...................................

------~--

N. GRAY

M. CALVIN

S.C. BHATIA

;I
Alkern Company (S) Pte Ltd
Singapore
COLLEGE OF ENGINEERING LIBRARY I
UNIVERSITY OF THE PHILIPPINES DILlMAN

Enzymes Biotechnology

ISBN: 9789810860882

First Edition: 2011

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Preface
Enzymes are biological catalysts, or chemicals that speed up the rate of reaction between
substances without themselves being consumed in the reaction. As such, they are vital to such
bodily functions as digestion, and they make possible processes that normally could not occur
except at high temperatures when they would threaten the well-being of the body. A type of
protein, enzymes, sometimes work in tandem with non-proteins called co-enzymes. Among the
processes in which enzymes play a vital role is in fermentation, which takes place in the
production of alcohol or the baking of bread and also plays a part in numerous other natural
phenomena, such as the purification of waste-water.
Enzymes are special kinds of proteins that are found in all living matter. Man, animals,
plants-all living cells produce and need enzymes to live and grow. However, enzymes
themselves are not living organisms. Enzymes are catalysts substances which speed up
chemical reactions by being present in very small amounts and without being changed in the
reaction.
Modern biotechnology techniques are utilized to improve microbial production strains to
increase to enzyme yields and to make minor amino acid changes that improve the
functionality of the enzyme. These changes are not known to increase the ability of enzymes to
cause allergies.
This reference textbook is divided into five sections. Section I discusses about principles of
industria! enzyme production and utilization. Chapter 1 in ~his section is devoted to basic
concepts of enzymes. Chapter 2 deals with general characteristics of enzymes, such as structure,
mechanism and kinetics of enzymes. Chemical reactions, including those catalyzed by
enzymes, depend on environmental conditions. The emphasis in Chapter 3 is on micro and
macro environmental effects on environmental activity, and micro as well as
macroenvironmental effects. The dominant environmental factors affecting enzyme action are
pH, water activity and temperature. Chapter 4 concentrates on modern methods of enzyme
expression and design in a simplified and understandable manner- genetically engineered
products and amplification of genetic engineering for new food related applications. Chapter 5
deals with fermentation process design. The task of the fermentation technologist is to provide
and maintain the environment required for carrying out a desired biological process. Thus, a
well designed growth environment is needed to obtain the maximum economic yield of the
product and this task resolves into environmental design, determining the environmental
requirements of the process, and fermentation engineering, providing the means for meeting
those requirements of the process, and fermentation engineering. Chapter 6 has a focus on
extraction and purification of enzymes. Various mediums of extraction and purification are
discussed in detail.

Section II concentrates on types of enzymes/ classification of enzymes. Chapter 7

concentrates on carbohydrate hydrolyzing enzymes. Chapter 8 is devoted to proteases which
are enzymes that digest proteins. Various types of proteases, such as plant, animal, microbial,
serine, etc. are discussed in detail. Chapter 9 deals with lipases which are enzymes those
catalyses the hydrolysis of fat or breakdown of lipoproteins. The chapter discusses sources of
microbial and industrial sources of lipases along with processes of application of lipases.
Chapter 10 concentrates on oxidoreductases which are enzyme catalyzing a reaction in which
two molecules of a compound interact so that one molecule is oxidized and the other reduced
with a molecule of water entering the reaction. Various types of oxidoreducatases along with
their general characteristics are discussed.
Section III is devoted to the application of enzymes in the industry. Enzymes are involved in
the formation of starch, gluten, hemicelluloses, and lipids during ripening and are the key to
dough fermentation and bread making, since they degrade these polymers. The activities of
enzymes in the flour used for bread-making depend on the climate, the type of grain, and the
harvesting conditions. Chapter 11 focuses on milling and baking. Chapter 12 and 13 discuss
starches, sugars and syrups and dairy products. Pectic enzymes occur in higher plants and are
produced by micro-organisms. Chapter 14 concentrates on pectic enzymes in fruit and
vegetable juice manufacture. Chapter 15 deals with enzymes associated with savoury flavour
enhancement. The focus of Chapter 16 and 17 is on wine and brewing. Chapter 18 is concerned
with fish processing and meat industry. Chapter 19 is concerned with textiles and laundry
detergents. Weaving textile fibers exposes the fabric's warp and weft strands to substantial
mechanical stress. To prevent breakage of these strands, they are generally reinforced with
sizing. Sizing can consist of various materials. Chapter 20 and 21 deal with pulp and paper as
well as tanning industry. Chapter 22 and 23 concentrates on clinical analysis and organic
synthesis. Chapter 24 deals with enzymes and bioremediation. Biological methods or
bioremediation promises to provide an eco-friendly solution to this menacing problem.
Section IV concentrates on instrumental techniques used in enzyme analysis. Chapter 25 is
devoted to instrumental techniques in enzymatic analysis. The course of enzyme-catalyzed
reactions may be monitored by a variety of techniques, of which the most common at the
present time are spectrophotometric, spectorfluorimetric and electrochemical procedures.
Chapter 26 and 27 deal with enzyme thermistors and enzyme electrodes for food analysis.
Section V focuses on special topics. Chapter 28 focuses on legal considerations. Regardless
of whether enzyme preparations are defined as food additives or technical aids, it must be
established that they are not health hazards. The purpose of this chapter is on legal aspects of
enzyme utilization and to inform the readers about the measures that the scientific community
and legislators have taken to provide maximum safety for both users and consumers of enzyme
preparation. It should be noted that regulations for enzyme preparations and their use are by no
means uniform throughout the world. Chapter 29 deals with economic considerations for the
use of technical enzymes. The economic benefits of using technical enzyme preparations lies in
lower process costs, often increasing product quality, reducing environmental impact, and

