Industrial Enzyme Applications E-Book

• Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 804

Veröffentlichungsjahr: 2019

Beschreibung

This reference is a "must-read": It explains how an effective and economically viable enzymatic process in industry is developed and presents numerous successful examples which underline the efficiency of biocatalysis.

Leseprobe

Table of Contents

Cover

Preface

Part I: Overview of Industrial Enzyme Applications and Key Technologies

1.1 Industrial Enzyme Applications – Overview and Historic Perspective

1.1.1 Prehistoric Applications

1.1.2 Growing the Scientific Basis

1.1.3 The Beginning of Industrial Applications and the Emerging Enzyme Industry

References

1.2 Enzyme Development Technologies

1.2.1 Introduction

1.2.2 Identification of Wild‐Type Enzymes

1.2.3 Enzyme Engineering

1.2.4 Impact of Enzyme Development Technologies Today and Tomorrow

Acknowledgments

References

1.3 Eukaryotic Expression Systems for Industrial Enzymes

1.3.1 Eukaryotic Enzyme Production Systems

1.3.2 Special Considerations for Working with Eukaryotic Expression Systems

1.3.3 Differences in Vector Design for Eukaryotic and Prokaryotic Hosts

1.3.4 Differences in Regulation of Gene Expression in Eukaryotes and Prokaryotes

1.3.5 Industrial Enzyme Production

1.3.6 Enzyme Production on Industrial Scale

References

1.4 Process Considerations for the Application of Enzymes

1.4.1 Biocatalyst Types Used in Industrial Processes

1.4.2 Enzyme Immobilization for Biocatalytic Processes

1.4.3 Reaction Medium Applied in Enzymatic Catalysis

1.4.4 Appropriate Reactor Types in Enzyme Catalysis

1.4.5 Assessment Criteria for Enzymatic Applications

References

Part II: Enzyme Applications for the Food Industry

2.1 Enzymes Used in Baking

2.1.1 Introduction

2.1.2 The Baking Process – The Baker's Needs

2.1.3 The Bread Quality – The Consumers' Needs

2.1.4 Trends and Opportunities for Baking Enzymes

2.1.5 Conclusion

References

2 Protein Modification to Meet the Demands of the Food Industry

2.2.1. Food Proteins

2.2.2. Processing of Food Protein

2.2.3. Enzymes in the Processing of Food Proteins

2.2.4. Food Protein Value Chain

2.2.5. Recent Enzyme Developments

2.2.6 Enzymes to Meet Future Needs

Acknowledgments

References

2.3 Dairy Enzymes

2.3.1 Introduction

2.3.2 Coagulants

2.3.3 Ripening Enzymes

2.3.4 Lactases

2.3.5 Miscellaneous Enzymes

2.3.6 New Developments

References

2.4 Enzymatic Process for the Synthesis of Cellobiose

2.4.1 Enzymatic Synthesis of Cellobiose

2.4.2 Cellobiose – Properties and Applications

2.4.3 Existing Routes for Cellobiose Synthesis

2.4.4 Enzyme Development

2.4.5 Process Development

2.4.6 Summary and Future Perspective

References

2.5 Emerging Field – Synthesis of Complex Carbohydrates. Case Study on HMOs

2.5.1 Introduction to Human Milk Oligosaccharides (HMOs)

2.5.2 Glycom A/S Technologies Toward Commercial HMO Production

2.5.3 Enzyme Development

2.5.4 Applications of the Optimized Enzymes for the HMO Profiles

2.5.5 Conclusion and Perspective

References

Part III: Enzyme Applications for Human and Animal Nutrition

3.1 Enzymes for Human Nutrition and Health

3.1.1 Introduction

3.1.2 Current Problems of Enzymes in Healthcare Business

3.1.3 Enzymes in Existing Healthcare Products

3.1.4 New Enzyme Developments in Healthcare Products

References

3.2 Enzyme Technology for Detoxification of Mycotoxins in Animal Feed

3.2.1 Introduction to Mycotoxins

3.2.2 Mycotoxin Mitigation Strategies

3.2.3 Enzyme Applications

3.2.4 FUM

zyme

®

3.2.5 Future Mycotoxinases

3.2.6 Conclusions

References

3.3 Phytases for Feed Applications

3.3.1 Phytase As a Feed Enzyme: Introduction and Significance

3.3.2 Historical Overview of the Phytase Market Development

3.3.3 From Phytate to Phosphorus: Step by Step Action of the Phytase

3.3.4 Nutritional Values of Phytase in Animal Feed

3.3.5 Phytase Application As Feed Additive

3.3.6 Effective Phytate Hydrolysis in the Upper Digestive Tract of the Animal

3.3.7 Kinetic Description of Ideal Phytases

3.3.8 Resistance to Low pH and Proteases

3.3.9 Temperature Stability

3.3.10 In lieu of Conclusion: Lessons from Phytase Super Dosing Trials

References

Part IV: Enzymes for Biorefinery Applications

4.1 Enzymes for Pulp and Paper Applications

4.1.1 Refining and Fiber Development Enzyme

4.1.2 Drainage Improvement Enzyme

4.1.3 Stickies Control Enzyme

4.1.4 Deinking Enzymes

4.1.5 Hardwood Vessel Breaking Enzyme

4.1.6 Native Starch Conversion Enzyme

4.1.7 Bleach Boosting Enzyme

4.1.8 Paper Mill Effluent Treatment Enzymes

4.1.9 Slushing Enzyme

References

4.2 Enzymes in Vegetable Oil Degumming Processes

4.2.1 Introduction

4.2.2 General Seed Oil Processes

4.2.3 Enzymatic Degumming

4.2.4 Enzymatic Degumming in Industrial Practice

4.2.5 Other Applications of Enzymes in Oil – Outlook

4.2.6 Conclusion

Acknowledgments

References

Part V: Enzymes used in Fine Chemical Production

5.1 KREDs: Toward Green, Cost‐Effective, and Efficient Chiral Alcohol Generation

5.1.1 Introduction

5.1.2 Ketoreductases

5.1.3 Cofactor Recycling

5.1.4 CodeEvolver® Protein Engineering Technology

5.1.5 Reduction of a Wide Range of Ketones/Aldehydes

5.1.6 Critical Selectivity Tools for Enantiopure Asymmetric Carbonyl Reduction

5.1.7 Examples of Improved KREDs for Improved Manufacturing

5.1.8 KREDs: Going Green and Saving Green

References

5.2 An Aldolase for the Synthesis of the Statin Side Chain

5.2.1 Introduction – Biocatalysis

5.2.2 The Aldolase DERA in Application

5.2.3 Directed Evolution and Protein Engineering to Improve DERA

5.2.4 Conclusions

Acknowledgments

References

Index

End User License Agreement

List of Tables

Chapter 1a

Table 1.1.1 Overview of enzyme classes used in different applications.

Chapter 1c

Table 1.3.1 List of common eukaryotic host microorganisms for the production of ...

Table 1.3.2 List of industrial host organisms for protein production and the res...

Table 1.3.3 Frequently used selection markers based on antibiotics for prokaryot...

