A Handbook on High Value Fermentation Products, Volume 2 E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Fachliteratur
- Sprache: Englisch
Written by a group of world-renowned experts, the second volume in this groundbreaking set continues where the first volume left off, focusing on fermentation products that contribute to human welfare across a variety of industries. Green technologies are no longer the "future" of science, but the present. With more and more mature industries, such as the process industries, making large strides seemingly every single day, and more consumers demanding products created from green technologies, it is essential for any business in any industry to be familiar with the latest processes and technologies. It is all part of a global effort to "go greener," and this is nowhere more apparent than in fermentation technology. This second volume in the groundbreaking new set, High Value Fermentation Products, focuses on industries that a concerned with human welfare, including the leather industry, textiles, pharmaceutical and medical, food processing, and others. Covering topics such as chitin and chitosan, microbial polyhydroxyalkanoates, propanediol, and many others, the editors and contributors have contributed to an extremely important facet of chemical and process engineering and how to move these industries into a much more sustainable and environmentally conscious direction. From converting waste into apparel to creating healthier foods and more effective medicines, this is truly a monumental work that is a must-have for any chemical engineer, scientist, or chemist.
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Veröffentlichungsjahr: 2019
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Contents
Cover
Foreword
Acknowledgements
Preface
About the Editors
Chapter 1: Challenges and Opportunities for the Production of Industrial Enzymes by Fermentation
1.1 Introduction
1.2 Production of Microbial Enzymes by Fermentation: A Process Perspective
1.3 Building Up Enzyme Catalysts for Process Applications
1.4 Conclusions
References
Chapter 2: Biotechnology of Leather: An Alternative to Conventional Leather Processing
2.1 Introduction
2.2 Structure and Significance of Leather Industry
2.3 History of Leather
2.4 Conventional Methods for Leather Processing
2.5 Biotechnology in Leather Industry
2.6 A Good Alternative to Conventional Chemicals in Leather Processing
2.7 Enzymes for Leather Processing
2.8 Importers and Exporters of Top Leather
2.9 Outlook
References
Chapter 3: Enzyme Catalysis: A Workforce to Productivity of Textile Industry
3.1 Introduction
3.2 Major Textile Enzymes, Mechanism of Action and Microbial Sources
3.3 Applications in Textile Industry
3.4 Technological Advancements to Enhanced Production of Textile Enzymes
3.5 Conclusion
Acknowledgement
References
Chapter 4: Current Trends in the Production of Ligninolytic Enzymes
4.1 Introduction
4.2 Ligninolytic Enzymes
4.3 Sources of Ligninolytic Enzymes
4.4 Production of Ligninolytic Enzymes
4.5 Purification of Ligninolytic Enzymes
4.6 Potential Applications of Ligninoytic Enzymes
4.7 Outlook
References
Chapter 5: Asava-Arishta: A Multi-Advantageous Fermented Product in Ayurveda
5.1 Introduction
5.2 Definition of Asava and Arishta
5.3 Method of Preparation for Asava Arishta
5.4 Role of Ingredients and Process
5.5 Advantages of Asava–Arishta Over Other Dosage Form
5.6 Future Perspective
Acknowledgements
References
Chapter 6: Production and Applications of Polyunsaturated Fatty Acids
6.1 Introduction
6.2 Biosynthesis of Polyunsaturated Fatty Acids
6.3 Sources of Polyunsaturated Fatty Acids
6.4 Different Fermentation Process for PUFA Production
6.5 Application of PUFAs
6.6 Future Perspectives
6.7 Conclusion
Acknowledgements
References
Chapter 7: Functional Foods and Their Health Benefits
7.1 Introduction
7.2 Fermented Functional Foods
7.3 Functional Whole Foods
7.4 Conclusion
References
Chapter 8: Industrially Important Biomolecules From Cyanobacteria
8.1 Introduction
8.2 Cultivation Methods for Cyanobacteria
8.3 Multifaceted Role of Biocompounds Found in Cyanobacteria
8.4 Industrial-Scale Production and Commercial Status
8.5 Future Perspectives
Acknowledgements
References
Chapter 9: Augmenting Bioactivity of Plant-Based Foods Using Fermentation
9.1 Introduction
9.2 Effect of Fermentation on Bioactivity of Plant-Based Foods
9.3 Different Fermentation Procedures
9.4 Factors Affecting Fermentation Process
9.5 Bioactive Properties of Fermented Foods
9.6 Conclusion and Future Perspectives
References
Chapter 10: Probiotic Intervention for Human Health and Disease
10.1 Introduction
10.2 Various Sources of Probiotics
10.3 Commercially Developed Probiotic Products
10.4 Global Market of Probiotics
10.5 Probiotic Production From Fermentation Process
10.6 Health Implications of Gut Microbiota Dynamics
10.7 Conclusions
Acknowledgement
References
Chapter 11: Saccharomyces - Eukaryotic Probiotic for Human Applications
11.1 Introduction
11.2 Advantages of Eukaryotic Probiotics Over Prokaryotic Probiotics
11.3 Probiotic Properties of Approved Yeast Strains
11.4 Pharmacodynamics of
S. Boulardii
11.5 Therapeutic Potentials of
S.boulardii
11.6 Fermentative Production of
S. Boulardii
11.7 Commercial Impact of Probiotic Products (Eukaryotic Probiotics)
11.8 Safety
11.9 Conclusion and Future Prospects
References
Chapter 12: Bioactive Polysaccharides Produced by Microorganisms: Production and Applications
12.1 Introduction
12.2 Classification and Structure of Polysaccharides
12.3 Fungal Polysaccharides
12.4 Bacterial Polysaccharides
12.5 Algal Polysaccharides
12.6 Microbial Polysaccharides versus Plant Polysaccharides
12.7 Biological Activities of Microbial Polysaccharides
12.8 Microbial Polysaccharides Production
12.9 Microbial Polysaccharides Recovery
12.10 Conclusion
References
Chapter 13: Shikimic Acid: A Compound of Industrial Interest with Respect to Swine/Avian Flu
13.1 Introduction
13.2 Shikimic Acid
13.3 Applications of Shikimic Acid
13.4 Conclusion
References
Chapter 14: 1,3-Propanediol: From Waste to Wardrobe
14.1 Introduction
14.2 Applications of 1,3-PDO
14.3 Microbial Production of 1,3-PDO
14.4 Challenges in Microbial Production of 1,3-PDO
14.5 Different Strategies for the Production of 1,3-PDO
14.6. Downstream Processing of 1,3-PDO
14.7 Economic Importance of 1,3-PDO
14.8 Future Prospects and Outlook
14.9 Conclusions
References
Chapter 15: Biomedical and Nutraceutical Applications of Chitin and Chitosan
15.1 Introduction
15.2 Different Forms of Chitin and CHS
15.3 Biosynthesis of Chitin and CHS
15.4 Biomedical Applications of Chitin and CHS
15.5 Nutraceutical Application of Chitin and CHS
15.6 Commercial Products
15.7 Conclusion
References
Chapter 16: Microbial Polyhydroxyalkanoates: Current Status and Future Prospects
16.1 Introduction
16.2 PHA Structure and Diversity
16.3 Physical Properties of PHA
16.4 Environment, Phylogeny and Diversity of PHA Producing Microorganisms
16.5 PHA Biosynthesis
16.6 Problems and Challenges in PHA Production on an Industrial Scale
16.7 Applications of PHAs in the Medical Industry
16.8 Conclusions and Future Prospects
Acknowledgements
References
List of Contributors
Index
End User License Agreement
Guide
Cover
Table of Contents
Begin Reading
List of Illustrations
Chapter 2
Figure 2.1
Value Chain of Leather Industry [12].
