of target protein. The medium temperature was kept at 37 or 46°C for 13 hours to reach the stationary growth phase (~16hr for DH5α). After 13 hours, expression cells were harvested by centrifugation at 10,000 x g for 10 minutes. The cell pellets were then suspended in 15-20ml lysis buffer (20 mM sodium phosphate, pH7.4, 500 mM NaCl, and 30 mM imidazole). Cells were broken by using sonication (Branson 450, Danbury,

CT) for four times for 60 seconds each time. The output of the sonicator was set at

85 level 6 (the maximum output of sonicator is 400 Watts) and the duty cycle of 90%

(pulse duration). Cell debris was centrifuged at 5,000 x g for 10 minutes and the supernatant collected was heated at 60 °C for 30 minutes to denature proteins which are not thermostable. The supernatant was collected after centrifugation at10,000 x g for 20minutes at 4 °C for further purification. The steps of obtaining crude protein described above are shown in Figure 4.2.

Plasmid pQE80L Glucoamylase gene with 6xHis-tag (SSO0990) from S ulfolobus solfataricus P2

Plasmid with gene pGKGA

Transfection into E.coli DH5

Growing E.coli Add IPTG DH5

Sonication

Centrifugation Crude soluble protein

Crude insoluble protein

Figure 4. 2 Production of thermostable GA by recombinant DNA technology.

4.2.4 Purification of recombinant GA

The crude enzyme was passed through a 0.22µm filter (MCE membrane,

Fisher Scientific 09719A) to remove small insoluble debris (avoid clogging of column), and then further purified by using two 1ml His-Trap Ni Sepharose™ columns in series (GE health). Firstly, the His-Trap column was pre-saturated with the lysis buffer (20 mM sodium phosphate, pH7.4, 500 mM NaCl, and 30 mM imidazole).

Then, the 40ml of crude protein solution was loaded to the columns with a flow rate of

0.6 ml/min. The proteins which have affinity to Ni2+ were retained in the column. The column was washed by approximately 120-150ml (at least 60 times of column volume) lysis buffer until the absorbance at 280 nm wavelength is close to that of lysis buffer (0.05-0.07) and stable. Finally, the thermostable GA with His6-tag was eluted by series of 10-15 column volume imidazole containing elution buffer (60mM,

200mM and 500mM imidazole in 20mM pH 7.4 sodium phosphate buffer with

500mM NaCl). The various protein fractions in each elution buffer (20 mM sodium phosphate, pH7.4, 500 mM NaCl, and 60, 200 or 500 mM imidazole) were concentrated and desalted by Amicon® ultra-15 15ml centrifugal filter with 50 kDa cutoff (Millipore, Billerica, MA, USA). After the first concentration, 6 times of 15ml

50 mM acetate buffers (pH5.5) were used as exchange buffer for desalting. The schematics of purification process is shown in Figure 4.3.

Crude protein

Heating

High temperature Centrifugation soluble protein

Low temperature soluble protein Filter 0.22µm

Immobilized Ni2+ ion affinity chromatography

Purified protein

Concentration Concentrated and desalting by protein centrifugal filter

Figure 4. 3 Purification of recombinant thermostable glucoamylase.

The photograph for the set up of the purification system is shown in Figure 4.4.

Figure 4. 4 Purification setup.

4.2.5 Gel electrophoresis and protein content

SDS-PAGE analysis was performed using 12% SDS-polyacrylamide ready to use gel (Bio-Rad, 16160) to assess the purity and molecular weight of the enzyme. 15 -

20µl of protein sample was mixed with Laemmli sample buffer (at least 1:1 ratio), then was boiled and centrifuged at 12,500 x g for 5 min. The gel was run in a Bio-Rad Mini

Protein electrophoresis device (Bio-Rad, Richmond, CA, USA) with 200 volts for 35 minutes. The protein bands were visualized by Coomassie brilliant blue. Protein concentration was determined by Bradford assay (Sigma, B6916, USA) with BSA as a

89 standard. Also modified Lowery reagent was used for protein determination with BSA as standard to verify the Bradford test results. Imidazole (>200mM) is incompatible substance for protein test by Bradford and modified Lowry methods. Use both methods can confirm the protein concentration in the elution.

4.2.6 Enzyme activity tests

The activity of GA was determined based on the initial glucose production rate by using various carbohydrates as substrates. 20 µl of GA (0.8mg/ml) was added into a preheated 180 µl of 50mM acetate buffer at pH5. 200 µl of this mixture was then added into 400 µl of preheated (to reaction temperature) 15mg/ml maltose, maltotriose or oligosaccharides (DP 4-7) solutions at pH 5 to reach a final concentration of 10 mg/ml at reaction medium. Samples were taken at 0, 0.5 and 1.5 minutes and the reaction was stopped by placing the sample in an ice-water bath. Then the samples were centrifuged at 12,500 x g for 5 minutes, and supernatant was used for activity test. The concentration of glucose was measured with a spectrophotometer at

340 nm by using glucose hexokinase (HK) kits (Sigma-Aldhich G3293). One U of enzyme is defined as the amount of enzyme that produced 1 µmol glucose per minute under assay conditions.

4.2.6.1 Influence of pH and temperature on activity of enzyme

Influence of pH was studied over a range from 4 to 7. 50mM sodium acetate buffer for pH range of 4 to 5.5 was used. 50mM sodium phosphate buffer was used for pH range of 6 to 7. Influence of temperature was studied from 75 to 110 °C with 5°C

90 intervals. For the temperatures higher than 95°C , a pressurized system was built and used (Figure 4.5). The lab air with approximately 20 psig was regulated to 10 psig and connected to a closed stirred batch tank reactor (with three openings on top). A thermocouple was also inserted into the reactor through one opening. The last opening is for adding enzyme into reactor.

V-1 V-2 Lab air Pressure Add enzyme Temp. supply regulator with long needle ~20PSI to 10 PSI syringe

V-3

Temperature

Stirred batch reactor

Hot oil bath

Feedback control hot plate

Figure 4. 5 A pressurized stirring tank reactor for enzyme activity test. 91

4.2.6.2 Effect of substrate on enzyme activity

The glucose production rates in various substrates solutions including maltose

(DP 2), maltotriose (DP 3), isomaltotriose (DP 3), maltotetraose (DP 4), maltopentaose

(DP 5), maltohexaose (DP 6), maltoheptaose (DP 7), D-(+) raffinose (DP 3), D-(+) melezitose (DP 3), stachyose (DP 4), Maltrin® M100 (DP 9-12) and soluble starch at a concentration of 10 mg/ml were determined at pH 5 and at 95°C . For enzyme reactions

200µl of GA (0.08 mg/ml) was placed in 400µl of 15mg/ml substrate in buffer solution at pH 5. The concentrations of enzyme and substrate in the reaction medium were 26.7 µg/ml and 10 mg/ml respectively.

4.2.6.3 Effects of ion on enzyme activity

The effects of various metal ions on GA activity were determined by adding

Ca2+, Mg2+, Cu2+, Na+ and Fe3+ to the reaction medium to obtain a final concentrations of 1, 5 or 10 mM in the reaction medium at 95°C and pH 5 using 10 mg/ml maltotriose as substrate.

4.2.6.4 Effects of chemical reagents on GA activity

The effects of different chemical reagents on GA activity were evaluated by adding EDTA, EDAC, Sulfo-NHS, 2-Mercaptoethanol, and SDS to the reaction medium to reach a final concentration of 10, 50, or 200mM in the reaction medium at

95°C and pH 5 using 10 mg/ml maltotriose as substrate. The mixtures were incubated at room temperature for 2 hours. Effect of a mixture of 200mM EDAC and 50mM

Sulfo-NHS with an incubation time of 15 minutes on enzyme activity was also tested.

4.2.7 Thermal stability of GA

Inactivation kinetics of thermostable GA was investigated. The GA was incubated at different temperatures between 75 and 110 °C . The activity at 0, 5, 8, 30,

60 minutes at each temperature were determined. For 75 and 80 °C , GA activities at

120 minutes were also checked.

At a constant temperature, the inactivation rate was determined from the slope of the following equation obtained by integrating Eq. 1.3,

Eq. 4.1

where E0 is the initial enzyme activity at time zero, and E(t) is the remained activity after heating for time t. kd is the denaturation rate constant. The Unit for GA activity was defined as the amount of enzyme that produces 1µmol of glucose per minute under assay conditions.

The temperature dependence of rate constant can be described by the Arrhenius equation,

Eq. 4.2

where Ea is the inactivation energy of enzyme, A is the Arrhenius constant, R is the gas

-1 -1 constant (8.314 J mol K ) and T is the temperature in K. The Ea and ln(A) was calculated by plotting the ln (kd) vs. 1/T.

The t1/2 , the half time of enzyme inactivation, defined as the time that 50% of initial activity of enzyme is retained at a constant temperature was determined.

4.2.8 Michaelis-Menten kinetics parameters

The Michaelis-Menten kinetics parameters, Km and Vmax ,were calculated by using different maltose (DP2) or maltotriose (DP3) concentrations with fixed enzyme concentration of 26.7µg/ml at pH 5 and at 95°C . The concentration range was studied varied from 0 to 100 mg/ml (0, 3.125, 6.25, 12.5, 25, 50, 75 and 100mg/ml).

4.3 Results and Discussion

4.3.1 The effects of E. coli growth factors on thermostable enzyme

production

The purpose of cloning gene from hyperthermphilic archaea to mesophilic bacterial cell was to grow and express more protein to acquire high protein yield. It is important to investigate the effects of different growth factors on the yield of target protein. The protein yield is related to the total mass of cells and the expression level in a single cell. The LB medium at the recommended level of 2% w/w, was inadequate for the growth of recombinant cells. To reach an OD (optical density) of 0.6-0.8 at 600 nm in 3 hr, it was necessary to increase the LB concentration to 4% w/w (Figure 4.6).

The recombinant E. coli overnight culture was inoculated in LB medium with a ratio of 1:100. The volumetric ratio between LB medium and air in flask (unbaffled) also affected the growth of recombinant E. coli when a shaking bath with 200 rpm rotation speed was used. The liquid to air volume ratio of 2:5, 1:5 and 1:10 was studied. It was found that the 1:10 had the highest growth speed. Compared to the 1:10 ratio, the

94 cloned cells grew even faster in the fermenter with 1.5L/min air supply rate and 200 rpm rotation speed (Figure 4.6).

4.5 4 3.5 3 2.5 2 1.5 1

Cell OD@600nm density Cell 0.5 0 0 3 6 9 12 15 18 21 24 27 30 Incubation time at 37°C [hr]

Figure 4. 6 Growth at different conditions: (▲) growth in fermenter with 4% LB, 1.5L /min flow rate and 200rpm agitation; (○) growth in flask with liquid volume to air ratio of 1/10, 4% LB; (♦) grow in flask with liquid volume to air ratio of 1/5, 4% LB; (+) grow in flask with liquid volume to air ratio of 2/5, 4% LB; (■) grow in flask with liquid volume to air ratio of 1/5, 2% LB.

The effect of liquid medium to air volume ratio in a fermenter can be explained by the oxygen transfer rate. Mcdaniel et al. (1965) showed that oxygen-supply rates can affect the growth of E. coli in the baffled or unbaffled flask. The major deficiency of using ordinary unbaffled Erlenmeyer flasks was that the oxygen transfer rates are low and could not meet the culture oxygen requirement when using typical growing liquid volume. The oxygen transfer rate may be particularly important when growing recombinant E. coli cells because the presence of plasmid in cells is considered as burden for occupying cellular resources to express plasmid genes (Geckil et al., 2001).

This study found that after adding IPTG, the growth rate of cells slightly decreased in log phase (Figure 4.7) because the intracellular protein expression may slightly affect the multiplication of E. coli. This result is similar to the finding of Geckil et al, (2001), who compared the growth rate of DH5α cell with other two recombinant DH5α cells.

Khosravi et al. (1990) reported that when the size of the plasmid increased, the growth of recombinant E. coli inversely decreased. They pointed out that the burden was shown as growth hindrance in whole cell level. In order to overcome the disruption by the extra energy demand of the plasmid expression, the cells could attempt to increase the oxidative phosphorylation rate which leads to the rapid consumption of oxygen

(Khosravi et al., 1990).

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4

0.2 Cell OD@600nmdensity Cell 0 0 1 2 3 4 5 6 7 Incuation time at 37°C [hr]

Figure 4. 7 Growth comparison with (○) and without (●) adding IPTG. Growth in flask with liquid to container ratio of 2/5, 4% LB. When simulating growth from a shaking flask to a stirred tank fermenter, the oxygen transfer rate can be compared by using empirical equations in literature.