Table 1.3.4 Frequently used auxotrophic systems based on data from Addgene [26] ...

Table 1.3.5 Selected examples of organisms with corresponding enzyme and yields ...

Table 1.3.6 Exemplifying list of organisms with corresponding enzymes produced, ...

Chapter 1d

Table 1.4.1 Classification of enzymes with respect to their class, availability,...

Table 1.4.2 Characteristics of resting and growing cells.

Table 1.4.3 Classification of biocatalytic systems with solid substrates and pro...

Table 1.4.4 Catalytic productivity (=turnover number, TON) for different product...

Chapter 2a

Table 2.1.1 Overview of the role of the enzymes in all processing and product qu...

Chapter 2b

Table 2.2.1 The information in this table was obtained from [2].

Table 2.2.2 Examples of enzymes active towards food proteins.

Table 2.2.3 Examples of bioactive peptides.

Chapter 2d

Table 2.4.1 Characterization of different SP and CP enzymes.

Table 2.4.2 Overview of the cellobiose phosphorylase libraries screened.

Table 2.4.3 SU/PU ratio compared to the wild‐type enzyme at different substrate ...

Chapter 2e

Table 2.5.1 Reaction kinetics of BiTF and its variants for transferase activity ...

Table 2.5.2 Characterization and comparison of different generations of engineer...

Table 2.5.3 Characterization of different candidates toward activity and selecti...

Table 2.5.4 Screened conditions for generation of the HMO profiles P2, P5, and P...

Chapter 3a

Table 3.1.1 Basic status of enzymes in each country (area).

Table 3.1.2 Population ratio of lactose intolerance.

Chapter 3c

Table 3.3.1 The main commercial phytases approved for animal feed, all from the ...

Table 3.3.2 Michaelis–Menten kinetic properties of recombinant appA phytase prod...

Chapter 4a

Table 4.1.1 Different enzymatic applications in pulp and paper manufacturing.

Table 4.1.2 Swelling effect of enzyme treatment on hardwood and softwood fiber.

Table 4.1.3 Enzyme laboratory evaluation results with softwood and hardwood pulp...

Table 4.1.4 Impact of enzyme usage on IGT printability values.

Table 4.1.5 Comparison of CSF and drainage test with different enzyme dosages on...

Table 4.1.6 Comparison of CSF and drainage test with different enzyme dosages on...

Table 4.1.7 Comparison of CSF and drainage test with different enzyme dosages on...

Table 4.1.8 Reduction of paper breaks in dryers and rewinder in 112 GSM.

Table 4.1.9 Reduction of paper breaks in dryers and rewinder in 125 GSM.

Table 4.1.10 Reduction of stickies and fluff accumulation at dryers.

Table 4.1.11 Different chemicals dosages with and without enzyme.

Table 4.1.12 Pulp brightness with and without enzyme at different stages.

Table 4.1.13 Common bleaching agents.

Table 4.1.14 Lab evaluation of bleaching enzyme.

Table 4.1.15 Benefits of bleaching enzyme application.

Table 4.1.16 Mill trial of effluent enzyme.

Table 4.1.17 Mill trial of slushing enzyme.

Table 4.1.18 Paper properties with slushing enzyme.

Chapter 4b

Table 4.2.1 Overview of the four most common phospholipids.

Table 4.2.2 Phospholipid composition of several common seed oils.

Chapter 5a

Table 5.1.1 Commonly used recycling systems to generate NAD(P)H. Most of the rec...

Table 5.1.2 KRED Substrates described in recent literature.

Table 5.1.3 PMI comparison table of developed production processes of some comme...

Table 5.1.4 Comparison of stereoselective alcohol incorporation for (

R

)‐tetrahyd...

List of Illustrations

Chapter 1a

Figure 1.1.1 Excerpt of Leeuwenhoek famous letter to the Royal Society, wh...

Figure 1.1.2 Excerpt of Kühne's original publication from 1876 where he in...

Figure 1.1.3 Historic events and estimated growth of enzyme business from ...

Figure 1.1.4 Jokichi Takamine's patent from 1894, the first enzyme patent ...

Figure 1.1.5 Overview of company roots starting with pioneering entreprene...

Chapter 1b

Figure 1.2.1 Sources of new enzymes. Wild‐type enzymes from nature are mad...

Figure 1.2.2 Selection criteria for industrial enzymes. Schematic illustra...

Figure 1.2.3 General strategies in directed evolution. (a) The evolutionar...

Figure 1.2.4 Strategies for single mutation libraries. In the full site sa...

Figure 1.2.5 Landmark inventions in enzyme technology and their impact on ...

Chapter 1c

Figure 1.3.1 Production hosts for industrially relevant enzymes classified...

Figure 1.3.2 Different possibilities for the construction of a eukaryotic ...

Figure 1.3.3 Comparison of prokaryotic and eukaryotic promoter regions (a,...

Figure 1.3.4 Genealogical tree of

T. reesei

.

T. reesei

QM6a as o...

Chapter 1d

Figure 1.4.1 Enzyme immobilization methods.

Figure 1.4.2 An overview of the applied reaction media in biocatalytic tra...

Figure 1.4.3 The schematic representation of parameters affecting the effi...

Figure 1.4.4 A 2LPS for the use of an organic phase as a substrate reservo...

Figure 1.4.5 (a) Dispersed phase: organic medium and continuous phase: aqu...

Figure 1.4.6 Solid–liquid biocatalysis involving solid substrate(s) and pr...

Figure 1.4.7 Fundamental types of reactors (

t

= time;

x

= place).

Chapter 2a

Figure 2.1.1 Schematic presentation of the various stages of mixing, ferme...

Figure 2.1.2 Mode of action of various starch degrading baking enzymes....

Figure 2.1.3 Schematic presentation of the action of phospholipases in bre...

Figure 2.1.4 Conceptual visualization of the structural consequences of th...

Chapter 2b

Figure 2.2.1 Protein intake per capita in least developed, other developin...

Figure 2.2.2 The food protein value chain according to the author's classi...

Figure 2.2.3 Flavor profiles for enzyme modified cheeses produced using ei...

Figure 2.2.4 The relative performance of papain and Promod™ 950L in yeast ...

Chapter 2c

Figure 2.3.1 Schematic representation of the cheese making process. Rennet...

Figure 2.3.2 Residual activity (%) of different Fromase® grades after incu...

Figure 2.3.3 Lactose digestion at lactase sufficiency (a) or lactase defic...

Figure 2.3.4 Lactase binds to the galactose moiety of lactose while expell...

Chapter 2d

Figure 2.4.1 Enzymatic synthesis of cellobiose from sucrose (SP, sucrose p...

Figure 2.4.2 Cellobiose production in a one‐pot reaction using SP wild typ...

Figure 2.4.3 Solubility curve of saccharides; *Data from Refs. [33,34].

Figure 2.4.4 Process scheme of the enzymatic production of cellobiose from...

Chapter 2e

Figure 2.5.1 General structure of HMOs. The schematic representation of mo...

Figure 2.5.2 Relative abundance of HMOs in human milk. Three of the more a...