making use of renewable resources in technology. Chapter 30 is devoted to patenting

biotechnology inventions. For biotechnology, patents are the most important form of
intellectual property. The chapter discusses number of reasons for issuing patents. For
examples, the patent holder obtains exclusive rights for the invention, rights that are a reward
for developing procedure, compound or apparatus. For a patent to be granted, an invention
must be novel, not obvious and useful.
Glossary and index have been provided at the end for quick references. Diagrams, figures
and tables supplement the text. All the topics have been covered in a cogent and lucid style to
help the reader grasp the information quickly and easily.
The book should serve as an essential reference textbook of enzymes biotechnology, and
useful reading for all students and teachers of engineering, environment and life sciences. The
researchers in analytical and instrumental techniques, chemists and industrialists will also find
it highly useful and informative.

N. Gray
M. Calvin
S.C. Bhatia

Contents at a Glance
Preface .................................................................................................................................................... v

SECTION I
Principles of Industrial Enzyme Production and Utilisation ..................................................... 1-99

Chapter

1. Basic Concepts of Enzymes .......................................................................................... 3-7

Chapter 2. General Characteristics of Enzymes ............................................................................ 8-31

Chapter

3. Environmental Effects on Enzyme Activity ............................................................... 32-47

Chapter 4. Modem Methods of Enzyme Expression and Design ................................................. 48-68

Chapter

5. Fermentation Process Design ..................................................................................... 69-85

Chapter

6. Extraction and Purification of Enzymes ..................................................................... 86-99

SECTION II
Types of Enzymes/Classification of Enzymes ....................................................................... 101-272

Chapter

7. Carbohydrate Hydrolysing Enzymes ........................................................................ 103-185

Chapter

8. Proteases .................................................................................................................... 186-222

Chapter

9. Lipases ...................................................................................................................... 223-231

Chapter 10. Oxidoreductases ........................................................................................................ 232-272

SECTION III
Application of Enzymes in Industry .......................................................................................... 273-508

Chapter 11. Milling and Baking .................................................................................................. 275-292

Chapter 12. Starches, Sugars and Syrups ................................................................................... 293-309
Chapter 13. Dairy Products .......................................................................................................... 31 0-317
Chapter 14. Pectic Enzymes in Fruit and Vegetable Juice Manufacture ..................................... 318-341
Chapter 15. Enzymes Associated with Savoury Flavour Enhancement ..................................... 342-357
Chapter 16. Wine .......................................................................................................................... 358-367
ix

x Contents at a Glance
Chapter 17. Brewing ..................................................................................................................... 368-381
Chapter 18. Fish Processing and Meat Industry ......................................................................... 382-389
Chapter 19. Textiles and Laundry Detergents ............................................................................. 390-399
Chapter 20. Pulp and Paper ........................... ,.............................................................................. 400-413
Chapter 21: Tanning Industry ....................................................................................................... 414-425
Chapter 22. Clinical Analysis ...................................................................................................... 426-456
Chapter 23. Enzymes in Organic Synthesis ................................................................................ 457-481
Chapter 24. Enzymes and Bioremediation ................................................................................... 482-508

