Novel enzyme technology for food applications

Novel enzyme technology for food applications


Related titles:
Modifying lipids for use in food
(ISBN 978-1-85573-971-0)
Any oil or fat should have the optimum physical, chemical, and nutritional properties
dictated by its end use. Modification of natural fats and oils is therefore important to
improve the quality of lipids for use in foods. When lipids are modified, though,
compromises have to be made as the physical, chemical and nutritional properties of lipids
are not always mutually compatible and this provides an important challenge for food
technologists. Edited by an eminent specialist, this collection shows how these challenges
have been met in the past, how they are being met today, and how they may be met in the
future.
Starch in food – Structure, function and applications
(ISBN 978-1-85573-731-0)
Starch is both a major component of plant foods and an important ingredient for the food
industry. Starch in food reviews starch structure and functionality and the growing range of
starch ingredients used to improve the nutritional and sensory quality of food. Part I
illustrates how plant starch can be analysed and modified, with chapters on plant starch
synthesis, starch bioengineering and starch-acting enzymes. Part II examines the sources of
starch, from wheat and potato to rice, corn and tropical sources. The third part of the book
looks at starch as an ingredient and how it is used in the food industry. There are chapters
on modified starches and the stability of frozen foods, starch–lipid interactions and
starch-based microencapsulation. Part IV covers starch as a functional food, including the
impact of starch on physical and mental performance, detecting nutritional starch fractions
and analysing starch digestion.
Proteins in food processing
(ISBN 978-1-85573-723-5)
Proteins are essential dietary components and have a significant effect on food quality.

Edited by a leading expert in the field and with a distinguished international team of
contributors, Proteins in food processing reviews how proteins may be used to enhance the
nutritional, textural and other qualities of food products. After two introductory chapters,
the book first discusses sources of proteins, examining the caseins, whey, muscle and soy
proteins and proteins from oil-producing plants, cereals and seaweed. Part II illustrates the
analysis and modification of proteins, with chapters on testing protein functionality,
modelling protein behaviour, extracting and purifying proteins and reducing their
allergenicity. A final group of chapters is devoted to the functional value of proteins and
how they are used as additives in foods.
Details of these books and a complete list of Woodhead’s titles can be obtained by:

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Novel enzyme technology
for food applications
Edited by
Robert Rastall

CRC Press
Boca Raton Boston New York Washington, DC

Cambridge England


Published by Woodhead Publishing Limited, Abington Hall, Abington,
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Contents

Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Part I Principles of industrial enzyme technology
1

Discovering new industrial enzymes for food applications . . . . . . . . . 3
Thomas Schäfer, Novozymes A/S, Denmark
1.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2
Where to screen for new enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3
How to screen for new enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4
Summary: which option to choose? . . . . . . . . . . . . . . . . . . . . . . . 13

1.5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2

Improving enzyme performance in food applications . . . . . . . . . . . .
Ronnie Machielsen, Sjoerd Dijkhuizen and John van der Oost,
Wageningen University, The Netherlands; Thijs Kaper and Loren
Looger, Carnegie Institution of Washington, USA
2.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2
Laboratory evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3
Examples of improving enzyme stability and functionality by
laboratory evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4
Rational and computational protein engineering . . . . . . . . . . . . .
2.5
Examples of improving enzyme stability and ability by rational
protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6
Examples of combined laboratory evolution and computational
design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

16
17
24

28
30
34


vi

Contents
2.7
2.8
2.9

3

4

5

Summary and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Sources of further information and advice . . . . . . . . . . . . . . . . . . 35
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Industrial enzyme production for food applications . . . . . . . . . . . . . .
Carsten Hjort, Novozymes A/S, Denmark
3.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2
Traditional sources and processes for industrial
enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3