Figure 2.2
Making of leather goods (
adapted from Acron - The Olde Hide House
...
Figure 2.3
Inflowand out flow diagram for leather processing [1].
Figure 2.4
Inflow and out flowdiagram for leather processing [1].
Figure 2.5
Comparative analysisin appearance of dehaired skin after chemical and enzymatic...
Figure 2.6
An overview of the leather production process, emphasizing steps that are at...
Figure 2.7
Dehaired skins and hides using proteolytic enzymes.
Chapter 3
Figure 3.1
Role of enzymes in different fields of life.
Chapter 4
Figure 4.1
Representative linkages present in lignin and basic structural units found in...
Figure 4.2
The catalytic cycle of lignin peroxidase (LiP); VA
·+
: veratryl...
Figure 4.3
The catalytic cycle of manganese-dependent peroxidase (MnP). Reprinted from...
Figure 4.4
The catalytic cycle of versatile peroxidase (VP). Reprinted from Journal of...
Figure 4.5
The catalytic cycle of laccase. Reprinted from Applied Biochemistry and...
Figure 4.6
Laccase mediator system (LMS) [49].
Chapter 5
Figure 5.1
Steps of fermentation - traditional vs industrial process.
Figure 5.2
Fermentation by yeast.
Chapter 6
Figure 6.1
Biosynthetic pathways of LC-PUFA from C18 PUFA precursors in vertebrates (Reactions...
Chapter 7
Figure 7.1
Kimchi (Source: https://i.ytimg.com/vi/0sX_wDCbeuU/maxresdefault.jpg).
Figure 7.2
Sourdough (a) Liquid (Source: https://breadtopia.com/wp-content/uploads/2016/08/starter.jpg)...
Figure 7.3
Nutmeg (
Myristica fragans
) (Source: https://media1.britannica.com/eb-media/...
Chapter 8
Figure 8.1
Multifaceted role of biocompounds found in cyanobacteria (BGA).
Chapter 9
Figure 9.1
Effect of fermentation on bioactivity of plant based functional foods.
Figure 9.2
Schematic diagram of production process of fermented plant extracts [Partially...
Figure 9.3
Bioactive properties of fermented plant extracts.
Chapter 10
Figure 10.1
Commonly used probiotic microorganisms.
Figure 10.2
Various health benefits of probiotics.
Figure 10.3
Various mechanisms used by probiotics for lowering cholesterol.
Chapter 11
Figure 11.1
Human intestinal system (Adapted from a graphic of David Carlson:...
Chapter 12
Figure 12.1
Polysaccharides from algae. Source [45].
Chapter 13
Figure 13.1
Pandemism of swine/avian flu over the globe (red zone showing the affected regions).
Figure 13.2
Antigenic re-assortment in the influenza virus.
Figure 13.3
A historical account of swine and avian flu.
Figure 13.4
Chemical structure of shikimic acid.
Figure 13.5
Synthesis of shikimic acid via Dials-Alder reaction [81].
Figure 13.6
Shikimic acid synthesis [83].
Figure 13.7
Chemical conversion of (-)-Quinic acid to (-)-Shikimic acid.
Figure 13.8
Shikimic acid pathway.
Figure 13.9
Applications of shikimic acid.
Chapter 14
Figure 14.1
Glycerol metabolism in
K. pneumoniae
showing the production of 1,3-PDO.
Figure 14.2
Production of glycerol from glucose.
Figure 14.3
Conversion of glucose/glycerol to 1,3-PDO by the expression of glycerol-3-phosphatase...
Figure 14.4
Metabolic engineering for the production of 1,3-PDO from D-glucose [147].
Chapter 15
Figure 15.1
Structure of Chitin (a) and Chitosan (b).
Figure 15.2
Forms of chitin: (a) α-chitin, (b) β-chitin and (c) γ-chitin.
Chapter 16
Figure 16.1
Transmission electron micrograph of
Pseudomonas
cells with intracellular...
Figure 16.2
General Structure, specific examples and common applications of PHAs [4].
Figure 16.3
PHA biosynthetic pathways [102] PhaA, 3-ketothiolase; PhaB, (R)-3-ketoacyl-CoA...
List of Tables
Chapter 1
Table 1.1
Commonly used industrial enzymes and their applications.
Table 1.2
Operations for the recovery of cell-bound enzymes [44].
Table 1.3
Main industrial enzymes producers.
Chapter 2
Table 2.1
Report of working group on leather & leather products twelfth five year...
Table 2.2
(Manual for oxazolidine leather tanning LIFE08 ENV/E/000140) [13].
Table 2.3
Sources and properties of some of the alkaline proteases used in dehairing.
Chapter 3
Table 3.1
Major enzymes used in the processing of textiles.
Table 3.2
Companies involved in commercial synthesis of textile enzymes and their products.
Chapter 4
Table 4.1
Ligninolytic enzymes and their main reactions [10-13],
Table 4.2
Characteristics of the main ligninolytic enzymes [14–16].
Table 4.3
Recent studies on ligninolytic enzyme production using different substrates under...
Chapter 5
Table 5.1
Contemporary intervention in fermentation processing.
Table 5.2
Testing parameter of Asava-arishta.
Chapter 6
Table 6.1
Algae as bioresources for PUFA production (adapted from spolaore
et al.,
...
Table 6.2
Microbial sources of PUFAs.
Chapter 7
Table 7.1
Fermented functional foods.
Table 7.2
Non-fermented functional foods.
Chapter 8
Table 8.1
Some common culture media used for the cultivation of cyanobacteria.
Chapter 9
Table 9.1
Fermentation conditions for certain herbal formulations.
Chapter 10
Table 10.1
Health benefits of probiotics isolated from diverse sources.
Table 10.2
Commercial probiotic products, sources and developer companies [33–35].
