PROGRAM SRI INTERNATIONAL

Menlo Park, California

Abstract

Process Economics Program Report No. 139

ENZYME TECHNOLOGY

(January 1981)

The report presents the technologies of enzyme production, recov-

ery, purification, immobilization, and application. The process

economics for these technologies are illustrated by:

l The production of a bran mold and a bran-amylase product by a surface cultivation process*

l The production of a bacterial alpha-amylase preparation by a submerged cultivation process.

l The immobilization of an enzyme by an entrapment/cross-linking process.

s The application of an immobilized enzyme in the production of high fructose syrup.

.

Included also is a brief review of some of the patents and publications

on enzyme-catalyzed chemical conversions.

PEP '79 WSF

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Report No. 139

ENZYME TECHNOLOGY

by WING SIEN FONG

January 1981

A private report by the

PROCESS ECONOMICS PROGRAM

Menlo Park, California 94025

For detailed marketing data and information, the reader is

referred to one of the SRI programs specializing in marketing

research. The CHEMICAL ECONOMICS HANDBOOK Program covers

most major chemicals and chemical products produced in the

United States and the WORLD PETROCHEMICALS Program covers

major hydrocarbons and their derivatives on a worldwide basis.

In addition, the SRI DIRECTORY OF CHEMICAL PRODUCERS services

provide detailed lists of chemical producers by company, prod-

uct, and plant for the United States and Western Europe.

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CONTENTS

1 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . 1

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2 SUMMARY...........................

3 INDUSTRY STATUS .......................

Product Forms. ....................... Applications. ........................ U.S. Governmental Regulations on the Use of Certain Enzymes. ..................... Major Producers .......................

4 TYPICAL PROPERTIES OF ENZYMES . . . . . . . . . . . . . . . . 27

5 ENZYME CATALYSIS ......................

Mechanism ......................... Specificity ........................ Inhibition. ........................ Cofactors ......................... Allosterism and Allosteric Enzymes. ............ Kinetics ......................... Effect of Temperature ................... Effect of pH ........................ Effect of Immobilization. .................

6 COMMERCIAL ENZYME PRODUCTION PROCESSES. . . . . . . . . . . . 59

Animal Enzymes. . . . . . .................. 59 Pancreatin. . . . . . . .................. 60 Pepsin . , . . . . . . .................. 60 Rennin (or rennet), . . .................. 61 Catalase. . . . . . . . .................. 61

Plant Enzymes . . . . . . .................. 66 Malt Amylases . . . . . .................. 66 Papain . . . . . . . . .................. 67 Bromelin. . . . . . . . .................. 68 Ficin . . . . . . . . . .................. 69

Microorganism Enzymes . . .................. 74 General Aspects . . . , .................. 74 Commercial Processes. . .................. 82 Surface Cultivation . .................. 85 Submerged Cultivation .................. 88

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12 13

16 17

39

39 39 40 41 42 43 49 50 50

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CONTENTS

7 ENZYME RECOVERY AND PURIFICATION. ............... 91

Precipitation ........................ 91 Salting Out ......................... 96 Addition of Organic Solvents. ................ 97 Addition of High Molecular Weight Substances. ........ 98 Addition of Metallic Ions or Complexes. ........... 98 Addition of Specific Agents ................. 99 Addition of Carriers. .................... 99

Crystallization ........................ 99 Extraction .......................... 100 Electrophoresis ........................ 100 Chromatography. ........................ 101 Adsorption .......................... 101 Ion Exchange............................ 101 Ultrafiltration (Gel Filtration). ............... 102 Biochemical Interaction (Affinity Chromatography) ....... 103 Lyophilization (Freeze Drying) ................ 103 Drying .......................... ..lO 4 Dialysis. ........................... 104 Activated Carbon Treatment. .................. 104 Concentration ......................... 104 Desalting.................................................. 105 Disruption of Microorganism Cells (For Intracellular Enzyme Recovery) .............. 105