SECTION IV
Instrumental Techniques Used in Enzyme Analysis ............................................................ 509-551
Chapter 25. Instrumental Techniques in Enzymatic Analysis .................................................... 511-523
Chapter 26. Enzyme Thermistors for Food Analysis ................................................................. 524-534
Chapter 27. Enzyme Electrodes for Food Analysis .................................................................... 535-551

SECTIONV
Special Topics ................................................................................................................................ 553-575
'

Chapter 28. Legal Considerations ................................................................................................ 555-565

Chapter 29. Economic Considerations for the Use of Technical Enzymes ................................ 566-569
Chapter 30. Patenting Biotechnology Inventions ......................................................................... 570-575
References ........................................................................................................................................... 577
Index ............................................................................................................................................... 579-586

...

Contents
Preface ................................................................................................................................................... v
Contents at a glance .. ............................. .. ... ........ .. ............. ... . .. .. ... ... ... ... .. ... .... .. . .. . .. .. .. .... .. ... .. ......... .. ..

IX

SECTION I
Principles of Industrial Enzyme Production and Uti/isation ..................................................... l-99

1.

Basic Concepts of Enzymes ...................................................................................................... 3-7

Introduction ................................................................................................................................... 3
Enzymes Defined as Catalysts ....................................................................................................... 4
History ofthe Development ofTechnical Enzymes ........................................................................ 4
Applications of Modem Enzyme Technology ................................................................................ 6

2.

General Characteristics of Enzymes ..................................................................................... 8-31

Introduction ................................................................................................................................... 8
Important Role of Enzymes ........................................................................................................... 8
Enzyme Structure and Action Mechanisms ................................................................................... 11
Enzyme Analysis and Enzyme Units ........................................................................................... 12
Enzyme Kinetics ........................................................................................................................... 17
Inhibition and Activation .............................................................................................................. 22
Non-Mi'r;haelis-Menten Behaviour ............................................................................................... 26
Kinetics of Multi enzyme Systems ............................................................................................... 28
Enzyme Utilisation Industry ........................................................................................................ 29

3.

Environmental Effects on Enzyme Activity ........................................................................ 32-47

Introduction ................................................................................................................................... 32
Effect of pH ................................................................................................................................... 32
Effect of the Aqueous Environment .............................................................................................. 34
Effect ofTemperature ................................................................................................................... 40
Effect of Other Environmental Conditions .................................................................................. 46

4.

Modern Methods of Enzyme Expression and Design ......................................................... 48-68

Introduction ................................................................................................................................... 48
Background ................................................................................................................................... 48
xi

xii

Contents
Genetic Engineering ..................................................................................................................... 51
Commercialisation of Genetically Engineered Products .............................................................. 61
Implications for the Future ........................................................................................................... 67

5.

Fermentation Process Design ............................................................................................... 69-85

Introduction ................................................................................................................................... 69
Process Design ............................................................................................................................... 69
Mode of Operation ....................................................................................................................... 72
Adaptive Control ......................................................................................................................... 82
Knowledge-based Control ............................................................................................................ 82
Statistical Optimisation ................................................................................................................ 83
Variability ..................................................................................................................................... 85

6.

Extraction and Purification of Enzymes ............................................................................ 86-99

Introduction ................................................................................................................................... 86
Extraction of Enzymes .................................................................................................................. 86
Purification of Enzymes .............................................................................................................. 90
Determination of Molecular Weights of Enzymes ....................................................................... 97

SECTION II

Type_s of Enzymes/Classification of Enzymes ....................................................................... 101-272

7.

Carbohydrate Hydrolysing Enzymes ............................................................................... 103-185

Introduction ................................................................................................................................... 103
Action of Enzymes ...................................................................................................................... 103
Amylases ..................................................................................................................................... 105
Carbohydrases Other Than Amylases ........................................................................................ 128
Cellulose Degradation ................................................................................................................ 141
Hemicellulases .....~ .................................................................................................................... 160
Pectin Degradation ..................................................................................................................... 169.
Pentosanases ............................................................................................................................... 179

8.

Proteases .............................................................................................................................. 186-222

Introduction ................................................................................................................................... 186
Plant Proteases ........................................................................................................................... 187
Papaya Proteases ............................................................................................................... 187
.Animal Proteases .. .. .. . .. . ... ..... .. . ... ... ... ...... .. ...... .... ..... ..... ...... ... .. ... .... .......... . .... .. ..... .. ...... .. .... .. .. .. 190
Microbial Proteases: Brief Overview ........................................................................................ 196
Ester Cleavage: Fat Hydrolysis ................................................................................................. 211

9.