Chapter 11
Table 11.1
List of approved probiotic strains by Food Safety and Standards Authority of India...
Table 11.2
Comparison of the properties of eukaryotic (Yeast) and prokaryotic (Bacteria)...
Table 11.3
Evidence based indications for eukaryotic probiotic
saccharomyces boulardii
...
Table 11.4
Commercially available eukaryotic probiotic products.
Chapter 12
Table 12.1
Classification of main homopolysaccharides according to the monomeric sugar...
Table 12.2
Fungal species and their corresponding bioactive polysaccharides.
Chapter 13
Table 13.1
History of known Flu Pandemics [32–34].
Table 13.2
Physicochemical properties of shikimic acid [77].
Table 13.3
Different columns and conditions used for the detection of shikimic acid by HPLC.
Table 13.4
Distribution of shikimic acid in the organs of various plants.
Chapter 14
Table 14.1
Structure and other general properties of 1,3-PDO.
Table 14.2
List of the 1,3-PDO concentration, yield and productivities reported by different...
Chapter 15
Table 15.1
Chitin extraction employing microorganisms and enzyme.
Table 15.2
Chitin and Chitosan based commercial products.
Chapter 16
Table 16.1
Comparison of properties of SCL-PHAs, MCL-PHAs and their copolymers with...
Table 16.2
PH As biosynthesis from renewable sources.
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Scrivener Publishing100 Cummings Center, Suite 541JBeverly, MA 01915-6106
Publishers at ScrivenerMartin Scrivener ([email protected])Phillip Carmical ([email protected])
High Value Fermentation Products Volume 2
Human Welfare
Edited by
Saurabh Saran
Vikash Babu
Asha Chaubey
This edition first published 2019 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2019 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com.
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Library of Congress Cataloging-in-Publication Data
ISBN 978-1-119-55483-7
Foreword
From last two decades we have witnesses unprecedented growth and development in biotechnology positioning the bioeconomy as a major indicator of advancement. Today, the global fermentation-based industry is already worth over 127 billion dollars. Based on the experience and expertise in this filed, we are trying to collect the different technologies advancement and products developed in biotechnology. This book ‘High Value Fermentation Products-Volume II (Human Welfare) is divided into various important sections related to Human Health like antibiotics, sugar & sugar alcohols, enzymes, nutraceuticals, metabolic engineered derived products, this will help the readers to understand the importance of fermentation derived product for the betterment of human health. This book will also help to overcome of various bottle necks of the Industry/ scientific community and shall be useful for the betterment of the society and environment. This book will also shares an insight into the recent research, cutting edge technologies, high value products, industrial demand which bring immense interest among young and brilliant researchers, cultivated scientists, industry personnals and talented student communities. The contents of the book have been designed in such a way that it is providing extensive coverage of new developments, state of the art technologies, current and future trends in biotechnology and fermentation. The reader will be introduced with basic and advanced methodologies on industrial microbiology and fermentation technology. The main goal of this book is to share and enhance the knowledge of each and every individual in this fermentation world.
Ram A Vishwakarma
Director, CSIR-IIIM
Acknowledgements
The Editors take this opportunity to gratefully acknowledge the assistance and contribution of the people who have faith in us in this undertaking for compiling of the Book “High Value Fermentation Products”-Volume II (Human Welfare).
We are in debt of Dr. Ram A. Vishwakarma, Director, CSIR-Indian Institute of Integrative Medicine, Jammu for his valuable and esteemed guidance to carry out this task. His scholarship and authorative knowledge has been a great source of motivation and inspiration.
First and foremost, it is not enough to express our gratitude in words to all the contributors for devotion and providing excellent matter of chapters on time.
The help and support provided by Mr. Chand Ji Raina, Mr. R.K. Khajuria and Mrs. Urmila Jamwal, was important and we acknowledge all of them with sincere thanks.
We are also thankful to the students of Fermentation Technology Division, CSIR-IIIM for their sincere efforts, dedication and determination to achieve objectives for the completion of this task in a given time.
Where emotions are involved, words cease to mean for our family members for the consistent motivation during the planning and edition of this book.
We avail the opportunity to express our heartiest thanks to ‘Almighty’ for pouring His care and blessings throughout and making this work a success.
Saurabh Saran
Vikash Babu
Asha Chaubey
Preface
Fermentation processes are being used for generations to meet the requirements for sustainable production of enzymes, food & dairy products and nutraceutical products etc. Thus, the modern biotechnology offers numerous opportunities for human welfare. Fermentation processes involve the proven scientific and engineering principles by biological agents. It uses variety of microorganisms such as bacteria, yeast and fungi for production of variety of value added products and biomolecules such as enzymes, antibiotics, hormones, organic acids, drug precursors and other metabolites.
The book entitled “High Value Fermentation Products” has been divided in two volumes namely, Human Health and Human Welfare. The Volume 1 of the book has 18 chapters focussed on basics to fermentation technology, antibiotics & immunosuppressants, antibodies, peptides & proteins, sugars & sugar alcohols and metabolic engineering derived products. The Volume 2 of the book with the theme ‘Human Welfare” has 16 chapters which primarily focus on enzymes, nutraceuticals, probiotics, biopolymers, and organic acids. The first chapter entitled ‘Challenges and opportunities for the production of industrial enzymes by fermentation’ aims to provide the insights on the industrial enzymes, their bioprocess and associated challenges. The second chapter on ‘Biotechnology of leather: An alternative to conventional leather processing’ provides the techniques and enzymatic methods involved in leather processing, an alternative green approach in leather processing. The third chapter on ‘Enzyme catalysis: a workforce to productivity of textile industry’ compiles the various enzymes involved in the textile industry. The fourth chapter entitled ‘Current trends in the production of ligninolytic enzymes’ highlights the recent biotechnology driven fermentation approaches for production of ligninolytic enzymes. Fifth chapter emphasizes on production of Ayurvedic preparations, i.e. Asava & Arishtas by fermentation. Production and applications of poly unsaturated fatty acids have been elaborated in the sixth chapter. Seventh chapter on ‘Functional foods and their health benefits’ provides information the importance of functions foods. The eighth chapter entitled ‘Industrially important biomolecules from cyanobacteria’ describes various types of biomolecules produced by cyanobacteria. ‘Augmenting bioactivity of plant based foods using fermentation’, the ninth chapter of the book elaborates the bioactivities of plant based fermentation products. The tenth chapter ‘Probiotic intervention for human health and disease’ emphasizes on the probiotics and their importance in human health and protection against diseases. Eleventh chapter of the volume elaborates the Saccharomycessp. based probiotics. Chapter twelve entitled ‘Bioactive polysaccharides produced by microorganisms: Production and applications’ elaborates various polysaccharides produced by microorganisms, their bioactivities and applications. Thirteenth chapter ‘Shikimic acid: A compound of industrial interest with respect to swine/avian flu’ is focused on an important compound, shikimik acid with special reference to swine flu. ‘1,3-Propanediol: From Waste to Wardrobe’ is the fourteenth chapter of the volume, which talks about another important compound with innumerable applications. Fifteenth chapter ‘Biomedical and Nutraceutical Applications of Chitin and Chitosan’ explains wide applications of chitin and chitosan. The last chapter of the volume 2 of the book is ‘Microbial polyhydroxyalkanoates: current status and future prospects’ which elaborates the features and importance of polyhydroxyalkanoates with wide industrial applications.