8 ENZYME PRODUCTION BY SURFACE CULTIVATION, A MOLD BRAN PRODUCT AND A BRAN-AMYLASE PRODUCT .............. 107

Process Description ...................... 107 MoldBran..........................lO8 Bran-Amylase Product. .................... 112

Process Discussion. ...................... 114 Cost Estimates, ........................ 115 MoldBran........................115 Bran-Amylase Product. .................... 116

9. ENZYME PRODUCTION BY SUBMERGED CULTIVATION, A BACTERIAL ALPHA-ADYLASE PRODUCT. .............. 129

Process Description ...................... 129 Process Discussion. ...................... 135 Cost Estimates. ........................ 137

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CONTENTS

IMMOBILIZATION OF ENZYMES. .................. 147

Immobilization Methods .................... 148 Adsorption ......................... 148 Entrapment ......................... 161 Microencapsulation ..................... 164 Covalent Binding and Intermolecular Cross-Linking. ..... 164

Immobilization Supports. ................... 170 Commercial Immobilization Processes. ............. 172 Commercial Applications of Immobilized Enzymes ........ 175 Production of High Fructose Syrup. ............. 175 Production of L-Amino Acids. ................ 175 Production of 6-Aminopenicillanic Acid ........... 178 Hydrolysis of Lactose in Whey. ............... 178

Commercial Applications of Immobilized Microorganism Cells . . 181 Enzyme Immobilization Based on Entrapment/ Cross-Linking Technology ................... 182 Process Description. .................... 182 Process Discussion ..................... 187 Cost Estimates ....................... 188

11 A COMMERCIAL APPLICATION OF AN IMMOBILIZED ENZYME, HIGH FRUCTOSE SYRUP PRODUCTION ............ 195

Review of Processes. ..................... 196 Process Description. ..................... 204 Process Discussion ...................... 210 Cost Estimates ........................ 210

12 CHEMICALS BY ENZYMATIC CONVERSION. .............. 217

Acrylic Acid or Acrylate from a Propionate or Propionic Acid (457294, 468001). ............... 217 Acrylsmide or Methacrylamide from Acrylonitrile or Methacrylonitrile (457318). ................ 218 Desulfurization of Petroleum (457345) ............ 218 Diepoxides from Diolefins (457295) .............. 218 Epoxides by Olefin Fermentation (457288) ........... 219 Epoxides and Glycols from Alkenes (457292) .......... 221 1-Epoxy-Alkanes from 2-20 Carbon u-Olefins or 4-20 Carbon a,w-Diolefins (457381) .................... 222 Hydrogen Sulfide from Gypsum (457296). ............ 224 Ketones and Secondary Alcohols from 3-6 Carbon Alkanes and Ketones from 3-6 Carbon Secondary Alcohols (488006). ..... 224 Others ............................ 225

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CONTENTS

APPENDIX A

APPENDIX B

DESIGN AND COST BASES. . . . . . . . . . . . . . . 227

SOME OF THE MORE COMMON TERMS, ABBREVIATIONS, AND SYMBOLS USED IN THE ENZYME INDUSTRY. . . . . . 231

APPENDIX C DEFINITION OF ENZYMATIC ACTIVITY AND SOME OF THE MORE COMMONLY USED UNITS. . . . . . . . . . 235

APPENDIX D ENZYME REPOSITORIES . . . . . . . . . . . . . , . 239

CITED REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . 241

PATENT REFERENCES BY COMPANY. . . . . . . . . . . . . . . . . . . . 253

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ILLUSTRATIONS

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5.1

6.1

6.2

6.3

6.4

6.5

6.6

6.7

6.8

6.9

6.10

8.1

8.2

8.3

8.4

8.5

I 8.6

8.7

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Graphical Determination of s and VMAE in the Michaelis-Menten Equation, . . . . . . . . . . . . . . . . . 47