(Sumino et al.,1992; Maier et al., 2004; Seletzky et al., 2007).

The oxygen transfer rate (OTR) is proportional to the mass transfer coefficient

KL, the specific transfer area AT, the oxygen solubility and the driving pressure or concentration difference across the gas–liquid interface as described by the following equation (Seletzky et al., 2007),

Equation 4.3

where the is oxygen concentration at gas–liquid interface, and with an estimated value of 1.09 *10−1 mol/m3 (Maier et al., 2004; Schumpe et al., 1982).

Also for the shaking flask KL*AT can be estimated by the following equation (Marie et al., 2003),

Equation 4.4 where the N is the rotational speed in rpm, Vl is the liquid volume in ml, d0/2 is the distance from shaking center to flask center in cm, and d is the maximum inner shake diameter of flask. For a 500ml flaks (d=20.5cm) which was filled with 100 or 50 ml

LB medium and was placed inside a shaking bath (d0=25cm), KL AT values were 0.076 s-1 or 0.136 s-1. The final OTR values were calculated by Equation 4.4 to be 0.030 mol/(l*hr) and 0.053 mol/(l*hr).

For the fermenter, Sumino et al.(1992) developed the following empirical equation,

Equation 4.5

where is the oxygen transfer rate (OTR) in mol/(l*hr) and Hl is the depth of liquid medium (m). Other parameters are described in Table 4. 1 for calculating the fermenter OTR used in this study. 97

Table 4. 1 Variables for fermenter OTR calculation

Variables and dimensions 2L Fermenter

Vessel diameter Dt m 0.13 Impeller diameter Di/Dt 0.41 Number of impellers N i 2 3 Liquid volume Vl (m ) 0.0015 Rotational speed N (1/min) 200

Gas flow rate Q/Vl (vvm)* 1 Superficial gas velocity Vs (m/h) 6.78 Pressure P (bar) 1.5

Oxygen content of gas Ro2 0.21 *vvm stands for volume per volume per minute, gas volume flow per unit of liquid volume per minute.

When 1.5 L of LB medium was placed inside the 2 L fermenter, the estimated

OTR value was 0.073 mol/(l*hr). The calculated OTR values explain that when increasing the liquid to flask volume ratio from 1/5 to 1/10 the cell growth rates are closer to the fermenter condition. It was estimated when the liquid medium to flask

(unbaffled) is 1/15 the growth rate could match with the growth condition in fermenter. It means that it needs to use approximately 22 L volume of flask to achieve a equal yield of 1.5 L recombinant E. coli growth in a fermenter. By using 0.55m diameter fermenter, Vl as 1/3 of the fermenter, and superficial gas velocity of 35.7m/h,

Sumino et al.(1992) reached a OTR of 0.2 mol/(l*hr). Also they reported an OTR value as low as 0.09 mol/(l*hr) when the liquid volume was increase (Sumino et al.,

1993).

Determination and optimization of OTR could be critical for the growth of recombinant cells which are important to achieve a better yield of activity.

4.3.2 Effects of IPTG concentration

An IPTG final concentration of 0.5mM in LB medium was first used to check whether there are over expression of thermostable protein or not. It was observed that there was slight over expression around 75kDa.

250kDa 100kDa 75K 75kDa 75K 50kDa75K 37kDa 75K 75K 25kDa

20kD75Ka

15kD75K a 10kDa 75K

75K

1 2 3 4 5 6 7 8 9 10

Figure 4. 8 SDS-PAGE gel of 0.5mM IPTG expression. From left to right column are: Standard, cell pellets from 1ml culture control (lane 2) and IPTG (lane 3) with dilution factor 5 (20µl sample plus 100µl sampling buffer), cell pellets control (lane 4) and IPTG (lane 5) with dilution factor 10, supernatant control and IPTG (lane 6 and 7), cell debris control and IPTG (lane 8 and 9), industrial GA (lane 10, 5µl+1ml sampling buffer)

The effects of different IPTG concentration are shown in Figure 4.9. It is difficult to tell the difference between different IPTG expression levels. 0.2mM,

0.5mM and 1mM appear to have the similar results. To verify this finding, the yield and activity of final purified enzyme by 0.2 and 0.5mM IPTG induction were compared. Both of them have a glucose production rate of 2.7µg/ml/min by using 10 mg/ml maltose as substrate. The yields of activity (activity percentage between purified and crude enzymes in terms of glucose production rate described by enzyme 99

activity, U) were 5.3% and 5.7% respectively. There was no significant difference

between two yield (p-value=0.1429>0.05 by two-sample t-test). For the cost saving,

0.2mM was chosen as the optimum concentration.

250kDa

100kD75Ka 75kDa K 75K 50kDa75K 3775KkDa

75K 25kDa

20kD75Ka

75K 15kDa 10kDa 75K 75K 1 2 3 4 5 6 7 8 9 10

Figure 4. 9 Effects of different IPTG concentration for protein expression of cell pellets. Marker (lane 1) Control (lane 2) 0.1mM (lane 3) 0.1mM (lane 4) 0.2mM (lane 5) 0.2mM (lane 6) 0.3mM (lane 7) 0.3mM (lane 8) 0.5mM (lane 9) 1mM (lane 10)

4.3.3 Effects of osmotic pressure (sorbitol) and increased growth

temperature

The major goal of protein expression in recombinant cell is often to achieve a

high amount of soluble product. But the metabolic system of the host bacterial cell,

such as E. coli, is not always favorable of producing target protein in soluble form

(Sorensen and Mortensen, 2005). Expression in E. coli sometimes can produce

100 inclusion body and decrease the yield of soluble protein. Oganesyan et al. (2007) reported that in the presence of osmolytes such as sorbitol, or exposing cell to heat shock (20 minutes) can improve level of expression in soluble form. Also Feng et al.

(2002) found sorbitol strongly stabilizes GA from A. niger.

Table 4.2 Show the temperatures (25°C , 37°C , and 46°C ), which have applied in literature, and sorbitol effects on the protein production and yield of activity. The biomass is the wet mass of pellets collected after the end of growth.

Table 4. 2 Effects of sorbitol and temperature for protein expression

Protein Protein Bio Temp.** Temp.*** before after Mass Yield Case* Sorbitol /Time /Time heat(mg) heat(mg) (g) % 1 no 37°C ,3hr 37°C ,13hr 44.9±3.1 18.2±1.4 1.93 3.5 2 yes 37°C ,3hr 37°C ,13hr 63.3±2.8 12.3±0.9 1.65 1.6 3 no 37°C ,3hr 46°C ,13hr 52.9±1.5 16.9±1.1 1.65 5.1 4 yes 37°C ,3hr 25°C ,13hr 80.1±3.7 10.4±1 2.74 4.0

*All four cases: Pre-culture with OD=5.5, t=3hr, OD=0.78, add IPTG 0.2mM,. 100ml LB with liquid to air ratio of 1:10 in flasks. Before heat treatment means: before crude protein was heated at 60°C for 30 minutes; Biomass contains water, yield is in terms of enzyme activity (U). ** Before IPTG addition *** After IPTG addition

Based on the results reported in Table 4.2, it is apparent that adding sorbitol increased the total soluble protein. However, the thermostable soluble protein did not increase. The optimum growth conditions for thermostable protein expression were considered as case 3.

4.3.4 Results of purification and activity yield at optimum growth

conditions

After growing in optimum conditions in fermenter, the crude protein was purified by Ni column. A series of elutions with varying imidazole concentration was 101 studied. 60mM or 200mM imidazole concentrations appeared to have the best elution effects. To assure all of the target protein will be eluted out, an imidazole concentration of 500mM was added as the final elution step. So three imidazole concentrations (60mM, 200mM and 500mM) were used in series for protein elution.

SDS-PAGE gel results of samples from each purification steps are shown in Figure 4.

10.

250kDa

100kD75Ka

75kDa75K 75K 50kDa

3775KkDa

75K

25kDa

2075KkDa

75K

15kDa

1 2 3 4 5 6

Figure 4. 10 SDS-PAGE of purification process. Lane 1 is marker. Lane 2 is the sample from cell pellets of initial growth stage without IPTG induction. Lane 3 is the sample of final stage of growth after IPTG induction. Lane 4 is the sample from crude protein after sonication and centrifugation. Lane 5 is the sample after 60°C 30 minutes heating. Lane 6 is the sample from 60 mM imidozol elution.

The SDS-PAGE gel indicated that the purification process was successful by

2+ achieving a single band using the His6-tag affinity to Ni . In elution, single band was around 75kDa. The MW higher than 65kDa, was reported by Kim et al., (2004), using 102 the same gene. Also, several GAs reported in literature from fungi (Li et al., 1998;

Nguyen et al., 2002; da Silva et al., 2009) or bacteria (Specka et al.,1991; Ganghofner et al., 1998) have similar MW around 75kDa. The theoretical MW of GA from S. solfataricus P2 was calculated to be 73kDa considering 622 amino acids account for

72kDa (amino acid sequence is shown in Appendix G and the MW estimation is by an online calculator http://www.encorbio.com/protocols/Prot-MW.htm) and His6 accounts for 1kDa. The theoretical estimated MW is close to the SDS-PAGE gel results (75kDa).

Figure 4.11 shows another interesting phenomenon of purification process. In elution 1(lane 6), a second band around 25kDa was also apparent. Nielsen et al. (2002) also found a 70kDa with two minor bands, 15kDa and 55kDa. They have shown that the smaller band (15kDa) is the starch binding domain (SBD), and the 55kDa band is the catalytic domain (CD). This proteolytic cleavage in the linker region, which is between the SBD and CD, was also observed in A. niger GA by Svensson et al.

(1986). Serour and Antranikian (2002) also reported two MWs when purifying GAs from several thermoacidophilic archaea.

103

250kDa

100kD75Ka 75kDa K 75K 50kDa75K 75K 37kDa

75K 25kDa

75K 1 2 3 4 5 6 7 8

Figure 4. 11 Two bands after purification. Lane 1 is marker. Lane 2 is the sample from cell pellets of initial growth stage without IPTG induction. Lane 3 is the sample of final stage of growth after IPTG induction. Lane 4 is the sample from crude protein after sonication and centrifugation. Lane 5 is the sample after 60°C 30 minutes heating. Lane 6 and 7 is the sample from 60 and 200 mM imidozol elution. Lane 8 is the sample pass through the Ni2+ column after loading of crude protein.

In fungal GA, normallly there are two domains, the smaller size domain is

SBD (Coutinho and Reilly, 1994; Juge et al., 2002; Rodriguez-Sanoja et al., 2005).

More often, SBD have two binding sites. So the 25kDa minor band could be the SBD of the thermostable GA. The cell pellet in gel (lane 3) shows an overexpression around

50kDa. The 75kDa is the total MW of subunit, if assume the 25kDa is the SBD then the 50kDa band could be the CD. The total, 25kDa plus 50kDa, is the MW of the major band.

Yield and protein contents from 1.5L culture grown in fermenter is summarized in Table 4.3

104

Table 4. 3 Purification steps and yield of recombinant GA

Total Total Specific Purification Activity Protein activity fold Yield U mg U/mg % Crude extract 15.55 222.66 0.07 1.00 100.00 This * Heat treatment 12.91 137.55 0.09 1.34 83.03 study Elution after His TrapTM 2.88 0.80 3.60 51.55 18.52 Concentrated purified protein 1.17 0.10 11.65 166.83 7.49

Cell extract 936 1345 0.696 1.00 100 Kim et Heat treatment 787 279 2.82 4.05 84 al., ** Butyl-Sepharose 150 13.13 11.43 16.4 16 2004 Superdex 200 HR 10/30 43.94 0.78 56.3 80.9 4.6

Crude extract 27.77 142.7 0.19 1 100 Dock et Heat treatment 37.53 65.05 0.57 2.9 135 al., *** Q-Sepharose 5.76 3.36 1.68 8.6 20 2008

Superdex 200 1.91 0.45 4.21 22 7

Crude extract 1390 238 5.84 1 100 Zheng Heat treatment 822 15.2 54.1 9.26 59.1 et al., **** Superdex 200 385 2.20 175 30 27.7 2010 (*1.5L growth in fermenter and all activity tests were performed at 95°C using maltose as substrate, ** activity tests were performed at 80°C using maltose as substrate, *** activity tests were performed at 75°C using amylopectin as substrate, ****activity tests were performed at

75°C using maltose as substrate)

Compared to results of Kim et al. (2004), a lower specific activity was observed in this study. Lower specific activity could be due to the adding of His6-tag, and different assay conditions for activity test. The addition of His6-tag to C-terminal is known to decrease the enzyme activity (Ledent et al., 1997). In this study, the His6- tag was added to N-terminal, the effects of adding His6-tag on thermostable GA 105 activity was expected to be low. The relative molecular weight of His6-tag is 780. This relatively small molecule rarely affects the protein activity; in many cases, it is not required to remove the His6-tag (Zachariou and Bailon, 2008).