In the Volume-2 of the book ‘High Value Fermentation Products’, editors have tried their best to compile contributions that provide applications and recent trends in the area of fermentation based processes for production of enzymes, biopolymers, probiotics and other nutraceuticals.
About the Editors
Dr. Saurabh Saran, PhD, is a Fermentation Scientist having experience in Industrial microbiology, Biotechnology and Fermentation Technology for more than 15 years. Dr. Saran has completed his PhD from Delhi University, India. Dr. Saran has got hands-on experience in working both industries and academic. He has worked in the industries like Reliance Industries Ltd., India. Later he was appointed as a Research Professor at Republic of Korea, South Korea. He has also worked as a Coordinator at the Technology Based Incubator, Delhi University South Campus, Delhi, Inida. Presently, he is working as a Senior Scientist, Fermentation technology division, CSIR-IIIM, Jammu, India. He has an expertise on the screening, isolation, production and scale up of Industrial Enzymes, Biochemicals & Biofuels. Expert in process development/engineering, scale up to 5L, 10L, 30L, 100, 300 L & 500L fermentation size, (batch, fed batch and continuation fermentation) strain engineering, downstream processing and applications of industrially important biomolecules. To my credentials, I have 3 patents and more than 25 international publications in peer reviewed international journals on fermentation technology.
Dr. Vikash Babu, PhD was born in Bulandshahr district of Uttar Pradesh, India on 1st September 1981. He did his Bachelor’s degree from I.P (PG) College Bulandshahr, India. After qualifying all India combined entrance exam for biotechnology conducted by JNU, New Delhi, India, he did his degree in Biotechnology from Kumaun University, Nainital. After completing his M.Sc degree, he qualified many national level competitive exams such as DBT-JRF- 2005, CSIR-UGC NET for lecturership- Dec. 2004 & June 2005 and GATE-2005. In Nov. 2005, he joined as a DBT-JRF in the Department of Biotechnology, Indian Institute of Technology, Roorkee under the superivision of Dr. Bijan Choudhury and registered for the Ph.D in the same department and Institute and completed his Ph.D degree in the year 2011. After finishing his Ph.D research work he joined Mangalayatan University, Beswan, Aligarh (India) as a lecturer. He left Manglayatan University in the year 2012 and joined Graphic Era University, Dehradun (India) as an assistant professor where he worked till June 2013. Currently, he is working as a scientist in CSIR-IIIM.
Dr. Asha Chaubey, Ph.D is Principal Scientist and Head of Fermentation Technology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India. She has about 15 years of research experience in the area of enzymology and fermentation technology. She is actively engaged in development of indigenous process development. Her research interests include exploration and exploitation of microorganisms for production of enzymes and bioactives in special reference to industrial applications. She has published research articles in the area of bioactives production, enzyme immobilization, biotransformation, kinetic resolution of racemic drug intermediates. She has also published several review articles and has been actively involved in the development of biosensors for health care and environmental monitoring and has several patents on lactate and cholesterol biosensors.
Chapter 1Challenges and Opportunities for the Production of Industrial Enzymes by Fermentation
Andrés Illanes
School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso Av. Brasil 2085, Valparaíso, P.O. Box 4059, Valparaíso, (Chile)
Corresponding author:[email protected]
Abstract
Enzymes have been used as industrial catalysts for over a century now. Plant and animal tissues and fluids have been gradually replaced by microorganisms as sources of enzymes, because of the advantages of intensive production by fermentation at high productivity and under controlled conditions. Genetic engineering has allowed the production of enzymes from any origin in microbial hosts, including higher organisms, non-culturable microbes and metagenomic pools. Complementarity, protein engineering tools allow producing enzyme variants with improved features as process catalysts. Enzyme production by fermentation is reviewed from a process perspective, and strategies for building up enzyme catalysts for process applications are presented considering the evolution of biocatalysis from rather simple reactions of hydrolysis to more complex reactions of organic synthesis where stringent conditions impose new demands for enzyme performance. Advances in the field as well as challenges both in the production and utilization of microbial enzymes are discussed.
Keywords: Microbial enzymes, biocatalysis, immobilized enzymes, protein engineering
1.1 Introduction
1.1.1 Sources of Enzymes as Process Catalysts
Enzymes are the catalysts of life. The metabolism of all living cell forms depends on enzymes since they allow the biochemical reactions to proceed at a sustained pace at the mild conditions required for cell integrity. Enzymes are complex molecules that have evolved to act with an outstanding molecular precision, being both specific in terms of substrate recognition and selective in terms of the reaction catalyzed [1]. These are outstanding properties that make enzymes attractive catalysts for chemical processes.
However, as process catalysts, enzymes should perform efficiently under conditions usually far apart from physiological. This is a major challenge and most efforts in the last 50 years have been devoted to making these metabolic catalysts robust enough to withstand the usually harsh conditions of an industrial process of biotransformation [2]. The industrial use of enzymes dates back to the early years of the last century. At that time, most enzymes used were extracted from plant tissues and from animal organs [3]. Some of these early industrial enzymes from plant and animal origin are still being produced in significant amounts. This is the case of the plant proteases papain and bromelain, which are used in food and beverage processing and also in cosmetic and pharmaceutical products [4, 5], and several lipases and proteases extracted from animal tissues that are used in food and leather processing, and also in some pharmaceutical products [6, 7]. Main industrial enzymes are listed in Table 1.1.
Table 1.1 Commonly used industrial enzymes and their applications.