Production of Pancreatin . . . . . . . . . . . . . . . . . . 62

Production of Pepsin . . . . . . . . . . . . . . . . . . . . 63

Production of Rennin . . . . . . . . . . . . . . . . . . . . 64

Production of Catalase . . . . . . . . . . . . . . . . . . . 65

Productionof Malt Amylases. . . . . . . . . . . . . . . . . 70

Production of Papain . . . . . . . . . . . . . . . . . . . . 71

Production of Bromelin . . . . . . . . . . . . . . . . . . . 72

Production of Ficin . . . . . . . . . . . . . . . . . . . . 73

Production of Fungal Enzymes by Surface Cultivation. . . . . 86

Production of Enzymes by Submerged Cultivation . . . . . . . 89

Enzyme Production by Surface Cultivation, A Mold Bran Product and a Bran-Amylase Product . . . . . . . 257

Enzyme Production by Surface Cultivation, A Mold Bran Product Effect of Bran Price and Changes in Labor, Capital, or Utilities Cost on Production Cost . . . . . . . . . . . . 119

Enzyme Production by Surface Cultivation, A Mold Bran Product Effect of Product Yield on Production Cost . . . . . . . . . 120

Enzyme Production by Surface Cultivation, A Mold Bran Product Effect of Operating Level and Plant Capacity on Production Cost . . . . . . . . . . . . . . . . . . . . . 121

Enzyme Production by Surface Cultivation, A Bran-Amylase Product Effect of Bran Price and Changes in Labor, Capital, or Utilities Cost on Production Cost . . . , . . . . . . . . 125

Enzyme Production by Surface Cultivation, A Bran-Amylase Product Effect of the Amount of Enzyme Concentrate Recovered from the Dry Bran Weight Loss on Production Cost . . . . . . 126

Enzyme Production by Surface Cultivation, A Bran-Amylase Product Effect of Operating Level and Plant Capacity on Production Cost . . . . . . . . . . . . . . . . . . . . . 127

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ILLUSTRATIONS

9.1

9.2

9.3

9.4

9.5

10.1

10.2

10.3

10.4

10.5

10.6

10.7

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Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product. . . . . . . . . . . . . . 259

Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Effect of Raw Material Prices on Production Cost . . . . . . 143

Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Effect of Gypsum Dilution on Production Cost . . . . . . . . 144

Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Effect of Changes in Utilities, Capital, or Labor Cost and in Enzyme Yield on Production Cost . . . . . , . . . . . 145

Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Effect of Operating Level and Plant Capacity on Production Cost . . . . . . . . . . . . . . . . . . . . . 146

Some of the More Common Covalent Binding Reactions Involved in the Immobilization . . . . . . . . . . 166

Flow Diagram for Continuous Production of L-Amino Acid By Immobilized Aminoacylase. . . . . . . . . . . . . . . . . 177

A Continuous Lactose Hydrolysis Process Using Immobilized Lactase. . . . . . . . . . . . . . . . . . 180

Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology. . . . . . . . . . . . . 261

Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Effect of Gelatin and Glutaraldehyde Prices and Changes in Labor, Capital, and Utilities Cost on Conversion Costs. . 192

Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Effect of Operating Level and Plant Capacity on Conversion Cost . . . . . . . . . . . . . . . . . . . . . 193

Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Effect of Soluble Enzyme Cost on Conversion Cost . . . . . . 194

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ILLUSTRATIONS

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11.1 A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production. . . . . . . . . . . . . . . 263

11.2 A Commercial Application of An Immobilized Enzyme, High Fructose Syrup Production Effect of Changes in Immobilized Enzyme, Miscellaneous Chemicals, Utilities, Capital, or Labor Cost on Conversion Cost. . . . . . . . . . . . . . 215

11.3 A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production Effect of Operating Level and Plant Capacity on Conversion Cost. . . . . . . . , . . . . . . . . . . . . 216

12.1 Epoxides of Olefin Fermentation . . . . . . . . . . . . . . 220

12.2 Epoxidation of Unsaturated Hydrocarbons by Immobilized Cells. . . . . . . . . . . . . . . , . . . . 223

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2.1

3.1

3.2

3.3

3.4

3.5

4.1

5.1

5.2

5.3

6.1

6.2

6.3

6.4

7.1

8.1

8.2

8.3

8.4

TABLES

Summary of Economics. . . . . . . . . . . . . . . . . . . .

