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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.
ii
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
3
11
12 13
16 17
39
39 39 40 41 42 43 49 50 50
iii
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
IV
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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
V
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
Vi
ILLUSTRATIONS
l
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
0
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
vii
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
.
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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Viii
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
iX
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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. . . . . . . . . . . . . . . . . . . . . .
xi
6
14
19
20
22
24
32
51
56
57
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
Xii
r
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
Xiii