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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

  • I I

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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 Produ

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