Applications of Enzymes . . . . . . . . . . . . . . . . . .

Production of Some of the Principal Enzymes in Japan . . .

Some of the Principal Enzymes Produced and their Major Producers in Japan. . . . . . . . , . . . .

Some of the Principal Enzymes Produced and their Major Producers in the United States. . . . . . .

Some of the Principal Enzymes Produced and their Major Producers in Western Europe . . . . . . . .

Description of Selected Industrial Enzymes . . . . . . . .

Properties of Free (Native) and Various Immobilized Aminoacylases . . . . . . . . . . . . . . . . .

Estimated Half-Life for Selected . Immobilized Enzyme Systems. . . . . . . . . . . . . . . . .

Activity and Activity Yield for Various Immobilized Aminoacylases . . . . . . . . . . . . .

Examples of Some of the Product Inducers and Analog Inducers . . . . . . . . . . . . . . . . . . . .

Examples of Catabolite Repression of Enzymes. . . . . . . .

Some of the Better Known Microorganisms Used in Enzyme Production . . . . . . . . . . . . . . . . .

Patent Summary of Some Microorganism Enzyme Production Methods . . . . . . . . . . . . . . . . .

Patent Summary on Some Enzyme Recovery and Purification Methods. . . . . . . . . . . . . . . . . .

Enzyme Production by Surface Cultivation, A Mold Bran Product Major Equipment and Utilities Summary . . . . . . . . . . .

Enzyme Production by Surface Cultivation, A Bran-Amylase Product Major Equipment and Utilities Summary . . . . . . . . . . .

Enzyme Production by Surface Cultivation, A Mold Bran Product Total Capital Investment . . . . . . . . . . . . . . . . .

Enzyme Production by Surface Cultivation, A Mold Bran Product Production Costs. . . . . . . . . . . . . . . . . . . . . .

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32

51

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75

76

79

83

92

109

110

117

118

TABLES

8.5 Enzyme Production by Surface Cultivation, A Bran-Amylase Product Total Capital Investment. . . . . . . . . . . . . . . . . . 122

8.6 Enzyme Production by Surface Cultivation, A Bran-Amylase Product Production Costs . . . . . . . . . . . . . . . . . . . . . 123

9.1 Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Stream Flows. . . . . . . . . . . . . . . . . . . . . . . . 132

9.2 Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Major Equipment and Utilities Summary . . . . . . . . . . . 133

9.3 Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Total Capital Investment. . . . . . . . . . , . . . . . . . 140

9.4 Enzyme Production by Submerged Cultivation, A Bacterial Alpha-Amylase Product Production Costs . . . . . . . . . . . . . . . . . . . . . 141

10.1 Patent Summary on Some of the Immobilization Methods for Enzymes and Microorganism Cells . . . . . . . . . . . . 149

10.2 Physical Properties and Applications for Controlled-Pore Ceramics and Glass. . . . . . . . . . . . . 173

10.3 Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Major Equipment and Utilities Summary . . . . . . . , . . . 185

10.4 Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Total Capital Investment . . . . . . . . . . . . . . . . . 190

10.5 Enzyme Immobilization Based on an Entrapment/Cross-Linking Technology Conversion Costs. . . . . . . . . . . . . . . . . . . . . . 191

11.1 Patent Summary on Some Glucose Isomerization Processes. . . 197

11.2 A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production Major Equipment and Utilities Summary . . . . . . . . . . . 207

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TABLES

11.3

11.4

11.5

12.1

A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production Bases and Assumptions. . . . . . . . . . . . . . . . . . . . 209

A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production Total Capital Investment . . . . . . . . . . . . . . . . . . 212

A Commercial Application of an Immobilized Enzyme, High Fructose Syrup Production Conversion Costs . . . . . . . . . . . . . . . . . . . . . . 213

Typical Nutrients for the Cultivation of a Methylotropic Microorganism . . . . . . . . . . . . . . 221

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