State of Art in Europe

INSTITUTE FOR PROSPECTIVE TECHNOLOGICAL STUDIES
SEVILLE
W.T.C., Isla de la Cartuja, s/n,
E-41092 Sevilla
IPTS Project
Modern Biotechnology and the Greening of Industry
BIOCATALYSIS:
STATE OF THE ART IN EUROPE
Economic and environmental benefits
of a process integrated technology
IPTS, 1998
1.1.1 Editors
Sørup, P.(IPTS), Tils, C. (IPTS), Wolf, O. (IPTS)
1.1.2 Project Co-ordinator

Enzing, C. (TNO)
1.1.3 Contributors from the ESTO network

Van Dalen, W (TNO-STB), de Hoop, B. (TNO-STB), Thomas, S. (SPRU),
Burke, J. (SPRU), Schmitt, A. (VDI-TZ), Heiden, L. (VDI-TZ), Viikari, L. (VTT)
EUR 18680 EN
EUROPEAN COMMISSION
JOINT
RESEARCH
CENTRE
Biocatalysis: State of the Art in Europe
IPTS 1998 2
Executive Introduction
Process integrated biotechnologies, especially the use of biocatalysts, are expected to

have an important contribution to cleaner production processes in a number of
industries. However, there are only a few - nowadays very well known - examples of
biocatalysts in industry; wider implementation seems to meet high barriers.
In order to gain further insight regarding specific factors that hinder or favour the
implementation of process integrated biotechnologies for environmental purposes, IPTS
has set up the project “Modern Biotechnology and the Greening of Industry”. The
overall goal of this framework project, is to generate information for policymakers in
the field of environment, biotechnology and R&D-policy.
In this report the results of one of the studies in this framework project are presented. In
the study an overview is made of the state of the art of biocatalysis in Europe. Three
specific aspects are addressed: economic and ecological benefits of biocatalysts in four
industrial sectors, Europe’s scientific and technical potential in biocatalysis, including
patents, and Europe’s economic potential of biocatalysis. Additionally, an inventory is
made of the future technical developments in biocatalysts.
It is concluded that Europe has a strong basis in biocatalysis and that environmental
benefits of biocatalysis in industry are in general an outcome, not an input. Methods for
the assessment of economic and environmental cost/benefits of (bio)technologies should
be developed, in order to take better informed decisions on process integrated
technologies by industry.
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Contents
EXECUTIVE INTRODUCTION....................................................................................... 2
1. INTRODUCTION ........................................................................................................... 5
1.1 BACKGROUND OF THE PROJECT: GREENING OF INDUSTRY ............................................... 5
1.2 CONTENT OF THE PROJECT: BIOCATALYSIS IN EUROPE ..................................................... 5
1.3 PROJECT TEAM ................................................................................................................. 6
2. TYPES AND FUNCTIONS OF BIOCATALYSTS...................................................... 8

2.1 INTRODUCTION ................................................................................................................. 8
2.2 BIOCATALYSTS ................................................................................................................. 8
2.2.1 Whole cell bioconversions........................................................................................ 9
2.2.2 Enzyme technology................................................................................................. 10
2.2.3 Industrial application of enzymes: bioprocessing.................................................. 11
2.2.4 Methods to improve biocatalysts............................................................................ 11
2.3 BENEFITS OF BIOCATALYSTS IN INDUSTRY ...................................................................... 13
2.3.1 Pharmaceuticals and Fine Chemicals.................................................................... 14
2.3.2 Food and Drinks and Animal Feed ........................................................................ 16
2.3.3 Textile industry....................................................................................................... 21
2.3.4 Pulp and paper industry......................................................................................... 22
2.4 ECONOMIC AND ECOLOGICAL BENEFITS OF BIOCATALYSTS............................................. 24
3. EUROPES SCIENTIFIC-TECHNICAL COMPETENCE IN

BIOCATALYSIS ........................................................................................................... 33
3.1 SCIENCE BASE IN LIFE SCIENCES AND BIOTECHNOLOGY: EU VS. US .............................. 33
3.2 EUROPEAN COMPETENCE IN BIOCATALYSIS .................................................................... 34
3.3 SCIENTIFIC COMPETENCE IN THE INDUSTRIAL SECTORS .................................................. 35
3.4 PATENT ANALYSIS OF BIOCATALYSIS .............................................................................. 37
3.4.1 Relevance of patent statistics ................................................................................. 37
3.4.2 Biocatalysis patents................................................................................................ 38
3.5 CONCLUSIONS ................................................................................................................ 44
4. AN OVERVIEW OF THE ECONOMIC POTENTIAL OF