Lipases ................................................................................................................................ 223-231

Introduction ................................................................................................................................... 223
Sources ofMicrobial Lipases ..................................................................................................... 223
Lipases in Food Manufacturing Processing ................................................................................ 227
Structured Lipid with the Aid ofLipases .................................................................................. 229

Contents

xiii

10. 0-xi.doreductases .................................................................................................................... 232-272

Introduction ................................................................................................................................... 232
Polyphenol Oxidase ....................................................................................................................... 232
Peroxidases ................................................................................................................................... 241
Lactoperoxidase ......................................................................................................................... 248
Catalase ...................................................................................................................................... 250
Sulphydryl Oxidase .................................................................................................................... 251 ..
Glucose Oxidase ........................................................................................................................ 254
Pyranose Oxidase ...................................................................................................................... 256
Xanthine Oxidase ........................................................................................................................ 257
Lipoxygenase ............................................................................................................................. 258
Dehydrogenases ......................................................................................................................... 264
Application ofOxidoreductases ................................................................................................. 265

SECTION III
Application of Enzymes in Industry .......................................................................................... 273-508

11.

Milling and Baking .............................................................................................................. 275-292

Introduction ................................................................................................................................... 275
Enzyme in Flour ........................................................................................................................ 276
Use in the Baking Industry ........................................................................................................ 282
Conclusion .................................................................................................................................. 292

12. Starches, Sugars and Syrups ............................................................................................. 293-309

Introduction ................................................................................................................................... 293
Starch Production ....................................................................................................................... 293
Starcb.froduction and Application .............................................................................................. 294
Sweetener and Syrup Production ................................................................................................ 296
Applications of Sweeteners and Syrups ...................................................................................... 307

13. Dairy Products ...............:................................................................................................... 310-317

Introduction ................................................................................................................................... 31 0
Natural Enzymes of Milk .............................................................................................................. 310
Milk-Clotting ............................................................................................................................... 311
Cheese Ripening ......................................................................................................................... 314
Lactase ....................................................................................................................................... 315
Hydrogen Peroxide and Catalase Treatment ............................................................................... 316
Miscellaneous Application of Enzymes in Dairy Processing ..................................................... 316

14. Pectic Enzymes in Fruit and Vegetable Juice Manufacture ........................................... 318-341
Introduction ................................................................................................................................... 318
Pectic Substances and Pectic Enzymes ...................................................................................... 318
Occurrence and Microbial Production of Pectic Enzymes ......................................................... 320

xiv

Contents
Food-Processing-Related Properties ofPectic Enzymes ............................................................ 321
Methods Used in Technological Pectic Enzyme Research ........................................................ 325
Use of Commercial Fungal Enzymes as Processing Aids .......................................................... 327
Quality Aspects of Clear Fruit Juices Obtained by Enzyme Treatment of Juices or Pulp .......... 333
Technological Functions ofEndogenous Pectic Enzymes ......................................................... 337

15. Enzymes Associated with Savoury Flavour Enhancement ............................................. 342-357

Introduction ................................................................................................................................... 342
Flavour Enhancers ..................................................................................................................... 342
Enzymatic Hydrolysis ofNuclear Material ................................................................................ 344
Commercial Production of Extracts Rich in Flavour Enhancers ............................................... 353
Yeast Extracts ............................................................................................................................. 353
Conclusion .................................................................................................................................. 356

16. Wine ................................................................................................................................... 358-367

Introduction ................................................................................................................................... 358
Endogenous Enzymes ..................................................................................................................... 358
Use of Commercial Enzyme Preparations in Wine Making ........................................................ 361
Future Developments ................................................................................................................. 366

17. Brewing ........................................................................................................................ ~:: ..... 368-381

Introduction ................................................................................................................................... 368
Malt and Its Reactions during Brewing ....................................................................................... 368
Use of Brewer's Adjuncts .......................................................................................................... 372
Brewing with Very High Adjunct Levels ..................................................................................... 374
Keeping Beer Clear with Chillproofing Enzymes ....................................................................... 375
Making New and Special Types of Beer with Enzymes ............................................................ 376
Improving Filtration with Enzymes ........................................................................................... 379
Extension of Shelf Life of ~ackaged Beer ................................................................................. 380

18. Fish Processing and Meat Industry ................................................................................ 382-389

Introduction ................................................................................................................................... 382
Traditional Processes ................................................................................................................. 382
New Applications of Enzymes in Fish Processing ..................................................................... 385
Enzymes in the Meat Industry ................................................................................................... 388