Figure 2.5.3 Schematic overview of engineered bacteria for 2′‐FL productio...

Figure 2.5.4 Reaction scheme for the transfucosidase and transsialidase as...

Figure 2.5.5 Diversification of HMOs into seven HMO profiles using differe...

Figure 2.5.6 Reaction mechanism of transfucosylase and illustration of the...

Figure 2.5.7 Effect of the single mutations on transfucosylated product fo...

Figure 2.5.8 Reaction catalyzed by Pd2,6ST in the target reaction.

Figure 2.5.9 Regioselectivity of PlST‐WT and variant PlST‐078 in a biotran...

Figure 2.5.10 Thermostability improvements of PlST variants in comparison ...

Chapter 3a

Figure 3.1.1 Enzymatic hydrolysis of lactose by lactase in stomach.

Figure 3.1.2 Enzymatic hydrolysis of oligosaccharides by α‐galactosidase i...

Figure 3.1.3 Healthcare products containing other enzymes. (a) Dextranase ...

Figure 3.1.4 Prevention of postprandial glucose absorption level by TG.

Figure 3.1.5 Enzymatic reaction of TG.

Figure 3.1.6 α‐Glucosidase inhibitors (α‐GIs). (a) Glucobay (acarbose) Bay...

Figure 3.1.7 Melanin's biosynthesis pathway.

Chapter 3b

Figure 3.2.1 The “Big Five” mycotoxins.

Figure 3.2.2 The pathway of fumonisin catabolism in

Sphingopyxis macrogolt

...

Figure 3.2.3 Sphinganine and sphingosine are key intermediates of sphingol...

Figure 3.2.4 The ratio of sphinganine to sphingosine (Sa/So ratio) in bloo...

Figure 3.2.5 X‐ray crystal structure of fumonisin esterase FumD with bound...

Chapter 3c

Figure 3.3.1 Rule for inositol numbering. Carbon atoms of phytate numbered...

Figure 3.3.2 Crystal structures of HAPhy. Surface (left) and cartoon (midd...

Figure 3.3.3 A model of stepwise dephosphorylation of the different phytas...

Figure 3.3.4 Impact of expression host and post‐translational modification...

Chapter 4a

Figure 4.1.1 Comparison of microscopic pictures of hardwood and softwood u...

Figure 4.1.2 Effect of enzyme on refiner load and tensile strength.

Figure 4.1.3 MD (machine direction) tensile strength and softwood ratio du...

Figure 4.1.4 Comparison of after refining CSF and refiners loads (kilowatt...

Figure 4.1.5 Comparison of breaking length (BL) and ash (base paper and co...

Figure 4.1.6 Polarized light microscopy image of fines and ultra‐fines; ma...

Figure 4.1.7 Flow chart of stock preparation of middle layer, where enzyme...

Figure 4.1.8 Improvement in CSF while using high fines (MW) furnish during...

Figure 4.1.9 Improvement in CSF in ONP furnish during enzyme usages.

Figure 4.1.10 Flow chart of stock preparation.

Figure 4.1.11 Paper formation before (a) and after (b) drainage enzyme usa...

Figure 4.1.12 A typical waste paper processing plant.

Figure 4.1.13 Visual analysis of microscopic pictures.

Figure 4.1.14 Fiber tester images of vessels for blank sample.

Figure 4.1.15 Fiber tester images of vessels of enzyme‐treated sample.

Figure 4.1.16 Structure of amylose.

Figure 4.1.17 Structure of amylopectin.

Chapter 4b

Figure 4.2.1 The 2016/2017 overall production of vegetable oils [1].

Figure 4.2.2 General flow scheme of seed oil processing

Figure 4.2.3 (a) Molecular structure of phosphatidyl choline (here L‐α‐1‐p...

Figure 4.2.4 (a) Cartoon of lamellar liquid‐crystalline state of phospholi...

Figure 4.2.5 Schematic representation of the relevant phospholipase reacti...

Figure 4.2.6 Active sites of (a) PLC showing the three zinc atoms coordina...

Figure 4.2.7 Intact phosphatidyl choline and phosphatidyl ethanolamine lev...

Figure 4.2.8 Reactions with PLC+PI‐PLC on different oils with only water o...

Figure 4.2.9 Levels of phosphorus‐containing compounds versus time in four...

Figure 4.2.10 Levels of phosphorus‐containing compounds versus in water‐de...

Figure 4.2.11 Enzymatic processes, degumming, refining, or deep degumming....

Chapter 5a

Figure 5.1.1 Approaches to asymmetric induction of ketones by reducing age...

Figure 5.1.2

Lactobacillus kefir

KRED crystal structure with bound NADP

+

...

Figure 5.1.3 Codexis' CodeEvolver® protein engineering technology. The tec...

Figure 5.1.4 Typical chemical reagents and catalysts for asymmetric reduct...

Figure 5.1.5 (a) Precursors to the preparation of chiral alcohols. (b) App...

Figure 5.1.6 Carbapenem synthesis Incorporating KRED‐mediated DKR. Reactio...

Figure 5.1.7 KRED binding pockets determine substrate orientation. (a) (to...

Figure 5.1.8 Application on small molecule intermediates. Small heterocycl...

Figure 5.1.9 Biocatalytic reduction of a duloxetine intermediate. A KRED b...

Figure 5.1.10 KRED route to ethyl (

R

)‐4‐cyano‐3‐hydroxybutyrate.

Figure 5.1.11 Chemical and enzymatic approach comparison to (

R

)‐3‐hydroxyt...

Chapter 5b

Figure 5.2.1 Regio‐ and chemoselectivity of enzymes: (a) Regioselective en...

Figure 5.2.2 Schematic representation of a multistep chemical process from...

Figure 5.2.3 Improvement of ecological footprint of a pharma chemical by c...

Figure 5.2.4 Structural comparison of (a) the two top‐selling statin drugs...

Figure 5.2.5 Overview of biocatalytic routes to statin side chains. PLE, p...

Figure 5.2.6 (a) Physiological reaction of DERA reversibly splitting 2‐deo...

Figure 5.2.7 Functional group transformations from CTHP

5

via hydroxy‐lact...

Figure 5.2.8 DERA X‐ray structure‐based model, highlighting solvent expose...

Figure 5.2.9 Reaction scheme from DERA‐synthesized CTHP

5

via hydroxy‐lact...

Figure 5.2.10 Reaction scheme of DERA‐enabled product tree to unsaturated ...

Guide

Cover

Table of Contents

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Industrial Enzyme Applications

Edited by

Andreas Vogel and Oliver May

Copyright

Editors

Dr. Andreas Vogel

c‐LEcta GmbH

R&D Enzyme Development

Perlickstr. 5

04103 Leipzig

Germany

Dr. Oliver May

DSM Nutritional Products Ltd

Wurmisweg 576

4303 Kaiseraugst

Switzerland

All books published by Wiley‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.:

applied for

British Library Cataloguing‐in‐Publication Data

A catalogue record for this book is available from the British Library.

Bibliographic information published by

the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d‐nb.de>.