Design of expression systems for industrial
enzyme production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4
Development of an enzyme production process . . . . . . . . . . . . . .
3.5
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6
Sources of further information and advice . . . . . . . . . . . . . . . . . .
3.7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immobilized enzyme technology for food applications . . . . . . . . . . . .
Marie K. Walsh, Utah State University, USA
4.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2
Immobilized enzyme technology for modification of
acylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3
Immobilized enzyme technology for modification of
carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4
Immobilized enzyme technology protein modification . . . . . . . .
4.5
Immobilized enzyme technology for production of flavor
compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Consumer attitudes towards novel enzyme technologies in food

processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Helle Søndergaard, Klaus G. Grunert and Joachim Scholderer,
MAPP, University of Aarhus, Denmark
5.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2
Theoretical approaches to how consumers form attitudes to
new food production technologies . . . . . . . . . . . . . . . . . . . . . . . .
5.3
Studies of consumer attitudes to enzyme technologies . . . . . . . . .
5.4
Implications of consumer attitudes to enzyme technologies . . . . .
5.5
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6
Sources of further information and advice . . . . . . . . . . . . . . . . . .
5.7
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43
43
44
46
54
56
56
57
60

60
62
68
73
75
77
78

85

85
86
88
94
95
95
96
96


Contents

vii

Part II Novel enzyme technology for food applications
6

7

8

Using crosslinking enzymes to improve textural and
other properties of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Johanna Buchert, Emilia Selinheimo, Kristiina Kruus, Maija-Liisa
Mattinen, Raija Lantto and Karin Autio, VTT, Finland
6.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2
Types of crosslinking enzymes . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3
Application of crosslinking enzymes in baking and pasta
manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4
Application of crosslinking enzymes in meat and fish
processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5
Application of crosslinking enzymes in dairy applications . . . .
6.6
Other applications of crosslinking enzymes in food
manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.7
Analysing the chemistry of crosslinks formed by enzymes . . . .
6.8
Effect of biopolymer crosslinking on nutritional properties
of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.9
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzymatically modified whey protein and other protein-based fat
replacers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Jacek Leman, University of Warmia and Mazury in Olsztyn, Poland
7.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2
Enhancing the fat mimicking properties of proteins . . . . . . . . . .
7.3
Applications in low-fat foods . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzymatic production of bioactive peptides from milk and whey
proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Paola A. Ortiz-Chao and Paula Jauregi, University of Reading,
UK
8.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2
Angiotensin I-converting enzyme inhibitory peptides . . . . . . . .
8.3
Other bioactive peptides and their health benefits . . . . . . . . . . .
8.4
Production of bioactive peptides from milk and
whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6
Sources of further information and advice . . . . . . . . . . . . . . . . .
8.7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

101

101
103
109
114
118
122
122
124
126
126

140
140
142
149
152
153

160

160
161
165
170
177
177
177

viii
9

Contents
Production of flavours, flavour enhancers and other protein-based
speciality products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stuart West, Biocatalysts Ltd, UK
9.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2
Production and usage of monosodium glutamate (MSG) . . . . . .
9.3
Chondroitin sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4
Production of aspartame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.5
Enzymes for vanilla extraction . . . . . . . . . . . . . . . . . . . . . . . . . .
9.6
Enzyme modified cheese as a flavour ingredient . . . . . . . . . . . .
9.7
Enzymes used in savoury flavouring . . . . . . . . . . . . . . . . . . . . .
9.8
Enzymes used in yeast extract manufacture . . . . . . . . . . . . . . . .
9.9
Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.10 Sources of further information and advice . . . . . . . . . . . . . . . . .
9.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 Applications of cold-adapted proteases in the food industry . . . . . .
A. Guðmundsdóttir and J. Bjarnason, University of Iceland, Iceland
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2 Use of proteolytic enzymes in food processing . . . . . . . . . . . . .
10.3 Application of cold-adapted serine proteases in food
processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.4 Modifying marine proteases for industrial use . . . . . . . . . . . . . .
10.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11 Health-functional carbohydrates: properties and enzymatic
manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Simon Hughes and Robert A. Rastall, University of Reading, UK
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2 Dietary fibre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3 Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4 Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.5 Transgalacto-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6 Gluco-oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.7 Alternansucrase–maltose acceptor oligosaccharides . . . . . . . . .
11.8 Resistant starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.9 Arabinoxylan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.10 Oligosaccharides from non-starch polysaccharides . . . . . . . . . .
11.11 Pectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.12 Oligodextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.13 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.14 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183
183
186