The error for protein test could be an issue, in Kim et al. (2004)’s method only

4µg of enzyme was used for reaction. Since the specific activity (U/mg) is the activity per mg of enzyme amount, a small error of enzyme amount could lead to a big difference in final results. In our case, 16 µg of enzyme were used for activity test.

Also the assay conditions were different. Kim et al. (2004) used 5mg/ml maltose at pH

6 and at 80 ºC for 10 minutes of reaction time. This study used 10mg/ml maltose at pH

5.5 and at 95 ºC for 1.5 minutes of reaction time. Moreover, the glucose determination methods were different. Kim et al. (2004) used the glucose oxidase method and this study used glucose HK method. All the above reasons could lead to a difference in the determination of specific activity.

On the other hand, in this study, the yield was improved from 4.6% (Kim et al.,

2004) to 7.49%. This could be promising for purifying larger amount of thermostable

GA for S. solfataricus P2.

106

Table 4. 4 Activity yields comparison in recent publications

Microorganism Total Sp ac T (ºC) Reference Remarks protein (U/mg) (mg) Sulfolobus 0.1 (1.5L) 11.65* 95 This study Archaea solfataricus Sulfolobus 0.78 56.3** 80 Kim et al., Archaea solfataricus 45.04 95 2004 Thermoplasma 0.45 4.21*** 75 Dock et Archaea acidophilum 0 95 al., 2008 Thermoanaerobactor 2.2 175**** 75 Zheng et Bacterial tengcongensis MB4 43.75 85 al.,2010 / 95 (*1.5L growth in fermenter and all activity tests were performed at 95°C using maltose as substrate, ** activity tests were performed at 80°C using maltose as substrate, *** activity tests were performed at 75°C using amylopectin as substrate, ****activity tests were performed at

75°C using maltose as substrate)

4.3.5 Effects of pH and temperature on enzyme activity

The activities of thermostable GA between pH 4.5 to 7 are shown in Figure

4.12. The optimum pH is 5-5.5 and there was a significant activity reduction from pH

5 to 4.5. Also when the pH increased to 6, the enzyme lost 50% of its activity. This result is similar to the data of Kim et al., (2004). Also the optimum pH value was consistent with GA from other archaea, such as Picrophilus torridus with an optimum pH of 5 and Thermoplasma acidophilum DSM 1728 with an optimum pH of 5, and close to Sulfolobus tokodaii with an optimum pH of 4.5 (Schepers et al., 2006; Dock et al., 2008; Njoroge et al., 2005).

107

120

100

60 Relative rate [%] Relative 40

0 4 4.5 5 5.5 6 6.5 7 7.5 pH

Figure 4. 12 pH effects on free GA activity at 95°C (relative activities are normalized by the activity at pH 5), use 10mg/ml maltotriose as substrate.

However, Uotsu-Tomita et al., (2001) reported the GA from

Methanocaldococcus jannaschii MJ1610 and Thermoactinomyces vulgaris R-47 have optimum pH of 6.5. Also Serour and Antranikian, (2002) reported extracelluar GA from thermoacidiphilic archaea such as Picrophilus oshimae, Picrophilus torridus and

Thermoplasma acidophilum have optimum pH of 2. Most of the GA from bacterial sources have an optimum pH of 4.5-5 and most of the GA from fungi have an optimum pH of 4-5.5 (Table 1.1). Thermostable AA has an optimum pH of 5.5. In order to use the thermostable AA and GA at the same time, an optimum pH of 5-5.5 is desirable.

108

The optimum reaction temperature was around 90-95 °C (Figure 4.13).

110

100

Relative rate [%] Relative 60

30 60 70 80 90 100 110 120 Temperature [°C]

Figure 4. 13 Effects of temperature on free GA activity at pH 5, use 10mg/ml maltotriose as substrate. The optimum temperature was consistent with the result reported by Kim et al.,(2004) and also similar to GA from other archaea, such as Picrophilus oshimae, Picrophilus torridus and Thermoplasma acidophilum (Serour and Antrankikian, 2002). The thermostable enzyme also shows high activity (>90%) around 100 °C . This characteristic is very promising for starch hydrolysis at high temperature. The activities at 90 and 95°C do not have any significant difference (p=0.1347>0.05, two- sample t-test), the activities of 95 and 100°C also have no significant difference

(p=0.4922>0.05, two-sample t-test). The activities of 90 and 100°C have statistical significant difference ( p=0.0428<0.05, two-sample t-test).

109

4.3.6 Substrate specific activity

The specific activities of GA with various substrates are shown in Table 4.5.

Table 4. 5 Specific activity of GA for various substrates

Substrate Reaction rate DP U/mg protein Maltose 11.95±0.44 2 Maltotriose 91.74±5.59 3 Maltotetraose 20.08±1.91 4 Maltopentaose 12.68±0.64 5 Maltohexaose 10.09±0.86 6 Maltoheptaose 8.27±0.56 7 Malto-dextrin 14.05±1.08 9-12 (88.1%) Soluble starch 2.54 ±0.62 Isomaltotriose 0 3 D-(+)raffinose 0 3 D-(+)melezitose 0 3 Stachyose 0 4

GA hydrolyzes the maltotriose most efficiently. This confirmed the finding that the maltotriose have the highest relative activity reported by Kim et al., (2004). The maltotetraose (DP4) is the second optimum substrate for GA from S. solfataricus P2

(Table 4.5). Thermostable GA hydrolyzes maltotetraose almost twice faster than maltose. Serour and Antranikian (2002) also observed a significant drop of activity when using maltose by a GA from archaea P. torridus. Zheng et al, (2010) found the maltotetraose was the optimum substrate among DP 4-10 oligosaccharides by a bacterial GA from T. tengcongenisis MB4. They also observed that GA showed minimal activity toward the soluble starch. Results of this study are in agreement with the literature that recombinant GA showed high activity on maltotriose and lower activity on soluble starch (Kim et al., 2004, Njoroge et al., 2005, Schepers et al., 110

2006). Table 4.6 compares the results obtained from this study with literature in terms of substatrate activities. Uossu-Tomita et al. (2001) also reported that the GA from archaea Methanococcus jannaschii, hydrolyzed oligosaccharides faster than starch.

However, Serour and Antranikian (2002) reported that GA from P. torridus hydrolyzed both soluble and native starch. It is important to point out that the reaction medium had a pH of 2 and a temperature of 90°C since the enzyme was from thermoacidophilic archaea. In addition to the enzyme, the acid and high temperature conditions would be expected to contribute to starch hydrolysis too.

Commonly, fungal glucoamylse hydrolyzes starch more efficiently than oligosaccharides. This preference is different for the GA from archaea and bacteria

(Zheng et al., 2010). Antranikian et al. (2005) pointed out that maltotriose preference of the GA from S. solfataricus P2 reported by Kim et al. (2004) differentiate archaea

GA from fungal GA.

111

Table 4. 6 GA substrate percent relative activity comparison.

Koc Njoroge and This Kim et et al., Schepers et Zheng et Metin, study al.,( 2004) (2005) al., (2006) al, (2010) (2010) Original microorganism Substrate Archaea Archaea Archaea Archaea Bacteria Fungi Maltose DP2 13 15 12 8 44 68 Maltotriose DP3 100 100 100 100 50 DP4 22 21 40 18.40 DP5 14 8 31 7.20 DP6 11 7 13 8.80 DP7 9 5 11 5.60 Soluble Starch 3 0 0 60 72 100 Maltooligosaccharides* 15 Raffinose DP3 0 42 Maltooligosaccharides* (DP 4-10) 100 *For maltooligascharides, this study use DP 9-12, Zheng et al. (2010) used DP 4-10.

It is apparent that maltotriose (DP=3) was the best substrate for GA. The GA from S. solfataricus P2 was highly specific for maltotriose. It can not hydrolyze isomaltotriose (DP=3), D-(+) raffinose (DP=3) and D-(+) melezitose (DP=3) (Table

4.4). GA sequentially hydrolyzes the alph-1,4 glycosidic bonds from the non-reducing end. Isomaltosriose, raffinose and melezitose have no alph-1,4 glycosidic bonds.

Therefore, these compounds are not expected to be hydrolyzed by GA.

The observed higher activity of thermostable GA with maltotriose in comparison with the maltose could be that the maltotriose has a perfect size matching the distance between two catalytic carboxylates and binding sites (Figure 4.14). The two catalytic carboxylates model, commonly accepted in fungal GA, was both

112 explained by Sauer et al. (2000) and Kumar & Satyanarayana (2009). Compared to the fungal GA, the structure and function information for bacterial and archaeal GA is limited.

Reaction mechanism in catalytic domain, two catalytic carboxylates model

Catalytic B domain

R Non-Reducing end Binding sites

R’

Figure 4. 14 Proposed explanation of maltotriose with best substrate activity. (The reaction mechanism is modified from Sauer et al, 2000). R is the residue of maltotriose, R’ is the residue of maltose, A and B are the two catalytic carboxylates. 113

4.3.7 Metal effects on enzyme activity

Table 4.7 shows the influence of various metal ions on the recombinant GA activity. Mg2+ slightly increases the activity over the range of 1-10mM, which was in agreement with the data reported in literature (Chen et al., 2005; Dock et al., 2008;

2+ Zheng el al., 2010). Ca shows minimal effect on the GA activity. This indicates that the GA does not have Ca2+ dependence, which is different than the effect observed for most AAs. Low Ca2+ dependence is preferable for starch hydrolysis because it can save the material (calcium chloride) and ion exchange cost for the subsequent isomerization step. Fe3+ and Cu2+ inhibit the GA activities, this inhibitory effects was also observed in another archeal GA (Dock et al., 2008). Cu2+ inhibition effects was consistent with the bacterial GA (Zheng et al., 2010) but was in conflict with fungal

GA (Kumar and Satyanarayana, 2003; Chen et al., 2005, Bhatti et al, 2007; Michelin et al., 2008) and bacterial GA (Specka et al., 1991) behavior. The Cu2+ promotes the activity of fungal GA.

Table 4. 7 Influence of ions on GA activity

Metal ion 1mM 5mM 10mM None 100 100 100 Ca2+ 101.1 ± 0.6 97.2 ± 1.3 90.1 ± 1.6 Mg2+ 75.4 ±1.5 103.5 ± 1.4 110.2 ± 1.1 Cu2+ 46.4 ± 0.7 2.4 ± 0.2 2.1 ±0.2 Na+ 74.2 ± 0.3 56.2 ± 0.5 75.2 ± 1.9 Fe3+ 2.14 0 0

Before the activity test, 0.08mg/ml GA were incubated for 2 hours at room temperature. The reaction was started by adding maltotriose with a final concentration of 10mg/ml in pH 5 acetate buffer at 95°C.

114

4.3.8 Effects of chemical reagents on enzyme activity

The influence of several chemical reagents on GA activity is shown in Table

4.8. The GA activity increased by 10mM EDTA but was reduced by EDTA above

50mM concentration after 2 hours of treatment at ambient temperature. The inhibitory effect of EDTA (5-10mM) on the GA was also reported in other studies (Kumar and

Satyanarayana, 2003; Dock et al., 2008, Zheng et al., 2010). However, Li et al. (1998) reported that the activity of GA from Thermomyces lanuginosus was not affected by

30mM EDTA. Michelin et al., (2008) also found that the 10mM EDTA did not affect the activity of fungal GA from Paecilomyces variotii.

EDAC and Sulfo-NHS inhibited the enzyme activity after 2 hours of treatment at a concentration above 10mM. The inhibitory effects were decreased with short time pretreatment (15 minutes). In the case of adding 200mM EDAC and 50mM Sulfo-

NHS simultaneously, the GA kept 94% of initial activity. The 2 hours of treatment was selected based on the literature studies (Zheng et al.; Dock et al., 2008). The 15 minutes treatment time was consistent with the covalent binding procedure applied in this study. 2-Mercaptoethanol did not affect the GA activity at ambient temperature.