19. Textiles and Laundry Detergents ..................................................................................... 390-399

Introduction ................................................................................................................................... 390
Desizing Enzymes ...................................................................................................................... 390
Enzymes in Laundry Detergents ................................................................................................ 394

20. Pulp and Paper .................................................................................................................. 400-413

Introduction ................................................................................................................................... 400
Process and Production of Pulp for Paper .................................................................................. 401

Contents

xv

Recent Advances in the Application of Enzymes for Paper and Pulp ......................................... 409
Prospects of Genetic Engineering in Bioconversion .................................................................. 411

21. Tanning Industry ................................................................................................................ 414-425

Introduction ................................................................................................................................... 414
Skin Structure ............................................................................................................................. 415
Enzymes for Leather Processing ................................................................................................. 415

22.

Clinical Analysis ................................................................................................................ 426-456

Introduction ................................................................................................................................... 426
Measurement of Substrate Concentration with Enzymes ............................................................ 428
Measurement of Enzymes .......................................................................................................... 435
Immobilised Enzymes for Measuring Substrate Concentrations ............................................... 438
Enzyme Immunoassay (EIA) ..................................................................................................... 444
Future ......................................................................................................................................... 456

23. Enzymes in Organic Synthesis ........................................................................................... 457-481

Introduction ................................................................................................................................... 457
Hydrolytic Enzymes .................................................................................................................. 457
Oxidative-Reductive Enzymes ................................................................................................... 466
Carbon-Carbon Bond-forming Enzymes .................................................................................... 471

24.

Enzymes and Bioremediation ........................................................................................... 482-508

Introduction ................................................................................................................................... 482
Xenobiotic Compounds .................................................................................................................. 483
Toxicity ...................................................................................................................................... 486
Adaptive Responses ................................................................................................................... 487
Physico-chemical Factors Influencing Biodegradation .............................................................. 487
Hydrocarboo Degradation .......................................................................................................... 488
Enzymology of n-Alkane Oxidation ........................................................................................... 490
Genetic Regulation of Alkane Oxidation ................................................................................... 493
Biodegradation ofAromatic Compounds .................................................................................. 496
Mechanism ofMonocyclic Hydrocarbon Degradation .............................................................. 498
Enzymes of the Lower Pathway ................................................................................................. 503

SECTION IV
Instrumental Techniques Used in Enzyme Analysis ............................................................ 509-551

25. Instrumental Techniques in Enzymatic Analysis ............................................................ 511-523

Introduction ................................................................................................................................... 511
Principles of the Available Detection Techniques ........................................................................ 511
Automation in Enzymatic Analysis ........................................................................................... 518

xvi

Contents

26. Enzyme Thermistors for Food Analysis ........................................................................... 524-534

Introduction ........................ ,.................................................................................................... .'..... 524
Measuring Principle .................................................................................................................... 525
Instrumentation .......................................................................................................................... 526
Applications ............................................................................................................................... 527

27. Enzyme Electrodes for Food Analysis .............................................................................. 535-551

Introduction ................................................................................................................................... 535
Fundamentals ofAmperometric Devices ...................................................................................... 536
Electrode Materials and Fabrication .......................................................................................... 537
Development of Analytical Devices ........................................................................................... 538
Examples of Enzyme Electrodes Applied to Food Analysis ...................................................... 542
Enzyme Electrodes for Alcoholic Compounds ........................................................................... 548

SECTIONV
Special Topics ................................................................................................................................ 553-575

28. Legal Considerations ......................................................................................................... 555-565

Introduction ................................................................................................................................... 555
Regulatory Requirements for Enzyme Preparations .................................................................... 556
Who is Involved with Enzymes? ............................................................................................... 556
Toxicity and Allergies ................................................................................................................. 557
Toxins ......................................................................................................................................... 557
Documentation .......~................................................................................................................... 559
Some National Regulations ........................................................................................................ 560

29. Economic Considerations for the Use of Technical Enzymes ........................................ 566-569
Introduction ................................................................................................................................... 566
Increasing Yields and Improving Raw-materials Utilisation ..................................................... 566
Lowering Costs .......................................................................................................................... 567
Altering Technical Properties ....................................................................................................... 567
Improving Preservation, Flavour and Cleansing Action ............................................................ 567
Market ofTechnical Enzyme Preparations ................................................................................. 569

30. Patenting Biotechnology Inventions ................................................................................. 570-575