188
190
191
193
198
199
200
202
203
205
205
208
209
211
212
212

215
215
215
217
219
222
223
224
226
228
230
232
234

237
237


Contents

ix

12 Flavorings and other value-added products from sucrose . . . . . . . .
Gregory L. Côté, United States Department of Agriculture, USA
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2 Di- and oligosaccharides from sucrose . . . . . . . . . . . . . . . . . . . .
12.3 Polysaccharides from sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4 Other products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6 Sources of further information and advice . . . . . . . . . . . . . . . . .
12.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243

13 Production of structured lipids with functional health benefits . . . .
Xuebing Xu, Janni B. Kristensen and Hong Zhang, BioCentrumDTU, Technical University of Denmark, Denmark
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2 Production of diglyceride oils . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3 Production of healthy oils containing medium chain fatty acids
13.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14 Lipase-catalyzed harvesting and/or enrichment of industrially and
nutritionally important fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . .

George J. Piazza and Thomas A. Foglia, US Department of Agriculture, USA; and Xuebing Xu, BioCentrum-DTU, Technical University
of Denmark, Denmark
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2 Lipase selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3 Fatty acid harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4 Structured triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5 Single reaction step process for the production of STAG . . . . .
14.6 Multiple reaction step processes for the production of STAG . .
14.7 Nutritional and other uses of structured lipids . . . . . . . . . . . . . .
14.8 Summary and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243
244
257
260
261
262
262
270

270
271
278
282
282
282

285

285
286
294
295
301
307
307
308
309

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315



Contributor contact details
(* = main contact)

Editor

Chapter 3

R. A. Rastall
School of Food Biosciences
PO Box 226
Whiteknights
Reading
RG6 6AP
UK

C. Hjort

Novozymes A/S
Krogshoejvej 36
DK-2880 Bagsvaerd
Denmark

email:

Chapter 4

Chapter 1
T. Schäfer
Novozymes A/S
Krogshoejvej 36
DK-2880 Bagsvaerd
Denmark
email:

email:

M. K. Walsh
Utah State University
8700 Old Main Hill
NFS 318
Logan
UT, 84322-870
USA
email:

Chapter 5

R. Machielsen* and J. van der Oost
Hesselink van Suchtelenweg 4
6703CT, Wageningen
The Netherlands

H. Søndergaard*, K. G. Grunert and
J. Scholderer
MAPP
Aarhus School of Business
Halslegaardsvej 10
DK-8210 Aarhus V
Denmark

email:

email:

Chapter 2


xii

Contributor contact details

Chapter 6
J. Buchert*, E. Selinheimo, K. Kruus,
M. L. Mattinen, R. Lantto and K.
Autio
VTT
PO Box 1000

FI-02044 VTT
Finland
email:

Chapter 7
J. Leman
Faculty of Food Sciences
University of Warmia and Mazury in
Olsztyn
Heweliusza 1
10-718 Olsztyn
Poland

email:
secretarytheresac@
biocats.com

Chapter 10
A. Guðmundsdóttir*
Science Institute
University of Iceland
Læknagardi
Vatnsmýrarvegi 16
101 Reykjavík
Iceland
email:
J. B. Bjarnason
Dunhaga 3
107 Reykjavík
Iceland

email:

Chapter 8
P. A. Ortiz-Chao and P. Jauregi*
School of Food Biosciences
University of Reading
PO Box 226
Whiteknights
Reading
RG6 6AP
UK

Chapter 11
S. Hughes and R. A. Rastall*
School of Food Biosciences
PO Box 226
Whiteknights
Reading
RG6 6AP
UK
email:

email:

Chapter 9
S. West
Biocatalysts Limited
Cefn Coed
Nantgarw

Cardiff
CF15 7QQ
UK

Chapter 12
G. Côté
NCAUR/ARS/USDA
1815 N. University St
Peoria
IL 61604
USA
email:


Contributor contact details

Chapter 13
X. Xu*, J. B. Kristensen and
H. Zhang
BioCentrum-DTU
Technical University of Denmark
Building 227
DK-2800 Kgs. Lyngby
Denmark
email:

Chapter 14
G. J. Piazza* and T. A. Foglia
US Department of Agriculture
Agricultural Research Service

xiii

Eastern Regional Research Center
600 East Mermaid Lane
Wyndmoor
PA 19038
USA
email:
X. Xu
BioCentrum-DTU
Technical University of Denmark
Building 227
DK-2800 Kgs. Lyngby
Denmark
email:



Preface

Enzymes have been used in the food industry for many years. They have largely
been used as processing aids and they have many attributes that make them fit for
this purpose. They are generally non-toxic and speed up chemical reactions with
great specificity at low temperatures and pressures and at near-neutral pH. A large
industry exists to serve this need across the world.
One of the limitations of enzyme application in the food industry is the lack of
availability of enzymes with the required properties at an acceptable price. Whilst
desired enzyme activities are frequently known somewhere in the biological
world, they are often unsuitable for commercial application. In recent years,

however, there has been increasing sophistication in our ability to isolate novel
enzymes from biological sources and an expansion of the range of sources of
enzymes to include, for example, extremophiles. Such organisms frequently have
enzymes with higher pH and temperature optima and can extend the range of
processes in which enzymes can be used. We now have the ability to rationally
engineer or artificially evolve desired catalytic properties into enzyme molecules.
These new technologies will ultimately remove many of the limitations currently restricting the application of enzymes in the food industry and will open up
many more possibilities. Technological aspects are dealt with in Part I – Principles
of industrial enzyme technology. Chapters 1 and 2 deal with the discovery of novel
enzymes for food applications and the improvement of enzymes for food applications. Chapters 3 and 4 then examine the production of industrial enzymes and their
immobilisation in the context of food applications. Part I is concluded by Chapter
5 on consumer attitudes to novel enzyme technologies.
Concurrent with these technological developments has been the advance in our
knowledge of the role of specific food components in health and disease. This has
led to a significant increase in interest in functional food ingredients – compounds
that are specifically added (or whose levels are deliberately increased) in foods to


xvi

Preface

provide a specific health attribute beyond nutrition. Examples include prebiotic
oligosaccharides to improve gut health, bioactive peptides to help reduce blood
pressure, and nutritionally enhanced fats. Governments around the world are also
taking heed of modern nutritional knowledge and are increasingly looking to the
food industry to manufacture foods with a healthier profile. These nutritional
developments are starting to provide a new range of application areas for novel
enzymes and enzyme technologies and it is these applications that are discussed in
Part II – Novel enzyme technology for food applications. Chapters 6, 7 and 8 deal

with enzymatic modification of proteins to achieve cross-linking, to generate fat
replacers and to manufacture bioactive peptides respectively. Protein modification
also features in Chapter 9 on production of flavours and flavour enhancers and in
Chapter 10 on the application of novel cold-adapted proteases. The focus then
moves to carbohydrates, in Chapter 11 on health-functional carbohydrates and
Chapter 12 on value-added products from sucrose. Finally, the manufacture of
lipids with health and other functional attributes is discussed in Chapters 13 and 14.
This volume aims to give the reader an overview of recent developments in
enzyme technology in the food industry rather than an exhaustive account of
traditional applications. The aim is to increase awareness of and stimulate interest
in developing novel enzyme technologies to meet the new and changing needs of
the food industry.
Professor Robert Rastall
University of Reading


Part I
Principles of industrial enzyme
technology



1
Discovering new industrial enzymes for
food applications
Thomas Schäfer, Novozymes A/S, Denmark

1.1

Introduction

Enzymes have been exploited by humans for thousands of years. Traditional foods
and beverages like cheese, yoghurt and kefir, bread, beer, vinegar, wine and other
fermented drinks, as well as paper and textiles, were produced with the help of
enzymes which were present in starting materials as early as 6000 BC in China,
Sumer and Egypt. The epoch of classical biotechnology was marked by the
landmark discoveries of microbes by Leeuwenhook, of fermentations as biological
processes by Pasteur, of enzymes as proteins by Buchner and of the first enzyme
crystal structures by Sumner.
The modern era of industrial enzymology began in 1913 when Otto Röhm was
granted a patent for the use of a crude protease mixture isolated from pancreases in
laundry detergents. In the following years an increasing number of enzymes were
found in microorganisms and these microbes were cultured in large scale
fermentations to produce enzymes. However, the number of enzymes that could be
produced in this fashion was limited, because not all microbes are amenable to
large scale fermentation. The pioneering work of Avery and MacLeod, Hershey
and Chase, Watson and Crick, Cohen and Boyer and many others who introduced
the era of recombinant biotechnology revolutionized industrial enzyme screening
and production.
With the advent of genetic engineering, genes encoding interesting enzymes
could be transferred to and expressed in selected host microbes for production on
an industrial scale. Today, gene technology plays a major role in both the discovery