This finding was consistent with the results of Dock et al. (2008), Michelin et al. (2008) and da Silva et al. (2009). However, Kumar and Satyanarayana, (2003) reported an inhibitory effect of 2-Mercaptoethanol on a fungal GA from Thermomucor indicae- seudaticae. The SDS inhibited the GA activity significantly which was consistent with results reported by Kumar and Satyanarayana (2003) and Dock et al. (2008).

115

Table 4. 8 Influence of chemical reagents on GA activity

Reagent 10mM 50mM 200mM None 100 100 100 EDTA 103.8 ± 1.3 83.8 ± 2.9 53.9 ± 2.6 EDAC 76.6 ± 1.3 48.5 ± 2 4.6 ± 0.6 Sulfo-NHS 56.5 ± 1.6 65.4 ± 2.2 7.9 ± 0.7 2-Mercaptoethanol 105 ± 2.1 103 ± 1.7 101 ± 3.2 SDS 2 0 0 EDAC* 48.4 ± 1.8 Sulfo-NHS* 97.1 ± 1.3 EDAC(200mM) +Sulfo- NHS(50mM)* 94.7 ± 2.1 Before the activity test, 0.08mg/ml GA were incubated for 2 hours at room temperature. The reaction was started by adding maltotriose with a final concentration of 10mg/ml in pH 5 acetate buffer at 95°C. * Only 15 minutes treatment

Four chemical reagents (EDTA, 2-Mercaptoethanol, EDAC, Sulfo-NHS) were chosen to investigate because all of them were involved in the covalent binding procedure which was discussed in Chapter 5.2.1.

4.3.9 Thermal stability of recombinant GA

The thermal inactivation of enzymes occurs due to irreversible unfolding of protein secondary structure. This characteristic plays an important role for developing starch hydrolysis processing at high temperatures. The thermal stability of recombinant GA was evaluated between 75 and 100°C at pH 5 by using 10mg/ml maltotriose as substrate. The GA was stable at 75 °C for at least 2 hr (within 1% loss of activity in 2 hr). The thermal stability from 80 to 100°C is shown in Figure 4.15.

116

4.5

3.5

2.5

2 ln (E(t)/E0) ln 1.5

0.5

0 0 500 1000 1500 2000 2500 3000 3500 4000 Inactivation time [s]

Figure 4. 15 Inactivation of GA at different temperatures. 80 °C (■), 85 °C (▲), 90 °C (x), 95°C (○), 100 °C(♦).

The half-life time (t1/2) and denaturation rate constant (kd) calculated at various temperatures are shown in Table 4.9.

Table 4. 9 GA inactivation at different temperatures

Temperature t1/2 kd [°C ] [min] [min-1] 75 489 ± 7.7 0.0014 80 127.1 ± 4.6 0.006 85 35.5 ± 2.5 0.018 90 16.4 ± 0.4 0.042 95 5.3 ± 0.4 0.12 100 2.5 ± 0.1 0.294

At 75 °C the half time of GA was 489 minutes which was more stable than most of the fungal GAs (Table 1.1). Gomes et al., (2005) reported a half time of 480

117 min at 60 °C for GA from fungal Thermomyces lanuginosus. Chen et al. (2005) reported a half time of 20 min at 70 °C of GA from fungal Chaetomium thermophilum.

Michelin et al., (2008) reported a half time of 45 min at 60 °C of GA from fungal

Paecilomyces variotii. The half time at 80°C was close to the reported value by Dock et al., (2008), although 0.5 mM Ca2+ was added to achieve this thermal stability. The

Arrhenius model was fitted to the natural logarith of rate constant versus inverse temperature data (Figure 4.16). The activation energy of GA inactivation was calculated to be 218 ± 2 kJ/mol. Compared to the activation energy of GAs from fungi:

61 kJ/mol (Rao et al., 1981), 43 kJ/mol (Kumar and Satyanarayana, 2003), 69.1 kJ/mol

(Riaz et al., 2007) and 109 kJ/mol (Pavezzi et al., 2011), and bacteria: 38.3 kJ/mol

(Feng et al., 2002), the recombinant archeal GA has higher inactivation energy. This indicates the GA form S. solfataricus P2 is more stable than the GA from fungus and bacterial sources.

118

-5

-6

-7

)

ln(k -8

-9

-10 2.66 2.68 2.70 2.72 2.74 2.76 2.78 2.80 2.82 2.84 -1 1/T*1000[K ]

Figure 4. 16 Arrhenius plot of the thermal inactivation rates for GA in free form.

4.3.10 Michalies-Menten kinetics of recombinant GA

Maltotriose and maltose, in the concentration range of 0-100 mg/ml were hydrolyzed by GA. The Michalies-Menten model was fitted into the reaction rate vs. substrate concentration data by KaleidaGraph® software as shown in Figure 4.17. The recombinant GA kinetics follows the Michalies-Menten model in the range of 0-100 mg/ml substrate concentration. The Km for maltotriose and maltose were 2.86 mg/ml and 44.33 mg/ml respectively (Table 4.10). Feng et al. (2002) reported a Km of 0.9 mg/ml at 60°C in maltose buffer solution at pH 4.5 by using a bacterial GA from T. thermosacchrolyticum. Zheng et al. (2010) also reported Km values of maltotriose and maltose as 0.35 mg/ml and 2.41 mg/ml for a bacterial GA from Thermoanaerobacter

119 tengcongensis MB4. Their results were similar to Km values of an archaea from

Picrophilus torridus, 0.25 mg/ml and 0.88 mg/ml (Schepers et al., 2006). Lower Km means higher affinity. The calculated Km values of this study indicated that the GA had eighteen folds higher affinity for maltotriose than maltose. Other researchers reported 7 folds (Zheng et al., 2010) or 3.5 folds (Schepers et al., 2006). All of these results may imply that the GA from archaea or bacterial has higher affinity for maltotriose than maltose.

25 6

5 20

4 15

Reaction byrate using Maltose

Reaction byrate using Maltotriose [ mg mg [ glucose/(min*ml*mg enzyme) ]

5 mg [ glucose/(min*ml*mg enzyme) ] 1

0 0 0 20 40 60 80 100 120

Substrate concentration [ mg/ml ]

Figure 4. 17 Michalies-Menten model fitting with respect to substrates, maltotriose (Δ) and maltose (x).

120

The Vmax values (Table 4.10) for maltotriose and maltose are 25.24 and 8.22 mg glucose/(ml*min*mg protein) indicating that the GA can reach to a maximum reaction rate of 25.24 mg glucose per min per ml by using 1 mg of enzyme in maltotriose buffer solution with substrate concentration range above 20 mg/ml.

Table 4. 10 Substrates effects on GA kinetics parameters

Substrates Km Vmax kcat kcat/Km

[mM] [mM glucose/(s*mg s-1 [s-1*(mM)-1]

protein)]

Maltose 129.6±23 0.76±0.06 33.29±2.63 0.26±0.02

Maltotriose 5.7±0.89 2.34±0.06 102.05±2.43 17.90±0.46

Schepers et al., 2006 reported a Vmax value of 10U/mg and 0.2 U/mg for maltotriose and maltose at pH 5 and 50°C . The Vmax in this study was approximately

140U/mg, which was 14 times faster than the reported value of Schepers et al., (2006).

The difference could be caused by the reaction temperature since 95°C was used for this study. Zheng et al., (2010) reported a ratio of approximately 2.47 at pH 5 and

75°C for the turnover ratio between maltotriose and maltose. This study had a very similar value of 3.07 (kcat ratio between maltotriose and maltose).

121

4.4 Conclusion

A thermostable GA with an optimum temperature at 90°C and optimum pH at

5.0 was successfully expressed and purified from S. solfataricus P2 by adding His6- tag. The purification method with IMAC was effective and could reduce the purification cost of enzyme. The growth conditions were optimized by improving the

OTR and increasing growth temperature after IPTG addition to 46°C . The growth of recombinant cells was performed with 1.5L LB medium in a 2L fermenter with 200 rpm rotational speed and an air flow rate of 1.5L/min. The final yield of activity was

7%. The enzyme had a specific activity of 12 U/mg by using 10mg/ml maltose as substrate at optimum reaction conditions. The GA hydrolyzed the maltotriose most effectively with a specific rate of approximately 100 U/mg. The GA did not have Ca2+ dependence. Fe3+ and Cu2+ inhibited the enzyme while the Mg2+ promoted the catalytic reaction.

EDTA (10mM) did not have any adverse effect on the GA for 10 minutes incubation time before reactions at room temperature. Treatment with EDAC

(200mM) and Sulfo-NHS (50mM) simultaneously for 15 minutes incubation time at room temperature before the reaction retained approximately 94% of the enzyme activity.. 2-Mercaptoethanol did not have any adverse effect on enzyme activity at room temperature.

The half time at different temperatures (80-100°C ) was calculated. The GA had a half time of 16.4 ± 0.4 minutes at 90°C . The activation energy for inactivation of thermostable GA was also determined to be 218 ± 2 kJ/mol. Michalies-Menten 122 kinetics paramenters of free recombinant GA were also determined. The Km of maltotriose and maltose are 2.86 mg/ml and 44.33 mg/ml respectively. The Vmax values for maltotriose and maltose are 25.24 and 8.22 mg glucose/(ml*min*mg protein).

To our best knowledge this is the first reported data of reaction kinetics and inactivation energy for thermostable GA from S. solfataricus P2.

123

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Chapter 5 Co-immobilization of thermostable alpha-amylase and glucoamylase for starch hydrolysis

5.1 Introduction

A thermostable recombinant GA was used in combination with thermostable

AA for starch hydrolysis in free and in immobilized form. This chapter describes the development of a co-immobilized biocatalyst system in which the recombinant thermostable GA by entrapment and a thermostable AA (Liquozyme X) covalent binding was immobilized using calcium alginate as a support. The GA entrapment method was described in detail in Chapter 3. The covalent binding method was developed and applied to Liquozyme X by Tee and Kaletunc (2009).

Co-immobilization of enzymes can be achieved by a combination of entrapment, adsorption, and covalent binding. In this study, recombinant GA was entrapped inside a calcium alginate gel capsule first, followed by covalent binding of the AA on the surface of the gel capsule. The selection of covalent binding for the immobilization of AA was made so as to be able to hydrolyze starch at high temperatures, above 90°C . Compared to the surface adsorption method, the covalently bound AA was retained at higher levels at high temperature (Tee and Kaletunc, 2009).

The covalent binding involved use of several chemicals.

Ethylenediaminetetraacetic acid (EDTA) solution was applied to pre-treat the calcium

132 alginate gel capsules in order to increase the number of available carboxylic groups for

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDAC) binding. N- hydroxysulfosuccinimide (Sulfo-NHS), was added together with EDAC to improve the half-life of EDAC-activated carboxylic groups by forming a semi-stable amine- reactive NHS-ester with EDAC,. 2-Mercaptoethanol was used for quenching the excess EDAC (avoiding any adverse effect of EDAC on enzyme activity). It is necessary to study the effects of these chemicals and the process of the covalent binding procedure on the activity of entrapped thermostable GA. Several tests were performed before the co-immobilization, to determine effects of EDTA, EDAC, Sulfo-

NHS, and 2-mercaptoethanol on the activity of entrapped GA.

After examining the influence of the co-immobilization procedure on the activity of thermostable GA, co-immobilization was performed. The resulting co- immobilized biocatalyst system was tested in a batch reactor for reaction kinetics, thermal stability, and reusability.

There are few studies about immobilizing the AA and GA in the same system for one-step starch hydrolysis processing. Rao et al. (1981) produced a thermostable fungal GA with an ultimate goal of developing a stable immobilized glucoamylase system for the continuous production of glucose. Their purpose was focused on immobilization of one thermostable enzyme (GA). In industrial hydrolysis, saccharification step is performed at relatively low temperature (around 60°C), so the processing temperature needs to be cooled down after the liquefaction step.

Development of a thermostable GA can eliminate the cooling step. Legin et al. (1998)

133 pointed out that the greatest improvement for current industrial starch hydrolysis would be degrading the starch in one step. Leveque et al. (2000) also suggested that hydrolysis enzymes from thermophilic or hyperthermophilic archaea could be the best candidates for a one-step conversion of starch to glucose, which can decrease the cost of glucose production.