Enzyme
Source
Application
Glycosidases
α-amylase
mold
bakery, confectionery, brewery,first-generation bioethanol
α-amylase
bacteria
starch liquefaction, detergent, fabrics desizing, first-generation bioethanol
α-arabinofuranosidase
yeast, mold
wine making
β-amylase
plant, bacteria
glucose syrup, brewery
cellulase
mold
juice extraction and clarification, detergent, denim, second-generation bioethanol
β-galactosidase
yeast, mold
delactosed milk and dairy products, whey upgrading
β-glucanase
mold
animal feed supplement, brewery
β-glucosidase
yeast, mold
wine making
glucoamylase
mold
glucose syrup
invertase
yeast, mold
confectionery
naringinase
mold
juice debittering
pectinase
mold
juice clarification and extraction, baby foods, wine making
phytase
bacteria
animal feed supplement
xylanase
mold, bacteria
wood pulping and bleaching, bioethanol
Proteases
alkaline protease
bacteria
detergent, leather tanning and dehairing, stickwater treatment
aminopeptidase
mold, bacteria
protein hydrolyzate debittering
bromelain
Ananas comosus
stem
anti-inflammatory and burn healing preparations, drug absorption
chymosin
animal, recombinant yeast and mold
cheese-making
neutral protease
mold, bacteria
baking, protein hydrolyzate
papain
Carica papaya
latex
yeast and meat extracts, brewery, protein hydrolyzates, meat tenderizing, tanning, digestive aids, skin wound healing preparations
pepsin
animal
cheese-making
Other Hydrolases
lipases
animal, yeast, fungi, bacteria
flavor enhancer, detergent, biodiesel
aminoacylase
mold
food and feed fortification
penicillin acylase
mold, bacteria
β-lactam antibiotics
urease
bacteria
alcoholic beverages, urea removal
enzymes
Non-hydrolytic
glucose isomerase
bacteria, actinomycetes
high-fructose syrup
glucose oxidase
mold
food and beverage preservation
catalase
bacteria
food preservation, peroxide removal
nitrile hydratase
bacteria
acrylamide, nicotinamide
aspartate ammonia lyase
bacteria
aspartic acid
Most of the enzymes listed are hydrolases and even though most of these industrial applications are mature technology, technological improvements are still ongoing. Highly resistant enzymes for laundry (proteases, lipases and amylases), intensive use of enzymes in animal feeding (phytases, β-glucanases) and, above all, enzymes used for biofuel production (amylases, cellulases, hemicellulases, lipases), are major areas of development already having a strong impact on the enzyme market.
As seen in Table 1.1, most of these industrial enzymes are of microbial origin, mostly from bacteria, yeasts and molds, so its production is tightly bound to fermentation technology. The development of submerged fermentation and its success in the large-scale production of penicillin and other antibiotics triggered the production of enzymes by microbial fermentations that started to displace the former plant and animal enzymes, so that by 1960 30% of the enzymes were already produced from microbial strains, and two decades later the situation had reversed and more than 70% were produced intensively and independent of season and climate in well-controlled industrial fermentation processes.
Up to now, living cells are the only source of biocatalysts. The creation of synthetic molecules that mimic the active sites of enzymes (enzyme mimics) has been pursued in organic chemistry as a way of solving some of the restrictions of natural enzymes, like high production cost, narrow substrate specificity and propensity to degradation. Small molecular weight active site constructs (chemzymes) and catalysts based on molecular imprinting in synthetic polymers, and also in some inorganic matrices like silica and zeolites, with products and transition state analogues (abzymes) have been studied and evaluated as enzyme-like catalysts [8]. However, very few of them are able to catalyze reactions at the conditions in which enzymes perform and in such cases activity and stability are very low, but the wide range of catalytic activities that may be obtained and the continuing progress in the field will eventually represent a technological option to natural enzymes in the long term [9].
1.1.2 Advantages of Microorganisms as Hosts for Enzyme Production
Microorganisms are ideal hosts for producing enzymes. Reasons underlying are many: microbes are vigorous organisms with high specific growth rates and simple nutritional requirements that are rather easy to manipulate both genetically and environmentally, representing a huge reservoir of genetic material. These features are technologically meaningful, making the production of microbial enzymes more reliable, simpler and cheaper, using readily available raw materials in a controlled environment, so favoring process validation. Production of enzymes in microbial hosts is no longer restricted to the microbial genome since the development of genetic engineering that allows the expression into suitable microbial hosts of foreign genetic material, coming from any kind of organism, including higher organisms, non-culturable microbes, and even from metagenomic pools [10]. Cloning genes of extremophiles into suitable mesophilic microbial hosts has a striking importance for producing enzymes able to withstand the harsh conditions that may occur in biocatalytic processes [11, 12]; complementing the above, protein engineering strategies, like site-directed mutagenesis and directed evolution, are powerful tools for producing enzyme variants better suited for performing biocatalysis [13]. On the other hand, impressive advances in fermentation technology and process control allow obtaining high cell concentrations so that the volumetric productivity of enzymes can be greatly increased [14]. Only in the case of eukaryotic glycoenzymes (i.e., urokinase, tissue plasminogen activator) production in established animal cell lines is a better option because of the ability of these cells to perform post-translational glycosylation properly [15]; in this case, high costs associated to production can be absorbed because of the very high unit price of the enzyme [16].
Summing up, the advantages of using microorganisms for producing enzymes explain why now more than 90% of the enzymes marketed are from microbial origin and a significant fraction of them are produced with genetically manipulated microorganisms [17]. The case of chymosin clearly illustrates this: chymosin is a very specific aspartic acid protease that hydrolyzes the Phe105-Met106 peptide bond of κ-casein triggering its clotting in the presence of calcium ions to yield the curd [18]. It was traditionally produced by extraction from calf abomasum as a by-product of veal production; shortage prompted its replacement for cheaper and more readily available sources, so that now it has been replaced to a considerable extent by recombinant chymosin produced in suitable microbial hosts, mostly from the genus Kluyveromyces [19] and Aspergillus [20]; in addition, chymosin variants produced by protein engineering have been obtained with increased specificity and better pH profile than the native enzyme [21]. Improvements of the kind have been applied to ton scale industrial enzymes, as illustrated by the case of tailor-made proteases specifically designed to act efficiently under the harsh conditions of laundering [22]. Improving microbial enzymes by the rational modification of their structure and selection of variants with improved features by high throughput screening methods is already making an impact on enzyme biotechnology [23, 24].
1.2 Production of Microbial Enzymes by Fermentation: A Process Perspective
There is a broad spectrum of applications of enzymes as process catalysts, going from large-scale processes for the production of commodities, where enzymes are used mostly as barely purified preparations [25], to highly sophisticated uses in the chemical synthesis of bioactive molecules where higher purity is required [26]. Production process is very much dependent on the level of production and type of application. In fact, industrial enzymes for the detergent and textile industries, and also many of the enzymes used for food and feed applications, are usually produced in large quantities as rather crude preparations [27]. On the other hand, enzymes used in more sophisticated processes of chemical synthesis for the production of drugs and other specialties are usually required in smaller amounts and higher purity. Immobilized enzymes are increasingly being used in both small- and large-scale processes and in such cases increasing the purity of the enzyme starting material may be rewarded by the higher specific activity of the biocatalyst obtained [28, 29]. Localization of the enzyme is another key feature determining the production process: exported enzymes will be recovered from the spent fermentation medium, while cell-bound enzymes will be recovered from the biosolids that will be further subjected to extraction by cell disruption or permeabilization [30, 31].