Introduction ................................................................................................................................... 570
What Constitutes a Patent? ......................................................................................................... 571
Patenting in Different Countries ................................................................................................. 573
Patenting Multicellular Organisms ............................................................................................. 573
Patenting and Fundamental Research ......................................................................................... 574

References ........................................................................................................................................... 577

Index ............................................................................................................................................... 579-586

SECTION I
Principles of Industrial Enzyme
Production and Utilisation
1. Basic Concepts of Enzymes
2.
'

G~~eral

Characteristics of Enzymes

3
8

3. Environmental Effects on Enzyme Activity

32

4. Modern Methods of Enzyme Expression and Design

48

5. Fermentation Process Design

69

6. Extraction and Purification of Enzymes

86

Basic Concepts of Enzymes

INTRODUCTION

Life on earth is based on constant change. Inorganic matter forms the complicated structures that make
up the living world, which will again decay into lifeless matter. The lives of plants, animals and human
beings are included in this great cycle. The inert substances that form the foundation of life have a very
simple structure. Water. carbon dioxide and nitrogen are the basic ingredients that, with the ~nergy
supplied by sunlight, permit plants to synthesise the molecular moieties that support life. These compounds
are 1:1en used to provide more elaborate compounds required by animal life.
The use of enzymes and micro-organisms in processing raw materials from plants and animals has
oeen practiced for a long time. At one time, living micro-organisms were used predominantly. Traditional
processes, such as the production of alcoholic beverages and yeast-fermented doughs in baking bread,
are displayed in Egyptian wall paintings. Further examples are the processes for preserving food, such
as vegetable conservation by fermentation with lactobacilli, or preserving milk by making cheese.
Chemical !xperiments have shown how specific compounds. namely, the essential amino acids, can .
be generated from simple mixtures of carbon, nitrogen, oxygen and hydrogen when subjected to extreme
pressures and temperatures. Once formed, less extreme conditions can cause the degradation of these
complex structures as well. Under the current global conditions, such processes proceed only very
slowly. What, then, causes the rapid synthesis of these compounds that are essential for life and the
degradation ofthe high-molecular-weight and highly organised structures that make up life? The catalysts
for this assembly and disassembly were for a long time thought to be life itself, or rather a fundamental
principle of living cells.
The first observation of an enzymatic degradation reaction was by Spallanzani, a priest and naturalist
for Padua, Italy. After placing meat in small porous capsules, he examined the regurgitated pellets of the
hawks who had eaten this material and found the capsules to be empty, proving that the meat had been
rapidly liquefied by the stomach juices of these birds of prey.
Later, Kirchhoff found that barley contained a substance that was capable of liquefying starch paste
into sugar. He assumed that the reaction was caused by the gluten protein of the barley. Subsequently,
Payen and Persoz termed the working principle of this saccharification diastase (Greek for separation),
a term still used for the amylases in the brewing industry. The degradation of starch, which is composed
of sugar moieties and the subsequent process of alcoholic fermentation of the sugar into carbon dioxide
and alcohol occupied much of this nineteenth-century chemist's time.
I

Enzymes Biotechnology

Pasteur showed that fennentation is closelv associated with live veast. He distinguished between the
actions of 'organised ferments' (cellular) and the 'unorganised ferments (soluble). These soluble
ferments'. which are not bound to the living ceiL were labelled enzymes by KUhne. This tennis derived
from the Greek enzyme. meaning 'in sour dough. Concrete evidence for this assumption was provided
by E. Buchner, as he showed that the cell-free extract from yeast cells could also produce alcohol from
sugars. The enzyme active in fennentation was tenned zymase. The first book summarising the 'wonderful
mechanism of the enzymes known at that time and outlining the history of their discovery was written
by Green and is still read with interest today.
'

'

ENZYMES DEFINED AS CATALYSTS

Friederich Wilhelm Ostwald first coined the now-familiar definition of a catalyst. specifically. that a
catalyst is a substance that alters the rate of a chemical reaction without being present in the reaction
products. Thus. Ostwald recognised enzymes as catalysts.
Today, catalysts and catalysis are familiar terms. The catalysts in internal-combustion engines
accelerate the conversion of the pollutants carbon monoxide and nitrogen oxides into the less polluting
carbon dioxide and nitrogen, without any change in the catalysts during the process. According to the
basic research of Tammann and Van 't Hoff, a catalyst must be able to regenerate the initial substrate
from its products. Enzymatic reactions also have an equilibrium. An example is the fonnation an(
scisson of an ester that forms on reaction between an alcohol with an acid:
Acetic acid + alcohol