Park et al. (2005) also applied a covalent binding method to immobilize AA and GA onto diethylaminoethyl (DEAE) cellulose and then entrapped the resulting system inside calcium alginate beads. The optimum reaction temperature was improved from 50°C (free AA and GA) to 60°C (co-immobilized system). Their findings also demonstrated that co-immobilization of GA and AA can produce glucose from starch in one step. However, they also used glutaraldehyde as a crosslinker for covalent binding of GA and AA to DEAE cellulose.

Yang et al. (2010) co-immobilized AA and GA on magnetic chitosan beads and used them for starch hydrolysis up to 15 cycles. However, the reaction temperature was only 50°C andglutaraldehyde was used as a crosslinker.

This study demonstrated the development and application of a co-immobilized thermostable GA and AA biocatalyst system in a batch and a continuous stirred reactor for starch hydrolysis at high temperature.

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5.2 Materials and Methods

5.2.1 Materials

Starch from potato (Sigma, S2630), MES (4-morpholineethanesulfonic acid) sodium salt (Sigma, M5057), MES hydrate (Sigma, S8625), sodium acetate (Sigma,

S8625), maltose monohydrate (Sigma, M5885) were obtained from Sigma (St. Louis,

MO, USA). For enzyme immobilization, sodium alginate from brown algae (Sigma,

A2158), was purchased from Sigma (St. Louis, MO, USA); Liquozyme X

(Novozymes North America, NBP00038) was obtained from Novozymes, Inc.

(Franklinton, NC, USA). Liquozyme X will be referred to as AA in this chapter.

For the protein test, bovine serum albumin (Sigma, A0281) and Bradford reagent (Sigma, B6916) were purchased from Sigma (St. Louis, MO, USA). Stirring hot plate (Model Isotemp, Fisher Scientific, Pittsburg, PA, USA), modified

Erlenmeyer, water bath, peristaltic pump, condenser, thermometers, plastic tubing

(EW-06424-15) were used to set up a continuous stirred tank reactor (CSTR) system.

For covalent binding, EDTA was purchased from Fisher-Scientific (Pittsburg,

PA, USA); Sulfo-NHS (N-hydroxysulfosuccinimide) (24510) was bought from Pierce.

N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDAC, E6383) and 2-

Mercaptoethanol (M3148), were obtained from Sigma (St. Louis, MO, USA).

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5.2.2 Co-immobilization of AA and GA

The process of co-immobilization is explained in Figure 5.1. First, the recombinant GA was entrapped inside the calcium alginate capsule. Recombinant GA

(0.4 ml) was mixed with 1.2 ml CaCl2 to reach a final mixture of 0.2 mg/ml GA and

40 mg/ml CaCl2. Then the mixture was dripped into a 1% sodium alginate solution at

4°C through a 26G needle at a flow rate of 0.5 ml/min. The sodium alginate solution was stirred at a speed of 400 rpm with a magnetic stir bar. The distance from the needle to the alginate solution surface was set to 27 cm.

The GA entrapped capsules were then washed by washing buffer (50 mM acetate buffer at pH 5) and reinstated in a acetate buffer pH 5.0 containing 100 mM

CaCl2, . After 2 hours of curing, 5 grams of capsules were mixed with 10 ml of 10 mM

EDTA solution for 1 minute to increase the available sites of carboxylate groups for

EDAC reaction. After removing the EDTA solution, the capsules were washed by nano-pure water (5g capsules per 20 ml water) four times. The capsules were then pre- equilibrated in 15 ml of 50 mM MES buffer (activation buffer) at pH 5.5 for 20 minutes in a 50-ml beaker. After replacing the pre-equilibrated activation buffer with 5 ml fresh activation buffer, 2.5 ml of 200 mM EDAC and 2.5 ml of 50 mM Sulfo-NHS were added into the 50-ml beaker. After incubation with constant stirring at 120 rpm for 15 minutes, 1.4 ml of 2-mercaptoethanol (10-fold in excess of EDAC) was added to quench the EDAC reaction for 10 minutes. The capsules were then filtered and rinsed with 50 mM MES buffer (coupling buffer) at pH 5.5, containing 5 mM CaCl2.

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1% Alginate Glucoamylase Calcium Alginate Capsule 2+ with entrapped o Ca t t ce en rfa Glucoamylase tm su rea he t n t TA 2+ o ED a e C H ov rem N+ H N+

O O N + O N O NH2 C Unstable EDAC Intermediate N O O O S O O N O O HO O S O N O O Sulfo-NHS O O Semi-Stable Intermediate + N H3N H α-Amylase Immobilized Amylase with Entrapped Glucoamylase

Figure 5. 1 A covalent binding procedure for the co-immobilization of GA and AA in sodium alginate capsules. Revised from Tee and Kaletunc (2009).

After the attachment of a crosslinker, the surface of capsules was activated by forming an amine-reactive Sulfo-NHS ester intermediate on the surface (Figure 5.1)

137 for binding of AA. Capsules were immersed into 10 ml of 32 ± 2 µg/ml AA buffer solutions for 15 hours of immobilization at 25°C . After immobilization, the capsules were filtered and washed in 7.5 ml of coupling buffer. After washing, capsules were placed in acetate buffer at pH 5.5 containing 100 mM CaCl2, for 30 minutes for reinstatement of capsule strength All the washing and reinstating solutions were collected for protein content analysis to calculate the encapsulation efficiencies.

5.2.3 Mixed enzyme capsules preparation

In order to prepare the mixed enzyme capsules, 5 grams of the calcium alginate gel capsules with entrapped recombinant GA were prepared as described in 5.2.2.

Another 5 grams of blank capsules were prepared by dripping 40 mg/ml CaCl2 into a 1% sodium alginate solution (4°C ) through a 26G needle at a flow rate of 0.5 ml/min. The blank capsules were then washed by washing buffer (50 mM, pH 5 acetate buffer) and reinstated in a 100 mM CaCl2, pH 5.0 acetate buffer, which was used for covalent binding of AA. By utilizing the covalent binding procedure, the gel capsules with only covalently bound AA were prepared.

Equal amounts of gel capsules containing only entrapped GA were mixed with gel capsules containing only covalently bound AA to form mixed immobilized enzymes system.

5.2.4 Evaluation of the effect of covalent binding procedure on GA activity

During the covalent binding procedure, the entrapped GA was exposed to several chemical treatments including 10 mM EDTA, 200 mM EDAC, 50 mM Sulfo-

138

NHS, and 2-mercaptoethanol. In chapter 4.3.7, the effects of these chemical regents on free recombinant GA have been evaluated. None of these treatments inactivated the

GA more than 4% of initial activity at room temperature in 15 minutes.

The effects of these chemicals on entrapped GA activity were determined as described in Figure 5.2. One batch of capsules with only entrapped GA was exposed to

10 mM EDTA (Case 2) for 1 minute of treatment. One batch of capsules containing entrapped GA was exposed to chemical treatment steps necessary to prepare capsules for AA binding (Case 3). Also one batch was exposed to all chemical treatments followed by the binding of AA (Case 4). Activity of GA was determined at 95°C, and at pH 5.5 using 10 mg/ml maltotriose as substrate. The results were compared to the activity of the control (without any treatment, Case 1).

139

Case 1 GA entrapped capsules Control

10mM EDTA 1min

GA entrapped capsules Case 2 EDTA treatment

Amine-reactive ester activation (2.5ml 50mM Sulfo-NHS 2.5ml 100mM EDAC 5 ml activating buffer) 15min

GA entrapped capsules

2-Mercaptoethanol

Case 3 All GA entrapped capsules chemicals treatment

AA solution for 15 hrs

Case 4 effects of AA presence on GA activity

Figure 5. 2 Evaluation of chemical effects on entrapped GA. 140

5.2.5 Encapsulation efficiency of co-immobilized biocatalyst system

The immobilization efficiency of GA entrapment and AA covalent binding were calculated individually. The indirect calculating method described in Chapter

3.2.5 was used for loading efficiency calculation. The final ratio between GA and AA in co-immobilization system was calculated based on the amount of immobilized GA and AA.

5.2.6 Effects of pH and temperature on immobilized GA

The influences of pH and temperature on entrapped GA were tested to compare the activities of the free and immobilized GA in order to understand the effects of immobilization.

Influence of pH was studied between 4 and 7. Influence of temperature was studied between 80 and 110°C . For temperatures higher than 95°C , the pressurized batch reactor was used. Activity of immobilized GA (17µg/ml) using 10 mg/ml maltotriose as substrate was studied at various pHs and temperatures.

5.2.7 Thermal inactivation of entrapped GA

The inactivation rate of the entrapped GA from 80 to 110°C over a period of 1 hour using 10 mg/ml maltotriose as substrate was studied. The half time and inactivation energy were calculated as described in Chapter 4.2.7. These data were then compared to the data for the free enzyme to understand the effects of immobilization on thermal stability of recombinant GA.

141

5.2.8 Optimum pH and temperature of co-immobilized GA and AA system

The co-immobilized capsules with entrapped GA and covalently bound AA were placed in starch solutions from pH 4 to pH 7 with 0.5 intervals. The activity of the co-immobilized enzyme system as mg glucose/ml reaction medium per mg protein was determined by taking samples at 5 and 8 minutes. The sampling time was decided by constructing a progress curve of glucose production versus time. The reaction was stopped by immersing samples into ice water. The optimum temperature was determined by conducting experiments at 85, 90, 95, 100, and 110°C .

5.2.9 Michaelis-Menten parameters for immobilized GA and AA system

The Michaelis-Menten reaction kinetics in terms of glucose production rate were determined in a range of 0-75 mg/ml (maltotriose for GA entrapped capsules or soluble starch for the co-immobilized system) at the optimum reaction temperature of

95°C and at pH 5.5. Also the kinetic parameters of an equivalent amount of mixed thermostable GA and AA in different capsules were determined at the optimum reaction temperature (95°C ) and at pH 5.5.

5.2.10 Reusability and thermal stability of GA and AA system

The co-immobilized thermostable GA (17 µg/ml) and AA (12.5 µg/ml) was used for starch (10 mg/ml) hydrolysis in a batch stirring tank (1 gram of capsules in 3 ml of starch solution) at the optimum temperature (95°C ) and pH 5.5. The co- immobilized thermostable system was used for eight times. Each run was 10 minutes long.

142

The mixed individually immobilized enzyme system was composed of capsules containing only entrapped GA and capsules containing only covalently bound

AA. The mixed biocatalyst system had twice the amount of capsules as the co- immobilized system but the same total amount of enzymes. The mixed immobilized enzyme system was investigated under the same temperature and pH as the co- immobilized system.

A mixed free form of thermostable GA and AA, containing the same amount of each enzyme as was in the immobilized system was incubated at the optimum reaction temperature of the immobilized system. The activities of the enzymes incubated at 0, 10, 20, 30, 40, 50, 60, and 70 minutes were determined in order to construct a mixed free enzymes thermal stability curve at the optimum reaction temperature.

5.2.11 CSTR setup and operation

A continuous stirred tank reactor set up was constructed to test the immobilized enzyme system under continuous starch hydrolysis conditions. Four grams of the co-immobilized capsules or 8 grams of the mixed capsules containing 0.2 mg GA and 0.16 mg AA were each tested in a CSTR. The setup of CSTR and auxiliary units is shown in Figure 5.2.

A 500-ml wide-mouth Erlenmeyer flask was fitted with a silicone stopper containing three openings. A thermocouple probe was inserted into the flask through one of the openings for temperature control. A condenser was connected to the second

143 opening to prevent water evaporation and the inlet flowed through the third opening.

The flask was modified by adding an outlet at the 400-ml level in order to construct a continuous flow system. A special design (narrow neck) of the outlet pipe was modified to keep the immobilized capsules inside the reactor. The modified wide- mouth Erlenmeyer flask was placed on a stirring hot plate. A stir bar was placed inside the reactor so that a well mixed reaction medium with immobilized beads was obtained. The thermocouple probe connected to the hot plate controlled the temperature at approximately 95oC.

Two 1,000-ml bottles containing starch buffer solution (15 mg/ml or 100 mg/ml containing 0.25 mM CaCl2) were placed inside a water bath. The water bath was maintained at 95oC. A mixer was inserted into the bottle to prevent settling of starch in the bottle during the experiment. A peristaltic pump (Masterflex, Cole-

Parmer Instrument Co., Chicago, IL) was connected to the reactor inlet by the L/S 15 silicon (C-Flex®) tubing. The starch buffer solution from the 1,000-ml bottle was pumped into the reactor at controlled flow rate. Reaction products left continuously through the outlet pipe at the same inlet flow rate to maintain a constant solution volume in the reactor.