An enzyme production process can be divided into four stages: enzyme synthesis by propagation of the producing microorganism; enzyme recovery involving solid-liquid separation, concentration of the spent medium or cell disruption and cell debris removal; enzyme purification, consisting in one or more operations after enzyme recovery aiming to remove unwanted contaminants; enzyme product formulation including final polishing operations, stabilization and standardization.
1.2.1 Enzyme Synthesis
Microbial enzymes are mostly produced by submerged fermentations under highly controlled conditions; however, solid-state fermentation is a viable option when the producing microorganism is well conditioned for surface growth and a suitable solid substrate is available [32]. Submerged fermentation can be conducted in batch, fed-batch or continuous operation. Fed-batch is particularly appealing for the production of enzymes because it allows the control of metabolic responses of the producing cells and the operation is rather simple, thus it is often a preferred option at industrial level [33]. Continuous culture is in principle the most productive and controlled operation for producing microbial metabolites; however, industry is rather reluctant to adopt it because the prolonged operation times imply the hazard of washout of the producing strain by contamination or mutation [34], the latter being particularly critical when using recombinant microorganisms [35]. Besides enzyme localization, specific activity of the producing strains (units of enzyme activity per unit mass of microbial cells) is quite important, since it directly affects not only the cost of fermentation but also the cost of downstream operations; therefore, much effort has been spent in increasing it by both environmental manipulations (fermentation medium design, temperature and pH optimization, aeration and agitation rates) and genetic manipulations (genetic engineering and mutagenesis) [36]. Enzymes are subjected to different mechanisms of control, so that the corresponding triggering signals have to be considered for proper medium design. Most industrially relevant microbial enzymes are growth-associated, so increasing cell growth rate means increasing enzyme productivity. However, optimum conditions for growth seldom match those optimal for enzyme synthesis, so inevitably a compromise arises that should be judiciously solved in terms of enzyme production. Safety status of the producing strain is another key issue for selecting the enzyme and its usage at industrial scale. For instance, enzymes intended for food or pharmaceutical applications should be produced by microorganisms with the corresponding safety status. Obtaining such status may be costly and time-consuming so that in occasions it is a better option to clone the enzyme structural gene and express it in a safety host [37]. Regulatory issues with respect to enzymes produced from recombinant microorganisms [38], and genetic stability and safety are of paramount importance when producing recombinant enzymes [39].
1.2.2 Enzyme Recovery
After fermentation, solid-liquid separation is required to either collect the spent medium removing the cells in the case of extracellular enzymes, or the other way around in the case of cell-associated enzymes. Solid-liquid separation is a conventional unit operation that can be done by filtration or centrifugation, depending on the morphology of the producing microorganism [40].
Enzyme release is a highly desirable feature since the cell membrane acts as a powerful purification tool, so that further purification steps will be significantly reduced and in some cases unnecessary. In this case, a rather diluted liquid stream will be obtained; for a 20 g·L-1 cell concentration, extracellular protein is not expected to be much higher than 1 g·L-1 and even in the case of high cell density fermentations, extracellular protein will not exceed a few grams per liter, so that concentration of the spent medium will be required. It can be estimated that concentration should be increased by no less than one order of magnitude prior to the purification step, being a key determinant of the production cost of extracellular enzymes [41]. In this respect, the production of enzymes by solid-state fermentation is convenient since a much concentrated liquid can be obtained by pressing the fermented solids or recovering them with a reduced amount of extractant [42]. Vacuum evaporation and ultrafiltration are the most used operations for enzyme concentration, the latter being a preferred option for being gentler and readily scalable [43].
In the case of cell-associated enzymes, they should be recovered from the solid cell paste by extraction. The operation of extraction will be mostly determined by the location of the enzyme within the cell structure (periplasmic, cytoplasmic, or membrane bound) and the structure of the cell envelope. Operations for the recovery of cell-bound enzymes can be roughly divided into that producing cell disruption and that producing cell permeabilization by membrane damage. Periplasmic enzymes can be effectively recovered by permeabilization, while intracellular enzymes may require cell disruption. Microbial cells are hard to disrupt, especially bacteria and yeast, because of the resilient nature of their cell envelopes. Most used methods for recovery of cell-bound enzymes are in Table 1.2. Not all of them are amenable for scale-up at production level and the cost of this operation can represent a significant part of the enzyme production cost [44].
Table 1.2 Operations for the recovery of cell-bound enzymes [44].
Operation
Principle
Applicability at large scale
Cell rupture
Pressure
compression, shear stress
moderate
Homogenization
shear stress, cavitation
feasible
Milling
compression, shear stress
highly feasible
Sonication
cavitation
moderate
Decompression
decompressive explosion
moderate
Freezing-thawing
shear stress
unlikely
Dispersion in water
osmotic shock
unlikely
Thermolysis
cell wall rupture
moderate
Cell permeabilization
Alkali treatment
cell wall digestion
unlikely
Solvent treatment
membrane digestion
moderate
Enzymatic lysis
cell wall digestion and osmotic rupture
feasible
Autolysis
cell wall digestion and osmotic rupture
in some cases
Extracts, especially those obtained by cell disruption, are complex mixtures containing most of the intracellular components, so that downstream operations for enzyme purification become complex and costly, this being a major drawback of cell-associated enzymes. In this respect, gentle and more selective methods of enzyme recovery, involving permeabilization rather than disruption, are welcomed whenever possible [45, 46]. Whatever the method, enzyme extraction can be optimized in terms of recovery of active enzyme if sound models for the kinetics of protein extraction and enzyme inactivation at the conditions of extraction are developed [47]. A good method is one which is tough on cells and soft on proteins. After extraction, cell debris should be removed, which implies a second operation of liquid-solid separation. Besides the conventional methods, biphasic partition based on polymer incompatibility has been used successfully for producing particle-free extracts for the subsequent operations of purification [48]; the operation is readily scalable and equipment used is the same as used in liquid-liquid extraction [49].
1.2.3 Enzyme Purification
After recovery, the clarified concentrated broth containing the extracellular enzyme or the crude extract containing the intracellular enzyme is subjected to purification. Purification implies a series of operations aimed at removing the contaminants and producing an enzyme product of the desired purity. As said before, purification is much tougher for intracellular enzyme extracts. Leaving aside contaminants that can be rather easily removed, purification is focused mostly on removing contaminant proteins, so purification is essentially a protein fractionation stage. Purification is evaluated in terms of purification factor (PF) and yield of recovery (Y). Values for each purification operation (i) and for the whole purification stage composed by “n” consecutive operations are then defined by Eqs. 1.1 to 1.4
(1.1)
(1.2)
(1.3)
(1.4)
Where a is the specific activity of the enzyme (units of activity per unit mass of protein) and E is the total units of enzyme activity; subindex 0 denotes the initial values before purification.