© 2019 Wiley‐VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Print ISBN: 978‐3‐527‐34385‐0

ePDF ISBN: 978‐3‐527‐81375‐9

ePub ISBN: 978‐3‐527‐81377‐3

oBook ISBN: 978‐3‐527‐81378‐0

Cover Design Adam‐Design, Weinheim, Germany

Preface

Industrial enzyme applications are part of our everyday life since mankind discovered the benefits of transforming milk, grapes, and grains to durable, palatable, more tasteful products such as yoghurt, wine, beer, and bread. At that time, the microorganisms and enzymes that led to this transformation were used unconsciously. Since then, our knowledge about enzymes, and the development of technologies on how to adapt, produce, and apply them has improved dramatically, especially in the last few decades. This has led to a wide spectrum of enzyme applications that we come across in products from the food and drink, chemical, pharmaceutical, biorefinery, and the human and animal nutrition industry.

This book shall direct the perspective to a variety of enzyme types and their applications that are actually used in industrial processes. When we conceived this book, we asked the following questions: Which solutions can enzymes provide to address the needs of industries and consumers? Which enzymes provide which solutions, and why have these particular enzymes been chosen and are successfully competing with other solutions? What was the decisive advantage of the use of enzymes over the competitive process? What enzyme features were required and how were these obtained during enzyme development?

The book is written by experts from different industries who develop and apply enzymes in their teams to address various problems: from enabling “simple” cost reduction and process innovations for existing products (which often goes hand in hand with ecologic benefits) to product innovations, developing new products that provide additional value (e.g. sustainability, purity, nutritional) for consumers. We are very happy that we could attract authors from various industries that were willing to share insights into their work. Writing publications from an industrial position is often not the prime priority and is conflicted by company policies. Particularly for this reason, the contributions in this book provide unique and individual insights into industrial drivers and strategies for the implementation of enzymatic processes.

This textbook on Industrial Enzyme Application will serve as a reference guide for academic and industrial researchers and provides a unique, industrial perspective on the development and application of enzymes. It will help understand the industrial drivers in searching for and developing an enzymatic solution. We also hope the described examples inspire students and practitioners to develop new applications, providing our world with more sustainable solutions that enzymes are perfectly capable of providing.

c‐LEcta, Leipzig

DSM, Kaiseraugst

April 2019

Andreas Vogel

Oliver May

Part IOverview of Industrial Enzyme Applications and Key Technologies

1.1Industrial Enzyme Applications – Overview and Historic Perspective

Oliver May

DSM Nutritional Products, Wurmisweg 576, , 4303 Kaiseraugst, Switzerland

The field of industrial enzyme applications has been extensively reviewed, including its historic background [1–4]. Therefore this chapter will not repeat the excellent work of others but provide historic context in the following chapters and give a general overview of the field, including topics that we have chosen not to cover in detail in this book. It is also meant as a tribute to the many pioneering scientists that enlightened us with their brilliant minds about the miracles of life and how enzymes work (see Section 1.1.2) and to the visionary entrepreneurs shaping a growing multibillion enzyme business (see Section 1.1.3).

While we have come a long way in unraveling how enzymes work, we have still not captured all the details on how they achieve the huge rate acceleration of the reactions they catalyze, nor have we explored exhaustively their full potential in existing as well as new application fields. This leaves room for us and future generations standing on the shoulders of giants some of which are mentioned in the following, or are hidden in the prehistoric world that applied enzymes, even without knowing they existed.

1.1.1 Prehistoric Applications

Enzymes played an important role early in the history and development of humanity. The Neolithic Revolution of around 12 500 years ago marked the transition of lifestyles from hunting and gathering to agriculture and settlement. With this transition, farming practices were invented, leading to domestication of plants and animals. While safe storage of hunted or gathered food was certainly already important during the Neolithic times, the unconscious use of microbes and the action of their enzymes allowed to preserve the food and supply it to others who could focus on other activities outside the primary production of food. Certainly, people also enjoyed improved palatability or a desirable taste of food that was in contact with microorganisms that contributed to these desired properties.

While no one can really determine the exact date when the first products were made in which enzymes from microbes, plant, or animal tissues played such a beneficial role, the first scientifically proven evidence, based on residues found in pottery vessels for cheese making, dates back to 7500 years ago [5]. What a great invention to preserve milk without a fridge and make it palatable as well as enjoyable! Today's enzyme applications in dairy are covered in Chapter 2.3 and the achievements of Christian Hansen, an entrepreneur starting a very successful enzyme and starter culture business in 1874, are described in Section 1.1.3.

The first indication of enzyme‐assisted grain processing to produce an alcoholic beverage was found in the Neolithic village of Jiahu in China and dates back to 7000 BCE [6]. Based on chemical analyses of organics absorbed into ancient pottery the authors have shown that a mixed fermented beverage of rice, honey, and fruit (hawthorn fruit and/or grape) was being produced. The earliest proof of wine production dated to 5400–5000 BCE, at the Neolithic site of Tepe in Mesopotamia [7] where tartaric acid was found in an old jar, and around 5000 BCE from grape juice residues found in Dikili Tash in Greece [8]. For over 2500 years Aspergillus strains have been extensively used in China as starter cultures in grain (soy, rice) fermentation, a traditional practice for production of rice wine (sake) or other distilled products (shochu), which were imported from Japan by Buddhist monks [9]. The Japanese word Koji still uses the Chinese character () that means (wheat) grains fermented by fungi. Today, grain processing and malt production are separate industries whereas in the Western world malt production can be considered as the first sector that industrialized enzyme production (see also Section 1.1.3).

Next to being used for fermentative processes, Koji has been used as digestive aid, as first described 2500 years ago in a Chinese classic book entitled “Zuo‐Zhuan,” in the English Chronicle of Zuo or Commentary of Zuo [10]. In the description, wheat‐based Koji was used to treat digestion problems. This tradition was later turned into the first industrial application of a fungal enzyme by Jokichi Takamine, one of the pioneering entrepreneurs discussed in Section 1.1.3 and who also inspired the application of enzymes for nutrition and health, reviewed by Yoshihiko Hirose in Chapter 3.1. The good old tradition of fermented food is carried through to date and is estimated to provide about 20–40% of our food supply today [11].

The Egyptians, who already used yeast to brew beer, began to employ microorganisms as well as malt to make bread for which samples were found in different archeological sites dating to 2000–1200 BCE [12]. This ancient malt application in baking developed into a major field of enzyme application, which is reviewed by Putseys and Schooneveld in Chapter 2.1.

However, it is not only food that has benefited from enzymes long before their existence was known. One of the first technical (materials) applications can be found in leather processing, which provided ancient civilization with leather for water skins, bags, boats, or shoes as early as 7000 to 3300 BCE [13]. In this traditional process, the bating step that softens the material was a fermentative process. It relied on enzymes produced by bacteria found in pigeons or dog dung, which was added in this step. The replacement of dung by enzymes was a huge improvement that was started by Otto Röhm, whose pioneering work and the foundation of his enzyme company are described in Section 1.1.3.