A total of 400 ml of acetate buffer solution with 0.25 mM CaCL2, pH 5.5 was heated to the reaction temperature in the reactor. When the temperature equilibrium was established, the capsules that were pre-equilibrated in 5 ml acetate buffer at 95°C for 1 minute were added into the reactor. At the same time, the starch was also pumped into the reactor. The time when the starch solution was introduced into the

144 reactor was considered as the zero time. A 1-ml sample of the solution was collected through the outlet pipe. During the first 5 minutes, one sample was taken per minute.

After 5 minutes, one sample was taken every 5 minutes. The samples were cooled immediately in ice water, and 0.2 ml of each sample was added into 1 ml of 0.5N HCl for the starch hydrolysis test. The leftover sample was then centrifuged at 12,500 x g and 0.2 ml was taken for glucose analysis. The initial starch buffer solution, total product stream leaving the reactor, and the final solution in the reactor were also tested for starch and glucose concentration.

Figure 5. 3 CSTR setup.

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5.3 Results and Discussion

5.3.1 Effects of covalent binding procedure to entrapped GA activity

The entrapped GA without any treatment (Case 1) and those with treatments using the chemicals employed during the covalent binding of AA had a glucose production rate of 23 ± 1.2U/(mg of recombinant GA) when 10 mg/ml maltotriose was used as substrate at pH 5.5 and 95°C. The results indicate that the covalent binding procedure and the presence of covalently bound AA on a support do not have an adverse effect on entrapped GA activity. The entrapment in calcium alginate may have protected the GA from exposure to the adverse environment. The free form GA has a specific activity of 96 U/mg at 95°C when 10 mg/ml maltotriose is used as substrate.

This implies that the entrapped GA has a slower rate compare to free form. Compared to the free form of GA, the entrapped GA had the diffusion limitation of maltotriose into the capsules and starch hydrolysis products out of the capsules. Yankov et al.

(1992) reported an entrapped GA in polyacrylamide gel had 55% of the specific activity of free GA (47 U/mg) for first usage when using 4% (w/v) soluble starch as the substrate at pH 4.2 and 60°C.

146

5.3.2 Effects of pH on entrapped thermostable GA

The influence of pH on entrapped GA and free GA is shown in Figure 5.4.

120

100

60 Relative activity [%] Relativeactivity 40

0 3.5 4 4.5 5 5.5 6 6.5 7 7.5 pH

Figure 5. 4 pH influences on free (∆ normalized by activity at pH 5) and entrapped ( ᵡ normalized by activity at pH 5.5) GA at 95°C. The entrapped GA had a wider optimum pH range than the free GA. This is similar to the finding reported by Yankov et al. (1992) that the optimum pH increased upon entrapment of GA within polyacrylamide gel. Calcium alginate gel had a more protective effect above optimum pH. However, a similar pH dependence behavior for free and entrapped GA was observed below optimum pH. The pH change can affect the ionization of the active site of GA, which will affect the enzyme activity. Also the pH change can affect the proton transfer step of the catalytic site. Many enzymes employ transition-state proton bridging (general acid-base catalysis) as an important

147 strategy for catalytic acceleration (Schowen et al., 2000). The general acid catalyst and proton donor Glu179 and the catalytic base Glu400 in GA of A. niger have been reported as active sites (Sauer et al., 2000).

There is a significant activity (60%) drop at pH 4.5 both in free and immobilized GA. A similar behavior was also observed in the free enzyme results of

Kim et al. (2004) and in another thermostable GA from Streptosporangium sp. endophyte of maize leaves, with a 55% drop from optimum pH 4.5 to pH 4 (Stamford et al., 2002). This type of pH activity profile is different for GA from A. niger, which did not show a sudden drop in the pH 4-7 region (Tanriseven et al., 2002; Silva et al.,

2005; Bai et al., 2009). In the pH range of study, alginate is expected to be negatively charged due to the carboxylate groups (Tee and Kaletunc, 2009). The optimum pH of many enzymes shifts to the alkaline region if the support is anionic and to a more acidic region if the support is cationic because the pH will change the ionic degree of ionization of the active sites (Palmer and Bonner, 2007). Amino acid residues such as

Tyr, Thr, Ser, Ala, and Glu were involved in the active site of GA from fungi (Stoffer et al., 1997; Sauer et al., 2000). Glu has a negative charge in the pH range of 4-7 (PI =

3.1). Thermostable GA from S. solfataricus P2 theoretically has a negative charge at pH 7.0 (by theoretical calculation http://www.innovagen.se/custom-peptide- synthesis/peptide-property-calculator/peptide-property-calculator.asp).

The hydrogen ion concentration is higher in the alginate, which creates a microenvironment (more proton around the immobilized enzyme) that shifts the displacement of entrapped enzyme toward alkaline pH values (Tee and Kaletunc,

148

2009). In order to have the same ionization at active sites as the free form, the immobilized enzyme needs a higher pH. Also, Ca2+ can stabilize the GA (Kumar et al.,

2010). These two effects cause the protection of GA on the region higher than optimum pH.

5.3.3 Influence of temperature on entrapped thermostable GA

Optimum temperature shifted from 90°C for free GA to 95°C for immobilized

GA (Figure 5.5). The patterns of temperature effects on free or immobilized GA were similar as both activity versus pH and activity versus temperature relationships show a bell shape curve.

110

100

Relative rate [%]

30 60 70 80 90 100 110 120

o Temperature [ C ]

Figure 5. 5 Temperature influence on free (∇ normalized by activity at 90 °C ) and entrapped (● normalized by activity at 95 °C) GA.

149

The increase in optimum temperature was also reported in the literature (Arica et al., 1998; Wang et al., 2007; Bai et al., 2009). However, some studies did not observe an increasing optimum temperature upon immobilization (Tanriseven et al., 2002;

Silva et al., 2005). Arica et al. (1998) reported that the optimum temperature of a GA from A. niger increased from 50 to 55°C by covalent binding of the GA on Poly (2- hydroxyethyl methacrylate) (pHEMA) hydrogels. Bai et al.(2009) reported that the optimum temperature of covalently bound GA from A. niger on a hydrophilic support containing epoxy groups increased from 45 (free GA) to 55°C. Immobilization of enzyme can improve the thermal stability by decreasing the conformational flexibility through the specific interaction between support and enzymes (Unsworth et al., 2007).

The physical constraint of GA in the calcium alginate gel matrix could also prevent the conformational change that can improve the thermal stability (Yankov et al., 1992).

However, this improvement was not observed by Tanriseven et al.(2002) when using entrapped the GA on calcium alginate fiber and by Silva et al. (2005) when covalently binding the GA on polyaniline. Because the immobilization methods and supports were different among various researchers, the possible improvement of optimum temperature may not always happen.

150

5.3.4 Thermal stability and inactivation kinetics of entrapped GA

The inactivation of entrapped GA at various temperatures from 85°C to 110°C is shown in Figure 5.6.

0 ln (E(t)/E0) ln 0 500 1000 1500 2000 2500 3000 3500 4000 -1

-2

-3

-4 Inactivation time [s]

Figure 5. 6 Inactivation of entrapped GA at various temperatures. 85 °C (■), 90 °C (▲), 95 °C (ᵡ), 100°C (*), 110 °C (♦). Substrate: maltotriose 10mg/ml in pH 5 acetate buffer.

Table 5. 1 Inactivation parameters of entrapped GA at different temperatures

Temperature kd t1/2 [°C ] [min-1] [min] 85 0.018±0.0015 53.3±1.2 90 0.042±0.0004 15.9±1.1 95 0.114±0.0024 5.44±0.14 100 0.276±0.0002 2.72±0.15 110 0.534±0.0033 1.10±0.02

151

Inactivation behavior of entrapped GA follows the first-order kinetics for all of the temperatures investigated. The values for inactivation rate constant and the half life time defined as the time for the enzyme activity to reduce to half of the initial activity are given in Table 5.1. Comparison of half times of free form GA (Table 4.9) with those of immobilized showed that improvement due to immobilization was observed only at 85°C . Other temperatures did not have significant improvement by entrapment

(two-sample t-test p = 0.5004 > 0.05 at 90°C , p = 0.5978 > 0.05 at 95°C and p =

0.1021 > 0.05 at 100°C ). This may indicate that the stabilization effect of support cannot prevent irreversible unfolding between 90 and 110°C . Also, the capsule has a hollow structure with liquid inside, and this is similar to free enzyme in solution.

The Arrhenius plots for free and entrapped GA are shown in Figure 5.7.

The calculated average activation energies of enzyme inactivation for entrapped GA

(213 ± 4kJ/mol) and the free GA (218 ± 2kJ/mol) were calculated to be not significantly different (two-sample t-test p = 0.1249 > 0.05).

152

-5

-6

-7

)

Ln(k -8

-9

-10 2.66 2.68 2.70 2.72 2.74 2.76 2.78 2.80 2.82 2.84 -1 1/T*1000 [K ]

Figure 5. 7 Arrhenius plots of the thermal inactivation rates for recombinant GA in free (○) and immobilized (●) form. Temperature range is for 80-100°C, maltotriose 10mg/ml pH 5.

Some studies in the literature have reported that immobilization could decrease the activation energy for enzyme inactivation. Kucera et al. (1982) found that free and covalently bound GA from A. niger had an activation energy of 74.2 kJ/mol and 71.4 kJ/mol, respectively, in the range of 15-50°C. Celebi et al. (1991) also reported a less than 7.5% inactivation energy decrease of immobilized GA on silicon diatomite. The decrease of activation energy for immobilized enzyme was also reported for invertase

(decreased from 31.3 to 25.4 kJ/mol) (David et al., 2006), xylanase (decreased from

51.2 to 25.7 kJ/mol) (Allenza et al., 1986), and levansucrase (decreased from 5 to 4.55 153 kcal/mol) (El-Refai et al., 2009). They all attributed the decrease of activation energy to the internal diffusion limitation caused by immobilization.

Miyamoto et al. (1973) pointed out the reasons could be the intraparticle diffusion of the substrate or the conformational change of enzymes when they investigated the activation energy of immobilized GA on Amberlite CG-50. Bahar and

Tuncel (2002) immobilized invertase on crosslinked poly(p-chloromethylstyrene) beads and found that the activation energies of free (11.84kJ/mol) and immobilized

(11.79 kJ/mol) invertase are very close. They speculated that the intraparticle diffusion resistance is low. The observation of insignificant differences between inactivation energies of entrapped and free GA may indicate that the diffusion limitation is very low for capsules when maltotriose is used as substrate.

5.3.5 Effects of pH on the co-immobilized biocatalyst system

The activity of the co-immobilized biocatalyst system of thermostable GA and

AA was evaluated as a function of pH and temperature. The entrapment and covalent binding efficiency for thermostable GA and AA are 52.5 ± 2.7% and 53.2 ± 4.7%, respectively.

Four grams of capsules included approximately 0.16 mg AA and 0.2 mg GA.

The co-immobilized system was tested using soluble potato starch (10 mg/ml, in acetate buffer with 0.25 mM CaCl2) as a function of pH (Figure 5.8). The optimum pH for activity in term of glucose production was found to be 5.5. Activities at pH 5 and pH 5.5 appeared to be not significantly different (two sample t-test with a p-value =

0.32 > 0.05). The recombinant GA entrapped in calcium alginate capsules had an 154 optimum pH 5-5.5. The co-immobilized GA and AA showed a similar pH activity profile in comparison with free mixed enzyme except the optimum pH shifted 0.5 to the alkaline region. The pH shift of activity profiles depends on the charge of the support and on the immobilization methods (Arica et al., 2000). In the pH range of study, alginate is assumed to be negatively charged due to the carboxylate groups (Tee and Kaletunc, 2009). The hydrogen ion concentration is higher in the alginate, which creates a microenvironment that shifts the displacement of bound or entrapped enzyme toward alkaline pH values. This shift toward alkaline pH of immobilized enzyme on negatively charged support also has been reported by Arica et al. (2000), Tumturk et al. (2000), Kumar et al. (2006), and Tee and Kaletunc (2009). For entrapped GA, the optimum pH did not significantly shift to the alkaline region (pH 5 and pH 5.5 have close activities for both free and entrapped GA) as shown in Figure 5.4. For covalently bound AA, the optimum pH shifted from 5.5 to 6 (Tee and Kaletunc, 2009). For the co-immobilized GA and AA, the pH shifted from 5 to 5.5. These results indicate that the support (calcium alginate) charge plays an important role in enzyme activity change at various pHs.