EUROPE’S BIOCATALYSIS INDUSTRY ............................................................... 46
4.1 INTRODUCTION ............................................................................................................... 46
4.2 THE ECONOMIC CAPABILITIES IN EUROPE ....................................................................... 46
4.2.1 Structure of the industry......................................................................................... 46
4.2.2 The structure of the market .................................................................................... 48
4.3 INTEGRATION OF BIOCATALYSTS IN INDUSTRY................................................................ 50
4.3.1 The pharmaceutical industry.................................................................................. 50
4.3.2 The food and drink and the feed industries ............................................................ 50
4.3.3 The textile sector .................................................................................................... 51
4.3.4 The pulp and paper sector...................................................................................... 52
4.4 BOTTLENECKS ................................................................................................................ 52
5. FUTURE TECHNICAL DEVELOPMENTS IN BIOCATALYSIS ......................... 56

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5.1 INTRODUCTION ............................................................................................................... 56

5.2 MODERN ENZYMES AND OTHER NEW TYPES OF BIOCATALYSTS ...................................... 56
5.3 MODIFICATION OF PLANT COMPONENTS ......................................................................... 58
6. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS................................ 61

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2. Introduction
2.1 Background of the project: Greening of Industry
One of the most important questions in the field of life sciences which IPTS wants to
address is the greening of industries and more specifically the role of new process
integrated technologies in decreasing environmental pressures. In general, it is
recognised that specific biotechnologies have the potential to be used as integrated clean
technologies. However, the fact that the introduction of new promising biotechnics,
especially biocatalysis, has taken place is somewhat limited. For that reason IPTS, in
the framework of the project Modern Biotechnology and the Greening of Industry,
commissioned a number of feasibility studies in order to gain insight in specific issues
related to biocatalysis research and its industrial implementation (dynamics of
innovation, integral conceptual framework, identification of ‘hot spots’ in R&D). These
studies were the basis for the analysis and identification of influencing factors that
stimulate or hinder the introduction of biocatalysis as process integrated technology.
In the framework of the contract of IPTS with the ESTO-consortium (the European
Science and Technology Observatory) it was possible to make a next step in the overall
‘Greening of Industry’-project of IPTS. A special ESTO-Task C project was proposed
which had as its main goal to make a state of the art of biocatalysis in Europe and
compare it with the USA and Japan.
This rather ambitious project proposal was discussed in the IPTS Advisory Board
Modern Biotechnology and the Greening of Industry and with the members of the
Interservice Group of the EU in June 1997. The recommendations brought forward in
both discussions, finally resulted in the project proposal ‘Biocatalysis in Europe’. The
results of this project are presented in this report.
2.2 Content of the project: Biocatalysis in Europe
The driving force behind the overall IPTS project and hence behind this project is the
question how biotechnology - with its promises as an environmentally sound technology
- can have an optimal integration in products and processes of the European industry.
This project focuses on a specific part of biotechnology: biocatalysis. Biocatalysts must

be considered as a process integrated biotechnology, as most environmentally sound
biotechnologies are add-on i.e. bioremediation technologies. The main goal of this
project, as it finally materialised, is to give an overview of the state of the art of
European biocatalysis. On the basis of this overview state further steps can be taken
towards initiatives which stimulate the introduction of green process integrated
technologies, including biotechnology.
The scope of the project is limited to four sectors: the fine chemicals/ pharmaceuticals
industry, the food and feed industry the pulp-and-paper and the textiles industry. These
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sectors were already chosen by IPTS for the overall project Modern Biotechnology and
the Greening of Industries of which this Biocatalysis project is a part (IPTS, 1997).
The state of the art of biocatalysis in Europe for these four industrial sectors is made of
the following aspects:
• the types of biocatalysts, fields of application and the benefits of the use of
biocatalysis;
• Europe’s scientific and technical potentials of biocatalysis, including the patents;
• Europe’s economic potential of biocatalysis.
This state of the art was made on the basis of literature study, database analysis and
interviews with industry.
Structure of the report