SvntheSIS
~======:

acetate ester + water

Hvdrolys1s

If acetic acid and alcohol are mixed in equimolar amounts. some ofthe molecules will react to fonn
an ester and water (synthesis). Yet. there still will be significant quantities of acetic acid and alcohol in
the reaction mixture as ';'ell as the products generated, ester and water. A mixture of the same composition
can also be obtained when starting with water and an ester (hydrolysis). Even in the absence of a
catalyst. the reactions reach equilibrium between ester and synthesis and ester hydrolysis.
When forming and splitting butyl ester. it was found that the equilibrium was rapidly established
from either direction on the addition of porcine pancreatic powder (pancreatic lipase). Thus. for life to
exist, the biochemical equilibria of essential and exceedingly complex reaction systems must be
established rapidly. Therefore, enzymes are the vital catalysts in all life processes.

HISTORY OF THE DEVELOPMENT OF TECHNICAL ENZYMES

Enzyme preparations. from extracts of plants or animal tissue. were used well before much was known
about the nature of properties of enzymes. The development oftoday"s industrially important microbial
enzymes began with the Japanese Jokichi Takamine. Born in Japan and later a US immigrant. Takamine
studied the production ofenzymes from moulds. He obtained a process patent for making a diastatic
enzyme preparation from-fungi. He named the product Takadiastase. a mixture of carbohydrases and
proteolytic enzymes.
The production occurred either via a surface culture, a semisolid culture, or the koji process on moist
wheat bran with nutrient salts and buffers added. The sterile culture media. mixed with spores from
Aspergillus oryzae, was poured onto metal trays to a thickness of a few centimetres and incubated in
climate-controlled culture chambers. The culture media, permeated with fungal mycelia, are ground
and extracted with water. In a modified fonn, this process is still used by various manufacturers. although

Basic Concepts of Enzymes

1
1

it has largely been replaced by the development of the submerged fermentation process carried out in
large fermenters.
Auguste Boidin discovered a new process for the manufacture of alcohol from cereals. Known as
the amyloprocess, this method involves the extraction of cereal polysaccharides by boiling, then
inoculation with a fungus that, in turn. produces saccharifying enzymes and subsequently yeast
fermentation of the sugars. Boidin and Professor Jean Etfront who had studied enzymes involved in
alcohol production since 1900, founded the Societe Rapidase in 1920.
Enzyme preparations from animal organs also continue to play an important industrial role. Otto
Rohm discovered the effectiveness of pancreatic proteases in liming and bating of hides in leather
manufacture. Until then, slurries of dog feces had been used in bating. Rohm concluded that the bating
effect of dog feces was due to excreted pancreatic proteases and subsequently developed the first
standardised enzymatic bating agent that marketed under the name oropon', offered the tanner greater
control and reliability in the sensitive leather manufacturing process.
Otto Rohm developed additional uses for the pancre1tic enzymes he manufactured in Darmstadt.
including the degumming of raw silk. The importance of these enzymes for the silk industry at that time
can be shown by the following cost computation: 'One kilogram Degomr .. a S (pancreatic protease)
with a value of 4 [Reichsmark] can degum the same amount of silk as I 0 kilograms of expensive
Marseilles soap' (at that time 2-3 reichsmarks per kilogram). Rohm introduced the first commercially
enzymatic detergent. The product, named Burnus'. rapidly became known as a good prewash and
soaking agent. In addition to pancreatic enzymes, it contained sodium carbonate and sodium bicarbonate,
which held the pH in the wash water below 9. The pancreatic proteases are sufficiently active and stable
at low temperatures in this pH range. However, pancreatic enzymes are inactive in new detergent
formulations that contain sequestrants, anionic detergents and oxidising agents.
Jaag in Switzerland developed a detergent that contained, as active components, proteases from
Bacillus subtilis. The Novo Company [NO] in Copenhagen deserves credit for the subsequent
development of this enzyme. This will be discussed in more detail later. Plant enzymes, especially the
proteases fr8m papaya fruits (i.e. papain), are industrially important. Wallerstein was the first to use
papain for stabilising beer. Beer stabilisation. which prevents protein flocculation or hazing on
refrigeration, is particularly important in the United States, where beer is cold-stored.
Boidin and Effront obtained a process patent for manufacturing an enzyme preparation from cultures
of B. subtilis, grown on the surface of a liquid nutrient medium. The preparation primarily contained
amylase, which was able to replace the malt that previously had been used in textile sizing.
Rohm and Haas began producing fungal proteases with semisolid cultures. These enzymes produced
gentle bating preparations suited tor special applications in leather manufacture. About 1934, the first
pectinase preparations for clarifying fruit juices and improving juice extraction from grapes were produced
by the same method.
In 1958, Underkot1er published a list of commercial enzyme preparations. The most important products
were the amylases that were used in bakeries and breweries and in the manufacture of starch hydrolysates.
Second were the microbial proteases, which were used in leather manufacture and in removing ~azes
from cold beer. Other enzymes such as pectinases, lactases, invertases. lipases, and cellulases were.
however. produced only in small quantities. The years following World War II saw a rapid development
of submerged fermentation technology, triggered by the then-extant antibiotic manufacturing methods.
Bacterial and fungal amylases could now be manufactured inexpensively. This had a great influence on
the commercial development of enzymatic starch conversion to dextrins and glucose.