4

Novel enzyme technology for food applications

of novel enzymes and the optimization of existing proteins, and is basis for the
production of the majority of industrial enzymes. Food applications of enzymes

represent a wide and highly diverse field including baking, dairy, juice, vegetable
processing and meat. The enzymes are used to obtain a number of benefits, like
more efficient processes, leading to reduced use of raw materials, improved or
consistent quality, replacement of chemical food additives and avoidance of
potential harmful by-products in the food.

1.1.1 Technologies for discovery of industrial enzymes
Nature holds a wonderful diversity of organisms and the corresponding wealth of
enzymes and has often been the starting point for the identification of novel
enzymes. For a variety of applications even Nature’s assortment faces some
limitations or it is too time consuming and difficult to look into Nature’s diversity.
This imposes a challenge for scientists to optimize existing natural enzymes and to
generate additional ‘artificial’ diversity to tailor-make enzymes for a given application. Natural diversity approaches and optimization strategies are complementary
routes and both are equally important in developing a high-quality diversity of
enzymes (Nedwin et al., 2005).
Today, discovery of enzymes for the food industry is not only a
multidisciplinary effort involving a wide array of different screening technologies, but is also based on close interaction between food scientists who
understand or model the application and biotechnologists who can deliver enzymes for initial trials. Each screening project is new and challenging.
Accordingly, each project needs to be uniquely designed to solve the specified
application problems in a certain industrial application and for each project the
expert team needs to have members with exactly the competencies needed to
find a solution. It is obvious that major enzyme companies have to master a
variety of technologies which, often in combination with each other, lead to the
solution. For all approaches it is important to stress that it is not the broadest
possible diversity, but rather the highest possible quality of diversity which will
lead to the ultimate goal, namely a novel product that addresses exactly the
specific demands of the industrial application. In this respect selection/
deselection via perfectly designed assays is of utmost importance, indicating the
significance of linking process understanding to biochemistry.

1.2

Where to screen for new enzymes

One of the main questions which has to be answered in the very beginning of each
discovery initiative is ‘where to look for diversity?’ (Bull et al., 2000; Fig. 1.1).
There are various potential sources, as input to screening programs is basically
divided into (a) natural enzyme diversity and (b) artificial diversity, which are
comprehensively reviewed by Schäfer and Borchert (2004) and Aehle (2004).
Here the basic principles will be summarized.


Discovering new industrial enzymes for food applications

Fig. 1.1

5

Overview of the main approaches to diversity input in screening programs.

1.2.1 Nature’s diversity: an unlimited source of enzymes
The challenge is that Nature’s diversity is virtually infinite and that living microorganisms have inhabited virtually all ecological niches on planet Earth during 3.5
billion years of evolution. The number of described bacterial and fungal species is
huge, new isolations are added daily so that the actual number can only be
extrapolated roughly. From bioinformatics analysis of the genomes it can be
assumed that a bacterial genome on average contains about 4000 enzyme coding
genes, while for fungi the number of enzyme encoding genes can be up to 20,000
(Hirose et al., 2000; Dunn-Coleman and Prade, 1998). The art of screening
obviously consists of having the right tools to find the ‘needle’ in this ‘haystack’
of biodiversity; no scientist will start looking into the totally available biodiversity,

but will look into groups of carefully selected microorganisms. Considering these
numbers and using best practice, it is obvious that all screening efforts face a
limitation in that we are only scratching the surface. Microorganisms, namely
bacteria, fungi and archaea, which are normally stored in culture collections of the
groups performing the screening or in public collections, where the strains are
accessible for everyone who is interested, often comprise the biological starting
material.
On top of the cultivated diversity, complex gene libraries compiled from natural
material without prior cultivation (Handelsman, 2005) can be generated and used
to discover industrial enzymes (Short, 1997) and other natural compounds (Brady
et al., 2001). Today, it is generally accepted that only minor numbers among the
whole of the microbial diversity have been cultured or might even be amenable to
growth in the laboratory (Torsvik et al., 2002) thereby leaving not only a huge set
of questions concerning our understanding of the role of microbes in their habitats,
but also an enormous potential for yet undiscovered physiological and biochemical