The structure of this report follows the above mentioned division. Chapters 2, 3 and 4
deal with the four aspects mentioned. In addition, we also made an overview of the
future technological developments in biocatalysis (chapter 5) and finally in chapter 6
conclusions are drawn and recommendations for further steps, including EU policy, are
presented.
2.3 Project team
The project was performed by an international ESTO-project team with research groups
from four European countries. The project management structure was rather complex.
Three of the four groups did research on a (set of) industrial sectors. The results were
input for chapters 2, 3, 4 and 5, which were collected and processed by the chapter
managers. The fourth group worked on the patent research. The results are included in
chapter 3.
The preliminary results were presented to IPTS and discussed in the project team. On
the basis of this discussion, conclusions and recommendations were formulated.
The participants and their part in the project are:

Dr. Christien Enzing of TNO-STB, The Netherlands
project co-ordinator and responsible for:
• results dealing with the food and feed industries which are presented in chapters 2,
3, 4 and 5,
• environmental aspects of biocatalysis in the fine chemicals/pharmaceutical sector
which are presented in paragraph 2.4,
• chapter manager of chapter 3 and 4,
• author of chapter 1 and 6. and
• final editor of the report.
The sector research (food, drinks and animal feed) was done by ir. Wieger van Dalen
and ir. Bram de Hoop of TNO-STB.
Prof. Dr. Liisa Viikari of VTT Biotechnology and Food Research, Finland, responsible
for:
• results dealing with the pulp-and-paper and the textile sector which are presented in
chapters 2, 3, 4 and 5, and
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• chapter manager of chapter 2.

Dr. Sandra Thomas of SPRU together with Dr. Julian Burke, School of Biological
Sciences, UK, responsible for:
• results dealing with the fine chemical/pharmaceutical sector, producers of important
inputs in the patent part and of future technological developments which are
presented in chapters 2, 3, 4 and 5 and
• chapter manager of chapter 5.
Dr. Anette Schmitt, together with Dr. Lars Heiden, VDI-Technical Centre, Germany,
responsible for:
• the patent research study which is presented in chapter 3, paragraph 3.4.
Literature
IPTS, 1997. Modern Biotechnology and the Greening of the Industry WP97/03 Project
description IPTS Project, Chris Tils and Per Sorup, Sevilla, August 1997.
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3. Types and functions of biocatalysts
3.1 Introduction
Nature is extremely diverse in terms of the large number and many types of organic
molecules required for life. This diversity is made possible solely due to the wide
catalytic scope of enzymes. Enzymes have been used for thousands of years, without
scientific knowledge, to preserve food. Many of these old processes have survived, and
been put into efficient technical frames, such as winemaking, beer brewing and the
production of milk products. However, enzymes can be used for a great number of other
products too.
The recent advances in the field of biocatalysts has enabled the biological processes to
compete successfully with conventional chemical processing. Combination of chemical
and biocatalytic systems are being developed thereby utilising the most attractive
features of biocatalysts i.e. enzymes, namely high specificity with less side or waste
products and higher yields, mild reaction conditions and usually low environmental
impacts.
Various industrial fields thus already use biotechnical methods in their processes. In
many cases, a bioprocess is the most straightforward and economical way to produce
these products. However, in all biological processes, the synthesis of the desired
product is catalysed by enzymes. There are also examples where biocatalysts may
compete with traditional chemical technologies. For instance organic chemicals, such as
acids, alcohol’s or acetone are still produced by chemical synthetic routes. Depending
on the raw material’s availability and cost, biological production processes have
replaced the chemical routes.
This chapter describes the basic methodologies of biocatalysts, including enzyme

technology and production methods using living microbial cells, as well as systems to
produce and improve biocatalysts (2.2). Well established examples of biocatalysts
applied in the industrial sectors selected for this project - fine chemical /pharmaceutical,
food and drinks and feed, pulp and paper, and textile industries - are being reviewed
(2.3). Non-specific biological methods, such as waste water treatment and
bioremediation are widely used for the degradation of various waste compounds, but
they are not dealt with in this report.
Finally in paragraph 2.4 the central question on the most important driving forces for
using biocatalysts in these industries is addressed. Conclusions are drawn on the
economic and ecological benefits of biocatalysts and whether ecological benefits are an
input or an outcome.
3.2 Biocatalysts
From a chemical point of view, enzymes are catalysts operating in a chemical reactor,
the cell. Enzymes may be used as isolated entities, or inside whole cells - derived from
microbes, plants or animals, which in turn may be active or resting. Independently of the
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area, biotechnical processes are classified into two major groups. In whole cell
bioconversions, living organisms are used to perform the desired reactions, whereas in
enzyme technology, isolated or crude enzyme preparations are used as catalysts.
3.2.1 Whole cell bioconversions