Enzyme applications in organic synthesis (see Chapters 5.1 and 5.2), feed (see Chapters 3.2 and 3.3), textiles, detergents, and in biorefineries (see Chapters 4.1–4.2) obviously do not root back to ancient times and are inventions of the twentieth century, which are further discussed in Section 1.1.3. More comprehensive reviews of detergent, textile, and biofuel applications can be found here [14–17].

1.1.2 Growing the Scientific Basis

Why does science matter if people already enjoyed the benefits of enzymes for thousands of years as described above? Well, while humankind indeed enjoyed the desired effects of enzymes such as reducing spoilage of valuable raw materials, creating an appealing taste, or supporting better digestion of food products, the desired effects sometimes just did not happen, or turned in highly undesired directions. Science was therefore needed to reduce or prevent such failures. Applying the insight science provided was not only making existing applications more robust. Science also enabled the discovery, development, and efficient production of new enzymes with desired properties, which opened up new opportunities to broaden their application field. The following section highlights key achievements of many brilliant minds in history that created the scientific foundation on which today's commercial success is built.

“Seeing is believing,” and seeing microorganisms was not possible before the pioneering Dutch scientist and gifted craftsman Antoni van Leeuwenhoek developed an analytical instrument with so far unprecedented magnifying power. With his specially prepared lenses he created simple but still very powerful microscopes that could reach a 300‐fold magnification. With these microscopes, he could not only see living microbes, which he originally referred to as animalcules (from Latin animalculum = “tiny animal”), but also already made observations in 1675 that brought him very close to recognizing which “magic forces” are behind some of the desired effects created by microorganisms and their enzymes. Figure 1.1.1 shows a copy of his famous letter to the Royal Society, which was translated in English by Henry Olden, the Editor of the Journal Philosophical Transactions of the Royal Society, where he not only disclosed the discovery of “living creatures” but also commented on the “bubbling water” comparing it with fermenting beer [18].

Figure 1.1.1 Excerpt of Leeuwenhoek famous letter to the Royal Society, which was translated in English by Henry Olden, the Editor of the Journal Philosophical Transactions of the Royal Society.

Leeuwenhoek decided not to share the details of preparing the lenses with the scientific community and took this secret with him when he died in 1723. Because he kept his knowledge secret, his discoveries were doubted or even dismissed during the following century as other scientists could not reproduce his results.

After the discovery of living microbes, another important achievement was reported by Anselme Payen and Jean‐Francois Persoz. The French chemists were working at a sugar factory to improve the process of starch conversion. In 1833, Payen and Persoz reported that an alcohol precipitate of malt extract (probably the oldest and highest volume industrial enzyme product) contained a substance that converted starch into sugar [19]. They named the substance “diastase” after the Greek word διάστασις (diastasis) (a parting, a separation) as it caused the starch in the barley seed to transform quickly into soluble sugars and hence allowed separation of the husk from the rest of the seed. They were also the first to propose a nomenclature for such substances using the suffix “ase” after the root that indicates which substance was modified by the biomolecule. This was the beginning of a systematic enzyme nomenclature that is used to date, with the exception of proteolytic enzymes ending with “in,” such as subtilisin. Today, “diastase” refers to any α‐, β‐, or γ‐amylase (EC 3.2.1.1‐3) that can break down carbohydrates and are applied in many different industrial applications. An overview of several enzymes based on the EC nomenclature and their application fields, some of which are further discussed below, is provided in Table 1.1.1. It is perhaps interesting to note that these fundamental discoveries, which inspired many innovations, were made in the context of industrial research.

Table 1.1.1 Overview of enzyme classes used in different applications.

EC number

Enzyme name

Application

Function

Oxidoreductases

1.1.3.4

Glucose oxidase

Baking

Gluten modification/dough strengthening

Brewing

Oxygen reduction/shelf life improvement

Dairy

Milk preservation

Textile

Bleaching

1.1.3.5

Hexose oxidase

Baking

Gluten modification/dough strengthening

1.10.3.2

Laccase

Pulp and paper

Bleaching

Textile

Bleaching of dye to prevent backstaining

Various

Cork treatment

Various

Polymerization of lignin for production of wood fiberboards

1.11.1.6

Catalase

Brewing

Shelf live improvement

Dairy

Milk preservation

Textile

Hydrogen peroxide removal

Various

Wastewater treatment

1.11.1.7

Peroxidase

Baking

Dough improvement

1.13.11.12

Lipoxygenase

Baking

Whitening of breadcrumb

Transferases

2.3.2.13

Transglutaminase

Dairy

Texture improvement

2.4.1.5

Dextransucrase

Various

Production of dextrans

Hydrolases

3.1.1.3

Triacylglycerol lipase

Baking

Bread improvement

Dairy

Modification of cheese flavor

Detergent

Greasy stain removal

Pulp and paper

Pitch removal

3.1.1.11

Pectin methylesterase

Beverage

Yield increase for fruit (berry, apple) juices, citrus fruit peeling

3.1.1.26

Galactolipase

Baking

Emulsification

3.1.3.8

3‐Phytase

Beverage

Mashing in brewing

Feed

Phosphate release

3.1.3.26

6‐Phytase

Beverage

Mashing in brewing

Feed

Phosphate release

3.2.1.1

α‐Amylase

Baking

Antistaling

Beverage

Mashing (brewing), apple juice production

Detergent

Removal of starch containing stains

Starch processing

Starch hydrolysis for syrup production

Feed

Degradation of starch

Various

Viscosity reduction in oil drilling

Textile

Desizing

3.2.1.2

β‐Amylase

Beverage

Mashing (brewing)

3.2.1.3

Glucoamylase

Beverage

Mashing (brewing), apple juice production

Starch processing

Hydrolysis of maltooligosaccharide for syrup production

Various

Toothpaste

3.2.1.4

Endo‐1,4‐β‐glucanase

Detergent

Softening, color improvement

Textile

Cotton finishing, denim ageing

3.2.1.6

Endo‐1,4(3)‐β‐glucanase

Feed

Increased feed efficiency

3.2.1.8

Endo‐1,4‐β‐xylanase

Baking

Improved dough stability/handling

Feed

Increased feed efficiency

Textile

Flax retting

Pulp and paper

Pulp bleaching

3.2.1.55

Arabinosidase

Beverage

Apple juice production

3.2.1.60

Endo‐1,4‐β‐mannanase

Detergent

Removal of guar gum containing stains

Textile

Flax retting

Pulp and paper

Pulp bleaching

Various

Viscosity reduction in oil drilling

3.2.1.91

Exo‐cellobiohydrolase

Detergent

Softening, color improvement

Textile

Cotton finishing

Pulp and paper

Mechanical pulping

3.4.21.26

Prolyl oligopeptidase

Beverage

Improve stability of beer

Supplement

Reduction of allergen

3.4.21.62

Subtilisin

Detergent

Removal of proteinaceous stains

Feed

Improved feed efficiency

Various

Membrane cleaning

3.4.21.63

Oryzin

Feed

Improved feed efficiency

3.4.23.4

Chymosin

Dairy

Cheese clotting

3.4.23.18

Aspergillopepsin I

Feed

Improved feed efficiency

3.4.23.22

Endothiapepsin

Dairy

Cheese clotting

3.4.23.23

Mucorpepsin

Dairy

Cheese clotting

3.4.24.28

Bacillolysin

Feed

Improved feed efficiency

Lyases

4.1.1.5

α‐Acetolactate decarboxylase

Beverage

Diacetyl removal for beer flavor enhancement

4.2.2.2

Pectate lyase

Textile

Cotton scouring

4.2.2.10

Pectin lyase

Beverage

Fruit juice production, citrus peeling

Isomerases

5.3.1.5

Xylose isomerase

Starch processing

Conversion of glucose into fructose for high fructose corn syrup production

5.3.4.1

Protein disulfide isomerase

Various

Hair waving

Ligases

6.3.2.28

Dipeptide ligase

Bioconversion

Production of dipeptides

Source: Aehle 2007 [1]. Adapted with permission of John Wiley & Sons.