6

Novel enzyme technology for food applications

traits including enzyme genes in the so-called metagenome (Lorenz and Schleper,
2002; Rondon et al., 1999). It is estimated that 1 g of soil contains more than 4000
different bacterial genomes, that is about 16 million enzyme encoding genes. By
isolating the genetic material, be it DNA or RNA, directly from the soil and cloning
this into suitable host complexes, ‘environmental libraries’ can be constructed.
These gene libraries need to be screened as described below using either sequencebased techniques or activity assays including some novel constraints caused by the
complexity of the library.

1.2.2 Bioinformatics and genomics

Input to screening efforts can also come from genes or genomes described by
researchers worldwide over time. The updated status of established genomes and
those underway can be obtained by visiting the homepage of the TIGR institute
( or the homepage of the DOE Joint Genome Institute (http:
//genome.jgi.org/). The use of existing gene information can potentially shortcut
the flow to a new product candidate, although in most instances a gene is described
by a sequence only, that is, no biochemical information is available. By using
sophisticated software tools within the new discipline of bioinformatics those
genes can be aligned to existing ones, grouped into enzyme families in order to
predict ideally their putative biochemistry, that is, enzyme activity (Henrissat and
Bork, 1996). This is also where one of the major pitfalls lies, namely that the
original description of the enzyme can turn out to be incorrect. An additional
complexity is the fact that roughly 30% of all gene sequences from genomes are
new, that is they do not resemble any biochemical description of the corresponding
protein. Interesting hits found in this way can subsequently be analysed in more
detail but this requires cloning and expression of the gene (see below) followed by
purification and characterization of the corresponding enzyme, which is a tedious
and resource-intensive effort when many genes are of potential interest. Accordingly, this comprises one of the major bottlenecks in genomics as the protein can
only be characterized very late in the process and the chance of failure is high.
Searching of gene databases, both generated in-house and external ones, is a daily
complement to the work of a screening scientist. In addition to screening the
external world of sequence data for novel enzymes, the discovery scientist must
also determine whether any enzymes found are novel and whether their use is
protected by patents.
Whole genome sequencing combined with bioinformatics, array studies and
proteomics are novel key technologies for the targeted improvement of production
strains. This has illustratively been described for lysine production in Corynebacterium glutamicum (Ohnishi et al., 2002). Whole genome sequencing which
completely maps all genes is, however, not ideal for discovery of selected genes,
for example those encoding for enzymes and especially for those enzymes that
match defined application criteria. Assuming an average genome size for a

bacterium of about 4 Mb, for yeast of 13 Mb (Zagulski, et al., 1998) and for
filamentous fungi in the order of 30–40 Mb (Dunn-Coleman and Prade, 1998;


Discovering new industrial enzymes for food applications

7

Radford and Parish, 1997), the costs of sequencing programs of total genomes are
unreasonably high for discovery purposes, especially considering the wide diversity of microbes that are interesting for enzyme screening. From the 4100 open
reading frames (ORFs) of the Bacillus subtilis genome, only a fraction may be
relevant for industrial enzymes. For many industrial applications, extracellular
enzymes are of major importance and it is estimated that B. subtilis produces 150–
180 secreted proteins (Hirose et al., 2000), while the number of secreted enzymes
for filamentous fungi might be in the order of 200–400 corresponding to their
larger genome sizes. This indicates that only 2–5% of the ORFs in a complete
genome are of primary interest for enzyme discovery. Accordingly, whole genome
sequencing can hardly be justified for enzyme discovery purposes. As a consequence alternative approaches have been developed to mine selectively microbial
genomes for secreted enzymes. Those will be described in more detail in Section
1.3.4.