155

120

100

Relative rate [%] 40

0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Figure 5. 8 Influence of pH on the free mix (○ normalized by the activity at pH 5 ) and co- immobilized (●normalized by the activity at pH 5.5) system.

5.3.6 Effects of temperature on the co-immobilized biocatalyst system

The optimum reaction temperature for co-immobilized GA and AA is 95°C , as shown in Figure 5.9. The optimum reaction temperature for covalently bound thermostable AA is 95°C (Tee and Kaletunc, 2009). For thermostable immobilized

GA, 95°C was also found to be the optimum temperature. The combined effect leads to an optimum temperature of 95°C for the co-immobilized biocatalyst system. The covalently bound AA first degrades starch to oligosaccharides. Then the oligosaccharides diffuse through the capsule boundary and are hydrolyzed further by 156

GA inside the capsule. The free mixed thermostable GA and AA (0.16/0.2) showed an optimum temperature of 90°C in terms of glucose production (Figure 5.9). The optimum temperature of free mixed GA and AA is the same as the free thermostable

GA (Figure 4.12). This may indicate that the thermostable GA is the rate-limiting step for total activity with respect to glucose production at the GA and AA ratio of

0.16/0.2. Compared to the free mixed biocatalyst system, the co-immobilized system improved the optimum temperature from 90 to 95°C . Park et al. (2005) reported co- immobilized GA and AA on DEAT cellulose with a ratio of 3/1 increased the optimum temperature from 50 to 60°C . They speculated that the covalent bonding reduces the conformational flexibility of the enzyme and leads to the increase of optimum temperature.

157

110

100

Relative rate [%]

30 80 85 90 95 100 105 110 115

o Temperature [ C ]

Figure 5. 9 Influence of temperatures on the co-immobilized (● normalized by the activity at 95°C ) and free mix (○ normalized by the activity at 90°C ) enzymes (thermostable GA and AA) system. Immobilization of enzymes can improve the thermal stability by decreasing the conformational flexibility through the forced interaction between support and enzymes

(Unsworth et al., 2007).

5.3.7 Michaelis-Menten kinetics of co-immobilized thermostable GA and

Kinetic parameters of the co-immobilized system and the free mixed enzyme system were determined in the stirred batch tank reactor by using soluble starch (0-75 mg/ml) at pH 5 with 0.25 mM CaCl2. Michaelis-Menten kinetics model was fitted to data (R2 = 0.99) (Figure 5.10). The calculated Michaelis-Menten kinetics parameters

158 are shown in Table 5.2. The lower Km indicates the decrease of affinity or change of enzyme conformation during immobilization. The Km of the co-immobilized GA and

AA system was determined to be approximately 5.6-fold higher than the free mixed enzymes. Tee and Kaletunc (2009) have found that the Km value of first use of covalently bound AA is about 2.5-fold higher than free AA. If the substrates have low accessibility to the enzyme active sites, a Km increase is expected (Chaplin and Bucke,

1990; Arica et al., 2000). For the co-immoblized system, two enzyme affinities have to be considered, the affinity of AA to starch and the affinity of GA to oligosaccharides.

15 Reaction rate Reaction

10 [mg glucose/(min*ml*mg [mg enzyme)]

0 0 10 20 30 40 50 60 70 80 Soluble Starch [mg/ml]

Figure 5. 10 Michaelis-Menten kinetics of free mixed (○) and co-immobilized (χ) GA and AA (0.16mg/0.2mg) system in the substrate (starch) range of 0-75mg/ml. Govindasamy ea al.(1992) reported the oligosaccharide distribution using thermostable

AA (Termamyl 120L) for Sago starch hydrolysis at 40°C and 90°C . The hydrolysis products included DP2(64.6%), DP3(17.4%), DP5(9.33%), and DP9(8.68%) at 40°C

159 after 24 hours reaction and at 90°C after 5 hours, the product had DP2 (48.3%), DP4

(9.18%), and DP6 (42.5%), with an absence of DP3 and DP5. The oligosaccharides affinity change for GA due to immobilization may have contributed to the overall Km leading to a 5.6-fold decrease instead of 2.5-fold observed for only AA covalent binding.

Table 5. 2 Michaelis-Menten kinetics parameters in the substrate range of 0~75m/ml

Substrate (starch) Km Vmax [mg/ml] [mg glucose/(ml*min*mg protein)] Mix free enzymes 1.13 ± 0.17 21.24 ± 0.53 Co-immobilized 6.37 ± 0.75 4.29 ± 0.14 enzymes

For maximum starch degradation rate Vmax, Tee and Kaletunc (2009) reported that Vmax of first use for covalently bound AA was approximately 90.6% of free AA.

In this case and in terms of glucose production, the immobilization resulted in 20% of free form. For the co-immobilized system, the oligosaccharides (the product of starch and AA) have to diffuse through the membrane of the capsule and then access the active site of GA. Furthermore, the produced glucose needs to diffuse out of the capsule membrane. The two diffusion processes will slow down the reaction rate and the combined effect will appear as reduced Vmax. For the maltotriose, the diffusion limitation was expected to be low (the activation energy of entrapped GA and free form are similar). All of this may indicate that the co-immobilization system has a reaction-limiting step by GA for the step of converting oligosaccharide to glucose and most likely for the step of diffusion of oligosaccharides through the capsule wall. The

160 decrease of Vmax and increase of Km have been also reported for AA immobilization

(Arica et al., 1995; Tumturk et al., 2000; Hasirci et al., 2006; Tee and Kaletunc, 2009) and GA immobilization (Uhlrich et al., 1996; Arica et al., 1998; Arica et al., 2000;

Shkutina et al., 2005; Kok et al., 2011). The kinetic parameters Vmax and Km undergo change compared to the corresponding counterpart of the free form due to several causes, such as enzyme structure change caused by the specific interaction between the enzyme and the support during immobilization, substrate accessibility to active sites caused by the support, and diffusion limitation through the support (Uhlrich et al.,

1996; Kok et al., 2011). The active sites of enzymes (in this case AA and GA) need to be accessed by the substrate for catalytic reaction. When enzyme is immobilized on the surface, an orientation for access of substrate is created; the surface of the support creates a barrier, especially for the macromolecular substrates such as starch

(Bickerstaff, 1997).

5.3.8 Comparison of reusability of mixed immobilized and co-immobilized

GA and AA

Although the co-immobilized system has a lower maximum rate for glucose production compared to the free mixed enzyme system (the reaction rates of mixed free enzyme versus inactivation time are shown in Figure 5.11a), the advantage of the immobilized enzyme system is it can be used multiple times. The usability of co- immobilized thermostable GA and AA for high temperature starch hydrolysis up to eight times is shown in Figure 5.11b. Each use was 10 minutes long at 95°C , so after

161 eight usages, the total reaction time was considered to be 80 minutes. A mixed immobilized biocatalyst system was also tested for usability in the same time scale

(Figure 5.11c). The inactivation rate constant of free mixed GA and AA was approximately six times faster (based on the slope of first five usages) than co- immobilized and mixed individually immobilized biocatalysts. Co-immobilized and mixed individually immobilized GA and AA had similar inactivation rate constants.

Both of the co-immobilized and mixed individually immobilized enzymes showed a two- step inactivation behavior. The two-step behavior was also reported by Tee and

Kaletunc (2009). During covalent binding of AA, the support surface also adsorbed

AA. Mainly during the first three uses at 95°C , the adsorbed AA leaked into the reaction medium and was lost during washing between usages. The leakage of AA was confirmed by comparing the starch degradation rate for first and second usage of co- immobilized enzyme system by another set of data. It was found that AA activity lost

59.1 % between first and second usage. The result was consistent with Tee and

Kaletunc (2009) reporting a loss of 57% of relative activity between first and second usage of covalently bound AA on calcium alginate beads. The area between the two slopes (Figure 5.11 b) indicates the rate loss caused by AA leakage.

162

(a) 14 12

Reaction rate [mg glucose/(min*ml per mg enzymes] 8

6 0 20 40 60 80 Time [min]

2.6

2.4

2.2

2.0

1.8 (b) 1.6

1.4

1.2

Reaction rate Reaction [mg glucose/(min*ml per mg enzymes)] 1.0

0.8 0 10 20 30 40 50 60 70 80 Time [min]

2.6

2.4

2.2

2.0

1.8 (c) 1.6

1.4

1.2

Reaction rate per mg enzymes)] [mg glucose/(min*ml 1.0

0.8 0 10 20 30 40 50 60 70 80 Time [min] Figure 5. 11 Reusability and inactivation rates of free (▲) in Figure 5.11 (a), Reusability and inactivation rates of co-immobilized (●) and mixed individually immobilized enzymes (○) in Figure 5.11 (b)(c). 1 gram of capsules, 3ml of 10mg/ml starch at pH 5.5 and 95°C .

163

After 80 minutes, the free mixed AA and GA kept a rate of 7.3mg glucose/(min*min) per mg enzyme. The initial glucose production rate of free mixed

AA and GA was 17.52 mg glucose/(min*min) per mg enzymes. The glucose production rate of the first-use co-immobilized system was 2.27 mg glucose/(min*min) per mg enzymes. The inactivation rate of free mixed enzyme was faster than co-immobilized enzyme, indicating that the co-immobilized system was more stable. By using it eight times, the co-immobilized enzyme system produced approximately the same amount of glucose as one-time use of free mixed GA and AA.

After six uses, the co-immobilized GA and AA retained 61% of initial activity for glucose production. For covalently bound AA, 10% of initial activity (starch degradation) remained (Tee and Kaletunc, 2009).

5.3.9 Starch hydrolysis in a continuous stirred tank

Potato starch (15mg/ml, pH 5.5, 0.025 mM CaCl2, at 95°C ) was pumped into a

CSTR with a flow rate of 11 ml/min. The starch and glucose concentration change with time is shown in Figure 5.12.

164

g/ml]

µ [

30 (a) 20

Glucose concentrationGlucose 0 0 20 40 60 80 100 120 Time [min]

12 g/ml]

m 10

[

8 (b) 6

0 Starch concentration 0 20 40 60 80 100 120 Time [min]

Figure 5. 12 CSTR glucose concentration (a) change by using co-immobilized (♦) and mixed individually immobilized (◊) enzymes. Starch concentration (b) change by using co-immobilized (●), mixed individually immobilized (∇) biocatalysts (GA and AA) and blank capsules (■). 15mg/ml starch, 11ml/min flow rate at 95°C . 165

The glucose production of the co-immobilized enzyme system was higher than the mixed individually immobilized capsules. Using fewer capsules is preferable because it can improve the volumetric efficiency by increasing the activity per volume of support (Cao, 2005). Starch degradation and glucose production after 109 minutes of continuous processing are summarized in Table 5.3.

Table 5. 3 Summary of immobilized GA and AA performance in CSTR

Initial starch Flow rate Enzyme Starch Glucose Glucose concentration system degradation production yield/starch feed [mg/ml] [ml/min] [g] [mg] [mg/g] 15# 11 Co- 14.4±0.9 87.7±0.5 4.88±0.04 immobilized 15# 11 Mixed 13.1±0.3 84.7±0.7 4.71±0.06 immobilized capsules 100## 20 Co- 18.7±0.8 112.9±1.6 1.03±0.02 immobilized #17.98 g of starch theoretically can produce 19.98 g glucose, 109 minutes of reaction; ##110 g of starch theoretically can produce 122.2 g glucose, 55 minutes of reaction.

There were 0.16 mg of AA and 0.2 mg of thermostable GA in the CSTR. It would be expected that 0.16 mg of free AA would degrade about 59.34 grams of starch in 109 minutes according to the correlation between the AA concentration and starch degradation rate (Tee, 2008). For 0.2 mg of thermostable GA in a 400-ml reactor, the concentration was 0.005 mg/ml and the corresponding glucose production rate was 10.38 µg/(ml*min), according to the relation between the GA concentration and glucose production rate (Appendix E). It would be expected that the total glucose produced in 109 minutes using a 400-ml reactor would be 452.6 mg for 0.2 mg free thermostable GA when using maltotriose as substrate. In reality, a lower amount of

166 glucose might be produced because maltotriose had the highest substrate specific activity.

Both the co-immobilized and the mixed individually immobilized enzyme systems showed a glucose concentration peak. This could be the thermal inactivation of GA and AA or substrate inhibition to AA or GA or products inhibition to AA.

Figure 5.13 shows a starch-glucose concentration change for 100 mg/ml with 20 ml/min flow rate. This indicated that, when the starch concentration increased, the product concentration peak appeared early (around10-15 minutes). For 15 mg/ml starch, the glucose concentration peak was around 50 minutes (Figure 5.12 a). This could indicate that the product or substrate inhibition happens faster when the substrate or glucose concentration peaks in a shorter time.