Since (PF)i will be higher than 1, while Yi will be lower than 1, it is apparent that PF will increase while Y will decrease with the number of steps, so purity and recovery run into opposite directions; this means that the higher the purity required, the lower amount of enzyme recovered. Then, the criterion for establishing the final purity of the enzyme preparation is critical. For the case of so-called technical enzymes, which are produced at a large scale as commodities, the criterion of purification is the minimum compatible with its intended use. Purification at large scale is complex and costly and generally not justified by the benefit of producing a purer protein; in such cases Y rather than PF is the objective function and in practice it means few to none purification steps, in the case of extracellular enzymes. The situation is quite different in the case of specialty enzymes, where purity cannot be sacrificed in favor of yield. In the case of enzymes that will be further immobilized, there is an incentive in purification even for bulk enzymes, since higher mass activities can be obtained.
Methods for protein fractionation have been thoroughly developed at laboratory scale aiming to obtain pure proteins for structural and functional studies. Some, but not all of them, are applicable for the production of enzymes, and reported data on protocols for enzyme purification at laboratory scale are quite poor in terms of yield having little meaning for production purposes [50]. Besides PF and Y, throughput and rate of the operation have to be considered. Those methods that can be scaled up for production purposes can be divided into those based on differential solubility and those based on differential retention by interaction with a stationary phase, usually referred to as chromatography [47]. The former are based on the differential precipitation under non-denaturing conditions by the action of precipitating agents (salts, organic co-solvents, polymers). PF values attainable by fractional precipitation are rather modest, well below ten in most cases [51], and these operations are used rather as an initial concentration step since precipitates formed can be redissolved in a reduced volume of liquid so that further purification steps are conducted over a significantly lower volume. Chromatographic fractionation is a very powerful technique for protein purification, initially developed for analytical purposes, but preparative chromatography has been extensively used for laboratory scale enzyme purification. Several types of chromatography exist according to the principle of fractionation that have been applied for enzyme purification: size-exclusion chromatography (gel permeation) is based on the molecular size [52], ionic-exchange is based on ionic interactions among charged groups [53], hydrophobic chromatography is based on apolar interactions [54], and affinity chromatography is based on functional properties of the protein [55]; affinity chromatography is in principle the most selective but also the more complex and expensive method. Chromatography is quite powerful in terms of purification at laboratory scale; however, scale up to production level is not an easy task and a compromise exits between resolution and throughput. Despite the very high resolution of chromatography, because of hydrodynamic considerations, chromatographic columns cannot be scaled up by geometric congruence so that column diameter to bed height ratio increases with size and dispersion is increased reducing resolution [56].
Since enzyme purification is envisioned as a series of sequential operations, some guidelines have been proposed for the design of the purification stage. Operations should be selected to fully exploit the difference in physicochemical or functional properties of the enzyme with respect to the contaminant proteins, each operation should be based on a different property and simple though not very selective operations should be conducted first to early reduce the processing volume, using more complex and selective operations for a later stage to act on a reduced volume of product stream [57]. Rational design and optimization of protein purification processes have been developed and optimal operation sequences determined by using expert system [58, 59].
1.2.4 Enzyme Formulation
After purification, the preparation has to be formulated as a product to be stored and delivered to the customer in a format adequate to its intended use. This is a rather neglected aspect of enzyme production that is seldom reported in the open literature. However, this is important both for bulk industrial enzymes and specialty enzymes that should meet strict regulations, and represents a competitive edge for the enzyme producer. Health and safety aspects regarding enzyme production are a major concern, and regulations are determined by the end use of the enzyme and may vary considerably according to it; regulations may also vary from country to country [60]. Enzyme formulation involves final polishing operations, stabilization and standardization. Polishing operations include the removal of trace contaminants (i.e., pyrogens, endotoxins, viruses), and in the case of bulk industrial enzymes may include salt removal by diafiltration or size-exclusion chromatography, pH adjustment if produced in a liquid format and drying (usually spray-drying) if produced as a solid preparation. Additional polishing operations may be required in the case of enzymes for special uses. Production of the enzyme as a liquid or solid preparation is mostly dictated by the end use of the enzyme, but decision is not trivial. Concentrated liquid preparations present some advantages: containment is simpler, drying cost is avoided and dosage is simpler. Solid preparations are intended mostly when enzymes are included in solid products, like detergents, and have the advantage of easier handling and transportation, and in many cases extended storage stability. Stabilization is a key issue in enzyme formulation, since the enzyme product should withstand long periods of storage and transportation maintaining its catalytic integrity. Microbial degradation should be avoided and structural conformation preserved. The first goal may be achieved either by adding authorized preservatives or by sterilization using absolute filtration. Preservation of the enzyme conformation, avoiding aggregation, unfolding or any deleterious alteration of its three-dimensional conformation, is mostly important; proteins are more stable in concentrated solutions, so concentration also plays that role in the case of liquid preparations, but enzymes are usually formulated in the presence of structural stabilizers like neutral salts and polyols and sometimes more specific conformational protectants are used, like the enzyme cofactor, the enzyme substrate, a substrate analogue or even an inhibitor [61]. Enzyme products should be standardized to ensure a product of uniform quality to the customer or end user. Catalytic potency of the enzyme in terms of its specific activity should be warranted and since batch to batch variations are unavoidable, standardization is done by diluting the enzyme activity to a certified value using varying amounts of excipients to that purpose that may be the same substances used for preservation [62]. Enzyme producers have their own way of measuring activity which may or may not be relevant to the user, but this method has to be clearly described by the producer to be checked by the user. Storage stability is also important and should be clearly specified in the product sheet provided by the producer. Product sheets usually contain additional information about physical characteristics of the products, but information about excipients and other components is seldom reported. In some cases, additional information like pH and temperature profiles, and even values of kinetic parameters are also provided, which is welcomed. An enzyme product must comply with all requirements of quality and compatibility with intended used before being launched and be produced according to good manufacturing practices. Most enzyme producing companies have the corresponding ISO (International Organization for Standardization) 9001–2008 certificates warranting that the production process complies with the requirements for standardization and quality assurance. Enzymes are sometimes produced in immobilized format and in this sense immobilization could be considered as a final step of product formulation; however, immobilization has a much more significant meaning since it is oriented to improve the enzyme catalyst performance, so it will be presented in the next section.
1.2.5 Market Outlook for Industrial Enzymes
Enzymes are increasingly being used as industrial catalysts not only for conventional degradation processes but also for more sophisticated processes of organic synthesis [63].