1.2.3 Protein optimization of enzymes
In cases where enzymes found from natural sources cannot provide the performance needed for a given application, protein optimization offers an attractive
option. In many applications the enzymes are very much stressed by, for example,
high temperatures, extreme pH values or the presence of metal ions, which are
known to induce unfolding of the protein. Several strategies can be followed in
order to optimize the properties of enzymes found in Nature. A simplified way of
looking at protein optimization technologies is to divide the field into rational
protein engineering and random molecular evolution (Fig. 1.1). This is discussed
briefly below and in more detail in Chapter 3.

Rational protein engineering is based on the knowledge of a given enzyme
structure and the corresponding biochemistry, for example the substrate specificity,
the temperature tolerance, inhibition by metal ions, and so on, the combination
thereof comprising the structure–function relationship. This is the parameter that
will be modified as changes in structure will lead to changes in functionality. The
challenge is to introduce changes that lead to improved functionality rather than
inferior variants of an enzyme. A key is the ability to create protein variants with
designed and deliberate amino acid alterations at any desired position that provides
the capability for precise probing of structure–function relationships in proteins
(Bott, 2005).
The gene is mutated selectively at specified sites and the corresponding enzyme
is expressed and subsequently tested to verify the hypothesis behind the mutation.
Positive mutations are collected and analysed in more detail, for example which
amino acids in the enzymes were changed, at the position where the mutations are
located. These mutations are eventually combined to find the ultimate combination
of positive mutation events. As easy as this might sound, this approach represents
a considerable challenge for researchers and in many instances the experiments
have failed. Several years of hands-on experience of biochemistry, bioinformatics
and structure–function analysis are a prerequisite for success. Importantly, it must


8

Novel enzyme technology for food applications

be acknowledged that we still only have limited understanding of protein function
and that only a small part of the huge natural enzyme diversity has been analysed
on a structural level, resulting in severe limitations for rational protein engineering.
We are still far from predictability, that is, knowing which amino acid change will
result in which consequence. Years of trial and error have, however, increased our

knowledge, especially of selected enzyme classes which are of major importance
for industrial enzymes. Accordingly, engineered variants of a number of hydrolytic enzymes such as proteases, amylases, lipases and cellulases are commercially
available today.
In contrast, no prior knowledge of structure–function relationships is needed to
carry out molecular evolution experiments. Here the basic principle is to carry out
random introduction of mutations, thereby generating DNA libraries consisting of
up to millions of variant genes. The DNA variation is expressed into protein
diversity in a variant library, for example in Escherichia coli or Saccharomyces
cerevisiae, the library is subjected to a screening procedure using a functional
assay (see below) and the best performing mutants are collected. There is a tight
connection between the selected variant protein and its encoding gene, which
makes it easy to sequence the enzyme coding gene and detect the mutation, in this
case after the modified phenotype was detected.
Random mutation leads to millions of mutants of a given gene and smart
screening systems are needed to identify the best performing variants. Robotics
equipment for colony picking, colony transfer into screen-able formats, often
microtitre plates, and addition of assay components are a prerequisite for this
approach (Eijsink et al., 2005).

1.3

How to screen for new enzymes

After the question ‘where to screen?’ has been answered the next question is ‘how
to screen?’ (Bull et al., 2000). There are several possibilities for this and variations
of these themes (Fig. 1.2). The following paragraphs will describe some of the
most prominent screening approaches.

1.3.1 Functional biochemical assays
The most preferred screening route for novel enzymes is via functional screening

assays, where the biochemical activity can be detected. Ideally, this will also show
how well the positive hits meet application requirements to be tested; a detected
amylase can, for example, also be tested for activity at elevated temperature, high
or low pH or under other hostile conditions by using the same assay principle.
Biochemical assays allow the screening of living microorganisms, of gene libraries
constructed from cultivated microbes and from the environment (metagenomes),
of artificial evolution libraries, as well as of rationally designed protein variants for
a wanted enzyme activity. Many of the assays can be implemented on agar plates,
where growing colonies can be tested for activity, or on a smaller scale using