Enzymes Biotechnology

In 1960, Novo Nordisk [NO] began production of alkaline bacterial proteases from Bacillus
licheniformis for use in detergents. A flurry of activity followed that led to a wave of innovations in the
entire field of industrial enzymes.
The commercial development of alkaline bacterial proteases was briefly interrupted when cases of
lung disease, due to allergic reactions, were reported in the United States. These allergies occurred in
workers who had direct contact with dusty enzyme concentrates during detergent manufacture.
Granulating the concentrates helped avoid these risks. In 1971, The National Academy of Sciences
(USA) confirmed that the use of detergents containing enzymes posed no health hazard to the consumer.
Proteases from B. lichemformis are currently the commercially most important microbial enzymes.
They represent approximately 60 per cent of all the industrial enzymes, followed by the carbohydrates
making up about 30 per cent. The latter include the amylases, glucose isomerases, pectinases and
cellulases.
The development of other industrial enzymes will be discussed in subsequent chapters.
APPLICATIONS OF MODERN ENZYME TECHNOLOGY

Major targets of modern enzyme technology continue to be preservation of foods and food components
(e.g. vitamins), more efficient use of raw materials and improvement offood quality such astexture and
taste. Enzymes are also used in the food industry for process optimisation to reduce process costs,
including energy requirements. Other current objectives include the utilisation of new raw materials for
feeding humans and animals, the manufacture of dietetic foods and eliminating antinutritive substances
from certain nutritional raw materials.
Biotechnological methods have also replaced some of the traditional chemical processes. Enzymatic
methods, which include the most recent procedures with immobilised enzymes and immobilised microorganisms, constitute only one aspect of modern biotechnology. Biotechnology encompasses many
processes for producing biologically active compounds, such as amino acids and antibiotics as well as
processing waste-water..,
Examples of new biotechnological processes include:
1. The use of enzymes in nonaqueous media for the production of chiral compounds and the
synthesis of special polymers.
2. The use of enzymes for the synthesis of amino acids, peptides and antibiotics.
3. The use of enzymes for recycling food wastes and in waste-water treatment.
4. The production of sweeteners, such as aspartame, using combined microbiological and enzymatic
methods.
5. The production of cyclodextrins from starch with specific enzymes.
6. The use of cellulases and lipases as active components of detergents.
7. The production of rare and expensive mammalian enzymes by micro-organisms. For example,
pure calf stomach rennet is currently not available in sufficient quantities.
8. The production of tailored enzymes to serve as specific, process-adapted catalysts. These
procedures are part of the so-called protein engineering, which means the design of proteins
with specific functions.
9. Large-scale production of enzymes by gene technology is to be expected in future years;
recombinant chymosin was a pioneering first. It has since overcome the world shortage of
chymosin in the cheese industry. Thus, many limited or costly traditional enzyme sources will
be replaceable.

Basic Concepts of Enzymes

In the past, obtaining the desired metabolites and improved performance depended on the conventional
genetic methods of inducing mutations and hybridising sexually propagating micro-organisms. In
asexually reproducing micro-organisms, physical and chemical mutagenesis produced the necessary
variability. The sought-after characteristic or product was subsequently found by screening large numbers
of progeny. Mutations randomly alter the genetic or hereditary information. Success depends on whether
one of the random mutations occurs at the correct site within the genome; only then will it be possible
to develop better microbial strains. The extensive screening required makes it very expensive to determine
whether such randcru mutations have produced the changes sought in the production of the desired
substance. Now, gene technology permits site-specific changes and yields products of great purity under
economically feasible conditions .

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