60 180 160 50 g/ml] 140 µ 40 120 100 30 80 20 60 40 10 20

0 0 Starch concentration [mg/ml] concentration Starch 0 10 20 30 40 50 60 [ concentrationGlucose Time [min]

Figure 5. 13 CSTR starch ( ■ )and glucose ( ♦ ) concentration change by using co- immobilized enzyme in 100mg/ml starch with a flow rate of 20ml/min.

167

More research needs to be performed to understand the substrate and production inhibition in the co-immobilized GA and AA system, such as studying the starch concentration effects on AA, investigating the oligosaccharides profiles, studying the effects of the concentration of resultant oligosaccharides on GA, and investigating the effects of glucose concentration on GA activity.

5.3.10 Comparison of CSTR with batch reactor

Buchholz et al. (2005) suggested a space-time yield calculation to compare the

CSTR and batch reactor. However, many parameters were unknown and needed to be estimated. CSTR can be compared with a batch reactor by looking at glucose yield using the same amount of enzyme for the same reaction time. For CSTR, 87 mg of glucose was produced by 0.36 mg of total enzyme in 109 minutes. For the batch reactor, glucose production rates for consecutive batches of co-immobilized enzyme were reported in Chapter 5.3.6. Hence, the cumulative glucose in the batch reactor for

0.09 mg enzyme in 109 minutes can be estimated as shown in Figure 5.14. The final amount is 40.6 mg. Using the same GA and AA concentration as were used in the

CSTR system, the glucose yield of the batch reactor is estimated to be 3.69 mg glucose/g starch versus 4.88 mg glucose/g starch in CSTR (Table 5.3).

168

45 2.5 40 35 2 30 1.5 25 20 1

15 Reaction rate 10 y = -0.0175x + 2.3306 0.5 5 R² = 0.99

0 0

Cumulative glucose production [mg] production glucose Cumulative

[mg glucose/(min*ml) per mg[mg enzyme] per glucose/(min*ml) 0 20 40 60 80 100 120 140 Time [minutes]

Figure 5. 14 The cumulative glucose production of co-immobilized capsules in batch reactor, rate (♦ data were from Figure 5.11b) and cumulative production (■). The batch reactor calculations were performed based on the initial reaction rate. Therefore, the calculation may have overestimated glucose production. These results imply that the CSTR could be a better choice for starch hydrolysis at high temperature.

For batch system, between two usages, the capsules were reinstated in 100 mM

CaCl2 at room temperature. The reinstatement of the capsules could help the stabilization of the calcium alginate capsule system. However, for the continuous system, the calcium alginate gel capsules were used for 109 minutes without reinstatement. This may indicate that co-immobilized enzyme system may not need reinstatement after each batch.

169

5.3.11 Comparison of thermostable GA performance with industrial GA

For the co-immobilized enzymes in a batch reactor, the thermostable GA/AA and industrial GA/AA had the same enzyme to starch ratio (w/w) with a value of

0.0032 (mg/mg). The thermostable system (0.05 mg GA and 0.044 mg AA in 30 mg of starch) produced 32.58 mg of glucose in 72 minutes. Data are from Figure 5.11. The glucose yield was 155.14 mg glucose/gram of starch.

The industrial low temperature (65°C ) enzyme system (0.4 mg GA and 0.43 mg AA in 250 mg of starch) produced 30.14 mg in 72 minutes (Figure 3.14). The glucose yield was 20.27 mg glucose/gram of starch.

The results indicated that, for the same enzyme/starch ratio in a batch reactor, the co-immobilized thermostable enzyme system can have a 7.6-fold of yield compared to co-immobilized industrial low temperature enzyme system (as described in Chapter 3).

The recommended Liquozyme X (AA) dosage level for liquefaction is from

0.3 to 0.55 kg/ton starch; 0.55kg/1,000kg starch is equivalent to 0.5mg AA/gram of starch (Table 5.4). In the co-immobilized thermostable system, the ratio is 1.6 mg

AA/gram of starch for each usage. Considering six usages, the enzyme/starch ratio is

0.27 mg AA/gram of starch. This indicates the AA reaches the recommended dosage.

It may also imply the conversion of starch to oligosaccharides is fast enough to provide substrates to GA. However, the recommended dosage of GA is from 0.05 to

0.06 (% w/w) or 200 U/kg starch; it is equivalent to 0.5-0.6 mg GA/gram of starch.

For the CSTR, the ratio of GA to starch is 0.2 mg GA/17.98 gram of starch. It is 54

170 times less than the recommended level. The results indicate that the potential yield could be 263 mg glucose/gram of starch. With the recommended GA and AA dosage,

26.3% of conversion can be reached in 2 hours.

Table 5. 4 Enzyme/starch ratio effects on glucose yields

Initial Starch AA GA Starch Starch Glucose Glucose starch /starch /starch degrada conver production yield/starch concentr- -tion -sion feed ation [mg/ml] [g] [mg/g] [mg/g] [g] % [mg] [mg/g] 0.3- 0.5- 0.55 0.6 15# 17.98 0.009 0.011 14.4±0. 80 87.7±0.5 4.88±0.04 9 100## 110 0.0015 0.0018 18.7±0. 17 112.9±1.6 1.03±0.02 8 #17.98 g of starch theoretically can produce 19.98 g glucose, 109 minutes of reaction; ##110 g of starch theoretically can produce 122.2 g glucose, 55 minutes of reaction.

Compared to the AA recommended dosage, 0.009 mg/g AA used in this study was much lower. However 81% of the starch was degraded. This indicated that the rate limiting step was the glucose production by GA. Increasing the AA dosage could decrease the DE (degree of dextrin equivalent), which can improve the glucose production because the thermostable GA shows higher activity toward the low DP oligosaccharides. It was also observed that the starch degraded 100% in 5 minutes when using 1.6 mg AA/g starch. When the AA dosage decreased to 0.0015, the starch conversion was only 17%.

171

5.4 Conclusion

A method for co-immobilizing the thermostable GA and AA was developed and verified. The chemical reagents used during the co-immobilization process did not reduce GA activity. The encapsulation efficiencies for GA and AA were 52.5 ± 2.7 and 53.2 ± 4.7, respectively. The immobilization shifted all the pH values above optimum pH, with a value of 0.5 to alkaline region. Also, immobilization improved the optimum temperature of 5°C and led to a wider optimum temperature range. The GA and AA thermal stability can be improved by co-immobilization. The Michaelis-

Menten results show that the immobilization will decrease the enzyme affinity to substrate. Compared to the free enzyme, the maximum glucose production rates also decreased.

The co-immobilized thermostable GA and AA can be applied for starch hydrolysis either in a batch reactor or in aCSTR. It was found that the glucose yield for the CSTR is higher than the batch system.

The co-immobilized thermostable GA and AA in calcium alginate gel capsules represents a potential system for improving starch hydrolysis process by increasing temperature, reusing enzyme, minimizing process steps, and saving labor and costs.

172

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178

Chapter 6 Future work and Investigations

Future efforts can dedicate toward three directions: improving total activity yield, optimizing continuous reaction conditions and analyzing the structure-function relation.

Currently, the total activity yield is about 7%. Although this work improved yield a little bit compared to the work of Kim et al.,(2004), more work can be tried to increase the yield such as improving the OTR for growth, stimulating expression by chemicals, expressing the gene into an extracellular enzyme system (e.g. Pfenex system), and transferring the gene to animal (e.g. goat).

With respect to the continuous system, work can be done towards optimizing process parameters, such as flow rate, starch initial concentration, AA/GA ratio and series reactors.

In terms of structure-function, efforts can start from accumulating knowledge on structure of GA from archaea. Although there is still unclear what underlies the enzyme thermostability of hyperthemophilic archaea, work can towards the comparison of primary, secondary and tertiary structures among fungi, bacterial and archaea. Except the multiple sequence alignment for primary amino acid sequence, modeling of 3D structure and determining the active sites or possible binding sites

179 should help to explain the thermostabilty, specific activity and reaction mechanism of this recombinant GA from S. solfataricus P2.

Also increasing the capsule strength for longer operation at high temperature, shortening the reinstating time, and finding the proper stabilizer for recombinant glucoamylase are all attractive research aspects to reach the final goal of improving starch hydrolysis efficiency at high temperature.

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Appendix A: Starch iodine test calibration curve

1.8

1.6 y = 0.0514x + 0.0019 R² = 0.9997 1.4

1.2

0.8

Net Abs 620nm @Abs Net 0.6

0.4

0.2

0 0 5 10 15 20 25 30 35 Starch concentration [mg/ml]

210

Appendix B: Glucose test calibration curve

1.8 y = 0.0366x + 0.0182 1.6 R² = 0.9988 1.4 1.2 1 0.8 0.6 0.4

Net Abs @ 340nm wave length wave 340nm @Abs Net 0.2 0 0 10 20 30 40 50 Concentration in cuvett [µg/ml]

211

Appendix C: Bradford test calibration curve

0.35

0.3 y = 0.0289x 0.25 R² = 0.9884 0.2 0.15 0.1

0.05 Net Abs@595nm Net 0 0 2 4 6 8 10 12 Concentration [µg/ml]

Bradford semi-micro method (1ml sample+1ml reagent), for the range of 0-10 µg/ml

1.2 1 y = 0.7412x R² = 0.9882 0.8 0.6 0.4

0.2 Net Abs 595nm @Abs Net 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 Concentration [mg/ml]

Bradford standard method (0.1ml sample+3ml reagent), for the range of 0-1.5 mg/ml 212

Appendix D: Glucose production rate versus GA concentration

100 90 80 70 y = 3153x 60 R² = 0.9891 50 40

[µg/(ml*min)] 30

20 Glucose production rate rate production Glucose 10 0 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 GA concentration [mg/ml]

Glucose production rate versus GA concentration. GA is from Aspergillus niger, at pH 5.5, 65°C , 10 mg/ml maltose as substrate.

213

Appendix E: Glucose production rate versus thermostable GA concentration

9000

8000

7000 y = 20762x 6000 R² = 0.9991

5000

4000

Glucose rate [µg/(ml*min)] rate Glucose 3000

2000

1000

0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Thermostable GA concentration in reaction medium [mg/ml]

Tests were run at pH 5.5, 95°C , 10 mg/ml maltotriose as substrate.

214

Appendix F: Modified Lowery protein test calibration curve

0.08

0.07

0.06 y = 0.0054x R² = 0.9898 0.05

0.04

0.03

Net Abs Abs 750nmat Net 0.02

0.01

0 0 2 4 6 8 10 12 14 Protein concentration [µg/ml]

215

Appendix G: Amino acid sequence of thermostable GA from S. solfataricus P2

ORGANISM: Sulfolobus solfataricus P2

Archaea; Crenarchaeota; Thermoprotei; Sulfolobales; Sulfolobaceae; Sulfolobus.

1 mrvssigngr mlinfdekgr ivdiyypyig menqtsgnpi rlaiwdkdkk vasldedwet 61 tvlyideanm veirsdvkel glsllsynfl dsddpiymsi vkiannenns rnikvffihd 121 inlysnpfgd tafydplsls iihykskryl afkvfttvst lseynigkgd ligdiydgnl 181 glngiengdv nssmgieini dpnsylklyy vivadrnleg lrqkirkinf anvetsftlt 241 ymfwrnwlkk nklfrnnlmq dikrvydvsl fvirnhmdvn gsiiassdfs fvkiygdsyq 301 ycwprdaaia ayaldlagyk elalkhfqfi sniansegfl yhkynpnttl asswhpwyyk 361 gkriypiqed etalevwaia shyekyedid eilplykkfv kpalkfmmsf meeglpkpsf 421 dlweerygih iytvstvyga ltkgaklayd vgdeilsedl sdtsgllkgm vlkrmtyngr 481 fvrrideenn qdltvdssly apfffglvna ndkimintin eiesrltvng giiryendmy 541 qrrkkqpnpw iittlwlsey yatindknka neyikwvinr alptgflpeq vdpetfepts 601 vtplvwshae fiiainklln hi

The results (OSU DNA facility) is exactly match the database in protein bank (NCBI)

216

Appendix H: Protein purification with Zn column in FPLC

Fraction 1

Fraction 2

217

Appendix I: Thermostable GA cost level

Cost

Thermostable GA Concentration in Broth (g/L) Broth in Concentration

Price ($ per kg) C&EN July 1988

218