Basically three types of whole cell bioconversion processes can be distinguished:
• production of intra- or extracellular metabolites, where the yield from the carbon
source is not the major parameter (enzymes, pharmaceuticals),
• production of bulk products with a high yield from the most economical substrates
(antibiotics, biomass such as yeast , bulk chemicals), and
• bioconversion of the raw material leading to modification of the chemical
composition or structure of the substrate (food conversions, modifications of woody
materials).
The technique of growing microbial cells and their biosynthetic reactions are widely
used in the biotechnical industries for the production of various metabolites.
Commercial compounds produced by microbial living cells include pharmaceuticals,
enzymes, organic acids and solvents, food products, biopolymers, steroids and sterols,
antibiotics and pesticides etc. A wide variety of microbial cells including bacteria, yeast
and fungi, are being used in industrial processes. The use of cultured animal or plant
cells differs clearly from microbial cells being generally expensive compared to
microbes. Most of the development work in this area relates to high value - low volume
products i.e. to new types of products in the field of the pharmaceutical industry.
A distinction can be made between the fermentation processes with living and growing
micro-organism (first and second type) and bioconversion with resting i.e. dead whole
microbial cells for even one-enzyme-bioconversions (third type), especially if the
enzyme needs a cofactor. An example of the latter is the production of acrylamide by
resting cells of Rhodococcus rhodochrous. In this process acrylonitrile is used as a
substrate producing about 20,000 tonnes acrylamide per annum. This is done in a batch
process with a high substrate concentration and conversion rate. Vitamin C is also
produced by whole cell transformation by Acetobacter suboxydans. Finally, a full range
of steroids are produced by several whole cell transformations including progesterone
and predenisolone.
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3.2.2 Enzyme technology

The first enzyme produced industrially was the fungal amylase, employed as a
pharmaceutical agent in the US as early as 1894. The first patent on using enzymes in
laundry detergents was announced in 1915. The utilisation of amylases and
amyloglucosidases for the production of glucose from starch was the second major area
for industrial enzymes. Currently, more than 2000 different types of enzymes have been
identified. They are classified to six major classes (IUPAC). Several hundreds of
enzymes are commercially available as speciality reagents or bulk products.
The industrial bulk enzymes consist mostly of fairly simple enzymes, carrying out
mainly hydrolytic reactions, i.e. degrading different natural polymers. These enzymes,
such as proteases, lipases, amylases and cellulases, are used on very different industrial
areas. The major applications of these enzymes are in food industry and detergent
manufacture. Bulk enzymes are sold as liquid or dried products. Table 2.1 summarises
the most commonly used commercial bulk enzymes in five industrial areas. In addition
to these, several minor applications exist.
Table 2.1. Commercial bulk enzymes for different industrial fields
Industry Type of enzyme Benefits
Food Amylases Hydrolysis of starch

Proteases Processing of cheese and meat
Pettiness Clarification of juices
Lipases Modification of fats
Glucose isomerases Production of fructose
Feed Hemicellulases Digestibility of feed

Cellulases Increased nutritional value
Phytases Improved phosphate uptake
Pharmaceutical Penicillin acylases Production of penicillin derivatives

Lipases, Proteases, Production of optically pure compounds (chiral
Aminoacylase resolution)
Textile Amylases Starch removal; desizing

Cellulases Denim stone washing, depilling
Pectinases Treatment of flax and other fibres
Proteases Degumming of silk, detergents
Laccases Denim bleaching
Catalases Removal of residual hydrogen peroxide
Pulp and paper Hemicellulases Improved bleachability

Cellulases Paper manufacture
Lipases Removal of pitch components
Cellulases/Hemicellulase Deinking of recycled fibres
Laccase Bleaching, fibre treatments
Source: Godfrey and West, 1996
New production technologies have decreased the prices of enzymes and the
development of new, more targeted enzymes has widened the applicability of enzymes.
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New application areas are, in healthcare, diagnostics, synthesis and purification of

organic compounds and the production of commodity chemicals. Speciality enzymes are
industrially used in clinical diagnosis and food analysis, as tools in genetic engineering
(nucleases, polymerases) and in organic synthesis of fine chemicals.
3.2.3 Industrial application of enzymes: bioprocessing