Soon after the first preparation of diastase from a plant source, additional enzyme classes were isolated. Important examples were proteases (EC 3.4.X.X), which hydrolyze proteins and peptides. In 1836, Theodor Schwann studied the process of human digestion and succeeded with isolating an enzyme that he called pepsin [20]. This is the first enzyme prepared from animal tissue, which found early applications in the leather industry to remove hair and residual tissue from animal hides prior to tanning (a process that produces leather from hides). It was also used in the recovery of silver from discarded photographic films by digesting the gelatin layer that holds the silver compound [21]. Another important contribution of Schwann was his recognition that “All living things are composed of cells and cell products,” which became the foundation of what is known today as the cell theory [22].

Probably one of the most influential scientists of the nineteenth century was Luis Pasteur. He is best known to the public for his invention of the heat treatment technique of, for example, milk and wine to stop bacterial contamination, a process now called pasteurization. He was also very influential in the field of medicine by recognizing that partly inactivated microbes can result in immunity, which laid the foundation for vaccination that saved millions of people's lives [23]. Furthermore, Pasteur also laid the foundation for our understanding of molecular asymmetry (chirality). He was separating different crystal shapes from each other to form two piles of tartaric crystals: in solution, one form of these crystals rotated light to the left and the other to the right, while an equal mixture of the two forms canceled each other's effect and did not rotate the polarized light. He further observed that a solution of this tartaric acid derived from living things rotated the plane of polarization of light passing through it, whereas tartaric acid derived by chemical synthesis had no such effect although its elemental composition was the same. Today, we know that this phenomenon is attributed to the specific properties of enzymes. They can induce chirality of reacting with prochiral compounds or discriminate different enantiomers, which is a valuable property of enzymes for the synthesis of enantiomerically pure compounds as reviewed in Chapters 5.1 and 5.2.

Pasteur also studied alcoholic fermentation where he proved that living microorganisms (yeast) are indeed responsible for fermentation: he has shown that the skin of grapes was the natural source of yeasts and that sterilized grapes and grape juice never fermented. In 1862, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur along with Ferdinand Cohn and Robert Koch came to the conclusion that this fermentation was catalyzed by a “vital force” contained within the yeast cells, which were called “ferments” [24]. He then moved on to investigate the conversion of alcohol into vinegar and concluded that pellicle, which he called “the flower of vinegar,” served as a method of transport for the oxygen in air to a multitude of organic substances, laying the foundation for applying biocatalysts in chemical syntheses [25]. In 1886, Brown confirmed Pasteur's findings and gave the causative agent in vinegar production the name Bacterium xylinum. Brown also found that this bacterium could oxidize propanol to propionic acid and mannitol to fructose [26]. Such oxidative biotransformation steps were later applied in the production of L‐ascorbic acid (vitamin C) as described in Section 1.1.3.

Wilhelm Friedrich Kühne, a professor of physiology at the University of Heidelberg, was the first to propose the term “Enzyme” for “ferments” in a publication from 1876 as shown in Figure 1.1.2 [27]. Despite the fact that he was working on digestive enzymes (trypsin) from pancreas, the word enzyme is derived from a Greek word “ενζυμoν” meaning “in yeast.”

Figure 1.1.2 Excerpt of Kühne's original publication from 1876 where he introduced the word enzymes for the first time.

The connection between the function of fermenting yeast and enzymes was finally proved by Eduard Buchner in 1897. He studied the ability of yeast extracts to ferment sugar in the absence of living yeast cells. In a series of experiments at the University of Berlin, he found that sugar was fermented even when there were no living yeast cells in the mixture and named the enzyme (mix) that brought about the fermentation of sucrose “zymase” [28]. In 1907, he received the Nobel Prize in Chemistry “for his biochemical research and his discovery of cell‐free fermentation.”

Once the existence of enzymes was shown, research began to provide detailed insights into their unique properties and functions. The fact that enzymes are very specific can be concluded from Pasteur's discovery of chirality. While he observed the effect, Pasteur did not yet make the direct link between the phenomenon of chirality induced by living microorganisms and the specificity of enzymes that were discovered later, as just described. A starting point to describe enzyme specificity was provided by Emil Fischer, who suggested in 1894 that enzymes and their substrates possess specific complementary geometric shapes that fit exactly into one another. This model is referred to as “the lock and key” model [29]. While this model can be used to explain enzyme specificity, it fails to explain one of the core functions of enzymes as a catalyst, which is the rate acceleration obtained by stabilization of a transition state. In 1958, Daniel Koshland suggested a modification to the lock and key model, which is known as the “induced fit” model: since enzymes have a rather flexible structure, the active site is continuously reshaped by interactions with the substrate [30]. This brought us closer to an explanation for how enzymes function but a real breakthrough in our understanding was achieved by enzyme structure determination.

Lysozyme was the first enzyme crystallized by Edward Penley Abraham and Robinson in 1937 [31] enabling many years later the elucidation of the three‐dimensional structure of hen egg white lysozyme described by David Chilton Phillips in 1965. He obtained the first 2‐Å resolution model via X‐ray crystallography [32]. As a result of Phillips' determination of the structure of lysozyme, it was also the first enzyme to have a detailed, specific mechanism suggested, which provided an explanation for how enzymes speed up a chemical reaction.

Starting with the crystallographic data of Rosalind Franklin [33]; the elucidation of the DNA structure in 1953 [34] by, and the awarding of the Nobel Prize to, Watson, Crick, and Wilkins [35]; the unraveling of the genetic code for which Marshall Warren Nirenberg, Har Gobind Khorana, and Robert William Holley received the Nobel Prize in 1968 [36]; and the introduction of recombinant DNA technology in 1973 by Cohen et al. [37], the field of molecular biology revolutionized the cost‐efficient production of enzymes, which is described in Chapter 1.4. It also provided efficient tools for enzyme discovery and engineering, which are further discussed in Chapter 1.2.

Building on the above‐described work and many more scientific achievements, the following section highlights the pioneering work of entrepreneurs that marked the beginning of the industrial application of enzymes and the development of a multibillion Euro enzyme industry.

1.1.3 The Beginning of Industrial Applications and the Emerging Enzyme Industry

Looking back at the ancient enzyme applications and the scientific history described above, it is not surprising that the first industrial applications of enzymes did not make use of isolated enzymes but crude extracts (such as malt extracts), or even whole cell biocatalysts.