Global industrial enzyme market was estimated close to US$ 4.5 billion by 2015 and might well be over US$ 5 billion by now. So-called technical enzymes represent 34%, enzymes for food and beverages represent 27%, and enzymes for other uses represent 39% [64]. The first two categories are mostly hydrolases acting on carbohydrates (glycosidases), on proteins (proteases) and on lipids (lipases). The last category includes the specialty enzymes being used in high-added value processes of organic synthesis, and represent the most dynamic sector. Lipases are outstanding catalysts for organic synthesis, being well suited for performing in low-water environments, as usually required for organic synthesis; its molecular structure is well conditioned to such purpose, many lipases being activated at water-oil interfaces and in hydrophobic solvents where they can catalyze esterification, transesterification and interesterification reactions [65]. Other hydrolases have been also used in their synthetic capacity; worthwhile mentioning is the use of proteases in peptide synthesis (i.e., synthesis of the dipeptide aspartame with thermolysin) [66] and glycosidases in oligosaccharide synthesis (i.e., synthesis of prebiotic galacto-oligosaccharides with β-galactosidase) [67].
Market figures should be taken with caution since a significant fraction of enzymes are produced by their own users or by joint ventures with supplying companies, so that the real commercial value of enzymes is definitely larger that the figures above given. Hydrolases still represent the major share of the enzyme market (about 45%); lyases represent more than 15% due to the massive use of nitrile hydratase in the bulk production of acrylamide; isomerases represent over 5% but this is referred solely to the use of glucose (xylose) isomerase in the production of high-fructose syrups for the food industry; oxidoreductases are co-enzyme requiring specialty enzymes used mostly for the synthesis of bioactive molecules for the pharmaceutical and fine-chemicals sectors and have experienced the most significant increase in recent years representing now close to 30% of the global market [26, 68].
Enzyme market is covered by a rather small number of highly specialized companies with outstanding skills in screening for new and improved enzymes, in highly controlled fermentation, in enzyme purification at large-scale, and in enzyme product formulation [69]. A list of most relevant industrial enzyme producers is presented in Table 1.3.
Table 1.3 Main industrial enzymes producers.
Company
Country
Main industrial areas of application
Novozymes
Denmark
Food and feed, biofuels, pulp and paper, leather, detergents
DuPont (Genencor)
U.S.A.
Food and feed, biofuels, detergents, textiles
DSM
The Netherlands
Food and feed, biofuels, detergents, textiles
BASF (Verenium)
Germany (U.S.A.)
Biofuels, animal health, pulp and paper, textiles
Amano
Japan
Food, pharmaceuticals
Novozymes and DuPont (Genencor) together represent about two-thirds of the enzyme market, the remaining one third being covered mostly by DSM, BASF and several Japanese companies.
In quantitative terms, food and feed, detergents, pulp and paper and leather are the most enzyme-demanding areas, where hydrolases are still prevalent. Emerging areas like biofuel and several applications in organic synthesis for the production of bioactive compounds are rapidly gaining a prominent place within the enzyme market, where not only conventional hydrolases are being used, but also coenzyme-requiring more complex enzymes like oxidoreductases, lyases and transferases that are progressively coming into play [70]. On the other hand, China, India and South Korea are increasingly relevant countries for industrial enzyme production.
1.3 Building Up Enzyme Catalysts for Process Applications
Enzymes are physiological catalysts that are not necessarily well suited to perform under process conditions. Converting these physiological catalysts into robust process catalysts is undoubtedly the major challenge for enzyme biocatalysis. This is even more so now, when enzymes are increasingly being used in reactions of organic synthesis, which are usually conducted under rather harsh conditions. Several strategies to tackle this problem are available, considering biocatalyst design, medium engineering and also bioreactor design [71].
Nature is an endless source of bioactive molecules and new enzymes with adequate properties for process applications can be obtained by bioprospecting [72] and the metagenomic approach is gaining increasing importance for the discovery of novel enzyme functions [73–75]. However, wild-type enzymes are frequently not active or stable enough for process applications so that improving them by molecular redesign and other protein engineering techniques already mentioned are of paramount importance for building-up industrial enzyme biocatalysts, both for conventional degradation processes and for organic synthesis [76–79]. Enzyme stabilization has received considerable attention, being a key issue for developing industrially useful enzyme biocatalysts [80]. Enzyme immobilization is probably the most powerful tool for producing robust process biocatalysts, salient features of immobilized enzymes being their increased stability, reusability and flexibility for reactor operation [81, 82]. Many strategies for enzyme immobilization are envisaged considering both carrier-bound and carrier-free systems [83]. The former allows high flexibility in biocatalyst design, produce very robust catalysts with increased stability and susceptibility to reactivation, but the considerable amount of inert material implies additional costs and low specific activities [84]. On the other hand, carrier-free biocatalysts, especially cross-linked enzyme aggregates (CLEAs) have very high specific activities because a substantial portion of the catalyst mass is active protein, and are simple to produce [85]; however, their operational stability and handling at process conditions are drawbacks to be overcome. Optimization of enzyme immobilization is a complex task, since it is a multivariable process and a sound, but not obvious, objective function is required for evaluation, so that no rational guidelines have been developed and the best system and conditions have to be determined in each case [86]. Despite this, immobilized enzymes have had a profound impact on enzyme biocatalysis at industrial level [87, 88] and are being increasingly important as catalysts for organic synthesis where operation conditions can be harmful for maintaining the enzyme active configuration [89]. Advances in several fields, like molecular biology, bioinformatics and material sciences are allowing an increasingly rational approach to enzyme immobilization [90–92], and sometimes the concept of immobilization engineering has been used to refer to this more rational approach to enzyme immobilization [93, 94].
In the last two decades, enzyme biocatalysis has experienced a change in paradigm: enzymes were traditionally considered as catalysts for aqueous media and, in fact, most of the early enzymes used were hydrolases acting in high water activity environments, but a gradual but sustained trend of biocatalysis as an alternative for organic synthesis has moved the attention to non-hydrolytic enzymes and hydrolases acting in reverse. The former are complex, labile and coenzyme-requiring proteins, so technological development has been complex; however, the high added value of some processes of synthesis [95] and the compliance of biocatalysis with the principles of green chemistry [96], have been the driving forces for development. Despite their complexities, oxidoreductases [97], lyases [98] and transferases [99] are being increasingly evaluated as catalysts for the synthesis of sophisticated bioactive molecules, where the molecular precision of the reaction is a key feature [100]. Hydrolases, that are robust and readily available enzymes, over which biocatalysis was developed, can under certain conditions act in reverse catalyzing bond formation instead of hydrolysis. This is of paramount significance since hydrolases can act with very high molecular precision to