Conventional biocatalysis is performed by enzymes in aqueous solutions. Two types of
applications relevant for this project are: enzymes in non-aqueous solutions and
immobilisation of enzymes for continuous conversion processes.
Water is a poor solvent for nearly all applications in chemical industry. Most organic
compounds of commercial interest are very sparingly soluble and are often unstable in
aqueous solutions. Over the past decade the application of enzymes in organic media
has become an alternative to chemical synthesis and analytical applications. There are
numerous potential advantages in employing enzymes in organic as opposed to aqueous
media. In aqueous solutions, enzymes such as lipases, esterases, proteases and
carbohydrases catalyse hydrolytic reactions. In organic media, however, these enzymes
catalyse a variety of synthetic reactions in high yields including esterification,
transesterification, interesterification, lactonization, thiotransesterification and
aminolysis. All these reactions are possible only in the presence of low-water activities.
Various enzymes in non-aqueous media can catalyse reactions formerly limited to
expensive and tedious chemical catalysts.
Continuous operation of biocatalysts can be achieved by immobilisation. Both enzymes

and micro-organisms can be immobilised. Immobilisation has several economic and
technical advantages: e.g. reduction of enzyme’s costs, more efficient reactor
performances and less downstream processing. Furthermore, products are easily
separated from the biocatalysts, and in some cases the enzyme properties are altered
favourably by immobilisation. Most enzymes, when immobilised, show a higher
stability than the soluble forms. Advanced examples of industrial processes based on
immobilised biocatalysts include isomerization of glucose to fructose, production of
various amino acids, and hydrolysis of penicillin’s to 6-aminopenicillanic acid. More
complex reactions involving coenzymes have not yet been used to a great extent at an
industrial scale. This technology is in principle applicable to all soluble substrates. Solid
substrates, however, have to be treated in batch or semicontinuous processes.
3.2.4 Methods to improve biocatalysts

Enzymes of commercial interest may be produced by cultivating microbes (bacteria,
yeast or filamentous fungi), plant or tissue cells in well controlled conditions in a
process especially developed for the production of a certain enzyme. Some industrial
enzymes are still being extracted from plants (e.g. bromelain, a protease from
pineapple) or from tissues (e.g. lysozyme, a carbohydrate hydrolysing enzyme from egg
white). Microbes are, however, preferential sources of enzymes, as they are easy, rapid,
and cheap to handle and cultivate.
Most of the industrial hydrolytic enzymes are produced either by species of the bacterial
genus Bacillus or species of the filamentous fungal genera Aspergillus and
Trichoderma. The economic advantages of these producers are their efficiency in
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secreting the enzymes in high amounts into the cultivation medium (i.e. extracellular
enzymes) and the possibility to cultivate these organisms on cheap media. In addition
extracellular enzymes are generally very stable, even under more “unnatural”conditions.
It is obvious that relatively few species will be used as producers, since the most
efficient enzyme producers will be genetically modified for that purpose. Especially for
applications in the food industry, so called ‘food-grade’ enzymes are produced only by
organisms with the GRAS (Generally Recognised As Safe) approval. The list of
accepted producers is published by the organisation of enzyme producers AMFEP
(Association of Microbial Food Enzyme Producers). The costs for the official approval
of a new organism as a producer of an enzyme for such fields as medical, food or other
consumer applications are quite often prohibitive.
Enzymes are naturally produced in fairly low quantities and their properties do not
necessarily meet those required in industrial processes. Furthermore, the naturally
produced enzyme mixtures may contain enzymes, unnecessary for the desired
application, impairing the action of the target biocatalyst, or causing even harmful side
reactions. Using methods of modern molecular biology, the commercial production
process of a desired biocatalyst can, however, be developed within a fairly short period.
The prerequisite is that the enzyme shows promises also from a commercial point of
view, justifying the high development costs of new products.
The developments in genetic engineering during the last decade have made it possible to
change the spectrum of the enzymes produced by a microbial species or to make an
efficient and approved species to produce an enzyme that by nature it is not able to
synthesise. The degree of purity of commercial enzymes ranges from crude enzymes to
highly purified speciality enzymes and depends on their application.
The development of an economically competitive product, which is optimal with respect