This is also true for the synthesis of chemical compounds in the so‐called biotransformation processes. Vinegar production is perhaps the oldest and best known example of such an industrial scale biotransformation process in which oxidation of ethanol is catalyzed by acetic acid bacteria. Another pioneering example of an industrial biotransformation process is the production of L‐(−)ephedrine, which was introduced by Knoll AG (today BASF) in the 1930s [38]. The process is based on the discovery of Neuberg and Hirsch who showed that in presence of yeast, benzaldehyde can condense with acetaldehyde to optically active 1‐hydroxy‐1‐phenyl‐2‐propanone, which is then chemically converted to L‐(−)ephedrine [39]. This was the first example of an enzymatic C–C coupling reaction at an industrial scale. More recent examples of industrial applications of aldolases catalyzing C–C coupling reactions are reviewed in Chapter 5.2.

Kluyver and de Leeuw, two Dutch scientists from Delft (NL), demonstrated in 1924 that Acetobacter suboxydans can oxidize D‐sorbitol to L‐sorbose [40]. This became an important intermediate in the Reichstein–Grüssner synthesis of L‐ascorbic acid (vitamin C) [41]. This synthesis was turned into an industrial process by Roche (Roche Vitamins, now part of DSM) in the 1930s [42].

In 1953, Peterson et al. reported that Rhizopus arrhius can convert progesterone into 11‐α‐hydroxyprogesterone, which was used as an intermediate in the synthesis of cortisone [43]. This microbial hydroxylation simplified and considerably improved the efficiency of the multistep chemical synthesis of corticosteroid hormones and their derivatives. Although the chemical synthesis from deoxycholic acid developed at Merck (Germany) was in principle possible, it was too complicated and uneconomical: 31 steps were necessary to obtain 1 kg of cortisone acetate from 615 kg of deoxycholic acid. The introduction of microbial 11‐α‐hydroxylation of progesterone dramatically reduced the price of cortisone from $200 to $6 per gram. Further improvements have led to an estimated price of less than $1 per gram [44].

Another remarkable example of applying a whole cell biocatalyst is the production of acrylamide. In the mid‐1970s, Nitto Chemical Industries (now Mitsubishi Rayon) introduced its biocatalytic production using a nitrile hydratase in the form of Rhodococcus cells. This process is considered a milestone in industrial biocatalysis demonstrating the impressive efficiency of enzyme systems operating at product concentrations of up to 700 g/l for the production of a low‐cost commodity chemical. It also demonstrates that enzymes can be applied in a very crude form as whole cell biocatalyst, providing a more cost‐competitive solution. The enzyme process is also a “greener” solution by consuming less energy and avoiding heavy metal wastewater problems of the alternative chemical process [45].

At the end of the 1970s Toyo Jozo (Japan) in collaboration with Asahi Chemical Industry (Japan) pioneered the industrial production of 7‐aminocephalosporanic acid (7‐ACA) by a chemo‐enzymatic two‐step process starting from cephalosporin C. In the 1990s developments from Gist‐Brocades applied metabolic as well as protein engineering, which enabled further breakthroughs not only for the production of cephalosporin C but also for the chemo‐enzymatic synthesis of several derivatives as reviewed in Chapter 5.1.

In the early 1980s another pioneering process was introduced by Degussa (now Evonik Industries). In collaboration with Prof. Kula and Prof. Wandrey a racemic resolution process was developed for production of enantiomerically pure amino acids (e.g. L‐methionine) applying an L‐amino acylase (EC 3.5.1.14) from Aspergillus oryzae in an enzyme membrane reactor (EMR). Since then, many other racemic resolution processes have been developed [46]. A more recent example using a non‐animal‐derived pig liver esterase is reviewed in Chapter 5.1.

The same group from Jülich and Degussa that developed the abovementioned EMR process also introduced the first reductive amination process in the 1990s for the production of L‐tert‐leucine and L‐neopentylglycine. In this process, a leucine dehydrogenase (EC 1.4.1.9) is used for reductive amination of a keto acid together with a formate dehydrogenase (EC 1.2.1.2) for recycling the cofactor NADH. More recent examples of applying dehydrogenases are presented in Chapter 5.2. A more comprehensive overview of enzymes used in organic synthesis and industrial biotransformations is provided here [2,3]. While the above‐described application field of enzymes delivers highly valuable solutions for challenging synthesis problems for the chemical, fine chemical, and the connected pharma field, its impact on growing the total enzyme business is still very limited, as shown in Figure 1.1.3.

Figure 1.1.3 Historic events and estimated growth of enzyme business from 1875 until 2025, broken down into different application fields.

In the early history of brewing, malt production was an integrated part of the brewing process. In the malting step, the water content and temperature of grains from malt, sorghum, or wheat were adjusted to allow them to germinate. During this germination process, grains that already contain enzymes (e.g. β‐amylases) produce additional enzymes such as α‐amylases. After stopping the germination process by a heating step, the malt product is obtained and contains a mix of enzymes that convert the starch of the grains into a fermentable sugar solution. During the last centuries, this malting step was separated from the brewing process, making malt probably the first commercial and to date the highest volume enzyme product. Its success can be attributed to the fact that it enabled an easier, faster, and more consistent brewing process. However, the malt enzymes, first isolated by Payen and Persoz in 1833 as described in the previous section, do have some limitations. They can only work at certain temperatures, pH values, or contain unwanted side activities giving rise to many enzyme innovations for the beverage industry, which is covered in Chapter 2.2. Access to standardized and relatively pure enzyme preparations was also an important factor to introduce amylases in the baking industries, which started to be applied in the beginning of the twentieth century with many following innovations covered in Chapter 2.1.

The history of modern enzyme technology really began when Christian Hansen, in a joint venture with pharmacist H.P. Madsen, opened the first enzyme factory in 1873. They started the first Danish production of pepsin preparations for treating digestive problems.

Christian Hansen also began working with rennet, the enzyme‐rich substance extracted from the fourth stomach of ruminants. These extracts were used since thousands of years for the manufacture of cheese to make the milk coagulate. As demand rose sharply in the nineteenth century, Christian Hansen began a series of experiments on the production of rennet. In 1874 the Chr. Hansen's Teknisk‐Kemiske Laboratorium opened, which is, today, known as the company Chr. Hansen. In a converted metal workshop in Copenhagen he began the commercial production of standardized quality‐assured, liquid animal rennet for dairy applications, which are further described in Chapter 2.3. Just as in the previously described example for the brewing industry, simplification and standardization to reduce process/batch failures with a negative economic impact for the emerging cheese industry was again an important driver and success factor for the introduction of a commercial enzyme product.

In contrast to Western countries, which originally used malt in their brewing processes as described above, Asian countries used the traditional “Koji process.” In this process Aspergillus strains are used as starter cultures to ferment rice or soy for production of rice wine (sake), soy sauce (shoyu), soybean paste (miso), and distilled spirits (shochu) [47]. Believing that Koji made from A. oryzae could revolutionize the American distillery industry and outcompete malt, Jokichi Takamine adapted the Koji process for the production of diastase (amylase), which he patented in 1894. This is reported to be the first patent on a microbial enzyme protecting the production of diastatic enzyme by growing fungi on bran and using aqueous alcohol to extract the enzyme as described in the patent application shown in Figure 1.1.4 [48].

Figure 1.1.4 Jokichi Takamine's patent from 1894, the first enzyme patent protecting a process for production of diastase from a fungal source. From US patent office.

While his initial attempts to introduce microbial diastase in the conservative American brewing industry was not commercially successful, he also realized that this enzyme preparation has potential medical applications. Parke, Davis & Company of Detroit received the license on the amylase and marketed it successfully under the brand name Taka‐diastase as a digestive aid to treat dyspepsia, which is caused by incomplete digestion of starch. This example and the pepsin‐based product developed by Chr. Hansen can be seen as starting points for the application field of enzyme supplements, which are further described in Chapter 3.1 by Hirose. Takamine expanded his business operations in both enzymes and pharmaceuticals and founded three major companies: Sankyo Pharmaceutical Company of Tokyo, the International Takamine Ferment Company of New York, and the Takamine Laboratory of Clifton, New Jersey. With his death, the International Ferment Company of New York was dissolved. Takamine's son, Eben, continued running Takamine Laboratory. After his death in 1953, Eben's widow sold the company to Miles Laboratories ending up in today's enzyme business of DowDuPont as described in Figure 1.1.5.

Figure 1.1.5 Overview of company roots starting with pioneering entrepreneurs to todays' world leading enzyme suppliers.

Takamine's work on fungal amylases was succeeded by Boidin and Effront, who pioneered the work on bacterial enzymes using surface fermentation. One of their landmark patents was obtained in 1917 claiming the use of amylases in processes above 80 °C and under alkaline conditions, which is advantageous in industrial starch processing [49]. A few years later they founded the company Societé Rapidase in Seclin (France) commercializing not only enzymes important to the emerging starch industry but also pioneering enzymes for textile applications [50]. Prior to the introduction of enzymes (initially in the form of malt or pancreas extract) textiles were treated with acid, alkali, or oxidizing agents, or soaked in water for several days so that naturally occurring microorganisms could break down the starch that is used as sizing agent. However, both methods were difficult to control and sometimes damaged or discolored the material, which could be prevented by using enzymes. As shown in the overview of companies in Figure 1.1.5, Societé Rapidase was later acquired by Gist‐Brocades (now part of DSM).

Another pioneering work contributing to important industrial enzyme applications was accomplished by Otto Röhm. According to traditional leather processing, bating required the excrement of dogs, a fact that did not improve the image of tanning, which was considered a stinking and unpleasant activity. Röhm's theory was that these excrements exerted their effect by residual amounts of the animals' digestive enzymes. If this was so, he concluded it might be possible to use extracts of the pancreas directly for bating. Such extracts containing the protease trypsin were tested and produced the expected positive results. Röhm accepted these results as confirmation of the correctness of his theory, but later experiments showed that it was not the animals' enzymes that were active, but rather enzymes of bacteria growing in the intestinal tract of the animals. He introduced the application of pancreatic proteases for bating of hides, replacing the use of less defined and less pleasant animal dung in the tanning process [51]. He standardized the enzyme activity and launched the product OROPON™. In 1907, OROPON went into production, and Röhm together with the merchant Otto Haas founded the company Röhm and Haas for commercialization of enzymes and other products. Later, they established a branch in Philadelphia (USA) and started to export enzymes to the United States and in 1911 to Japan. Within only a few years, Röhm replaced the manure bate with his enzyme product. Other successful applications of Röhm's pancreatic enzyme extracts were the removal of sericin gum from silk with the action of proteases and desizing of cotton fabrics at weaving mills with pancreatic amylase. The latter selectively removes the sizing agent (starch) that is added to yarn to improve the weaving process without affecting the fibers.

Probably the most famous application triggered by Röhm was the introduction of BURNUS® in 1914, the world's first enzyme product in laundry applications. BURNUS was not a detergent in the traditional sense, but a pure stain remover applied before the actual washing process. The protease‐containing product released the dirt embedded by the protein, which would have penetrated the textiles through the high washing temperatures. The laundry was soaked in BURNUS for a few hours and then washed with only a little soap and much less harm to both the fibers and the person doing the washing.

Next to pioneering enzymes for leather, textile, and laundry applications, Röhm also introduced pectinase derived from Aspergillus niger for fruit processing. In the 1930s, the Swiss fruit juice industry discovered that pectinases increase the yield and quality of their products [52]. Supply issues during the war allowed Röhm's competitor Schweizerische Ferment AG founded in 1915 in Basel (Switzerland) to enter the market with their first microbial‐derived product Pectinex in 1945. Today, the German branch of Röhm Enzymes belongs to AB‐Enzymes and the United States‐based Rohm and Haas ended up with DowDuPont. The Schweizerische Ferment AG, which also pioneered malt and pancreas‐based enzyme products for baking, textile, and laundry applications, now belongs to Novozymes, as shown in Figure 1.1.5.

The root of Novozymes, today's enzyme business leader with enzyme sales of €1.9 billion in 2016, goes back to the foundation of Novo Terapeutisk Laboratorium in 1925, which produced insulin from pancreas. At the end of the 1930s, the company developed a process to extract both insulin and active trypsin, which was inactivated in the earlier process. This was the start of Novo's enzyme business with trypsin as the first product for tanning application. Around 1945 Novo started to work on bacterial amylase using submerged fermentation of Bacillus subtilis. In 1951, TERMOZYM® was launched as Novo's first microbial enzyme, followed by AQUAZYME® in 1954, which was the first liquid enzyme (amylase) formulation for the textile market. At that time, the textile industry was the largest market for industrial enzymes, with Novo offering pancreatic and bacterial amylases, Schweizerische Ferment AG malt‐based amylase, and Societé Rapidase bacterial amylase, as described above. Over time, other enzymes have been successfully introduced in the textile industry next to amylases, such as cellulases to enhance the appearance and feel of cellulosic garments, catalases to eliminate residual hydrogen peroxide following bleaching or in dye house wastewater recycling, and proteases for the softening and anti‐pilling of wool as well as for the degumming of silk [16].

As previously mentioned, amylases turned out to be very valuable products triggering innovations in the starch processing industry in the manufacture of specific types of syrup i.e. those containing a range of sugars that could not be produced by conventional acid hydrolysis. Another real breakthrough in this industry was reached early in the 1960s when an enzyme, glucoamylase, was launched for the first time, which could completely break down starch into glucose [53]. Within a few years, almost all glucose production changed over to enzymatic hydrolysis because of greater yield, higher degree of purity, and easier crystallization.

In 1959, Gebrüder Schnyder, a small detergent producer in Switzerland, launched BIO‐40 and in 1962 the company Kortman & Schulte (Netherlands) launched BIOTEX containing bacterial proteases. Compared to the trypsin used in BURNUS the proteases in BIO‐40 and BIOTEX were more active and stable under conditions used in detergent applications