to specificity, stability or activity under the sometimes extreme conditions existing in
the process industry, is quite demanding, and various technologies are required for the
development (Box 1).
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Box 1 Modern biotechnologies for the improvement of biocatalysts
Genetic engineering: this techniques allows the transfer of a gene coding a useful activity into an
organism well adopted for industrial use (the host). Furthermore the catalytic behaviour or other
properties of enzymes can be changed and even new catalytic features created.
Protein engineering: understanding of the protein function-structure relationship is based on the

understanding of catalytic mechanisms and on the exact three-dimensional knowledge of protein
structure. Thus a theoretical route exists from a given enzyme with known catalytic properties to
an altered enzyme, the properties of which are changed to give improved characteristics in a
given process. With genetic engineering specific alterations of gene structures and consequently
in the amino acid sequences of proteins can be made. With knowledge of the chemical
interactions in the protein and using advanced computational methods one may, in some cases,
put forth accurate predictions of the change in the protein structure. In practice predetermined
changes are not easily obtained but require both hard and imaginative experimentation.
Catalytic antibodies: a route to enzymatic catalysis of reactions without any biological functions is
obtained through the research in catalytic antibodies. Antibodies are proteins which have evolved
to recognise foreign molecules, such as components of infectious agents, entering the body.
Antibodies can, however, be raised against practically any organic molecule. It has been shown
that antibodies, recognising the transition state form of a reacting molecule (or in practice a stable
analogue thereof) will stabilise this state. Consequently the activation energy is decreased
leading to a specifically catalysed reaction. This is an important fact, and a number of cases have
shown that the concept is true and hold in practice. Catalytic antibodies have, however, not yet
entered the industrial scene.
Biochemical engineering: this field deals with unit operations of production and use of
biocatalysts, describing their scientific and engineering basis, determining their performance and
operating characteristics, studying the factors which influence their performance and aiding in
their integration into complete processes. Today, commercial bulk enzymes can be considered as
cheap products, and their production processes with engineered organisms are fairly simple. The
product is, however, often present at a low concentration in a mixture of a large number of other
components. An essential part of such process is separation and purification of the enzyme at
the extent determined by the application. Separation and purification costs may be 50 to 80 % of
total production and investment costs. With bulk products their share is 10 to 30 %. Usually a set
of subsequent unit operations are used, such as filtration, centrifugation, ultrafiltration,
precipitation, or chromatography. Fermentation techniques optimise the production of biocatalyst
by an organism and separation techniques (down-stream processing) to isolate and, when
necessary, to purify the product from the fermentation broth. The price effect of a biocatalyst in a
potential application is often prohibitively high. Knowledge on advanced biochemical engineering
in addition to molecular biology are the keys to improved economy of biotechnical processes. It is
clearly desirable to maximise the content of the appropriate enzymes in the biomass that
produces it. Genetic modifications are important to achieve high product yields or contents of
biological catalysts.
3.3 Benefits of biocatalysts in industry
There is a abundance of existing knowledge on actual and potential use of biological

processes within a variety of industrial fields. These include areas with strong
biotechnical background such as the food and pharmaceutical industry sectors as well as
the more unconventional areas of textile or forest industries. In this chapter a general
overview is presented on the use of biocatalysts in the sectors selected for this project.
The description will specially focus on the benefits of biocatalysts in these sectors: their
catalytic, economic and environmental functions.
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3.3.1 Pharmaceuticals and Fine Chemicals

Pharmaceuticals are an European success story. European multinationals in this sector
lead the industry world-wide. All of Europe’s major pharmaceutical companies are
using biotechnology to protect, and in some cases, enhance their global competitive
position. Although Europe has been the leading producer of pharmaceuticals and has
produced more NCEs (new chemical entitles) than any other region, it is not clear that
this position can be sustained. The European industry’s share of NCEs has fallen
sharply in comparison to the US and Japan (see Table 2.2).
Table 2.2. Share of new chemical entitles (NCEs, %)
EU US Japan Total
1961-65 65 24 11 100
1986-90 39 32 29 100
Source: EFPIA, 1995.
The rising costs of R&D, the increasing cost containment measures in European health
spending and a slowdown in the growth rate of the European market have put enormous
pressure on the industry, particularly in Europe. Although the European multinationals
have been slow to establish biotechnology in-house as part of their R&D programmes
and manufacturing processes, nowadays most kind of biotechnology including
manufacturing processes - biocatalysis - are entirely integrated into R&D programmes.
Although modern biotechnology has been introduced relatively recently into the
pharmaceutical industry, the impacts on the economic performance of the
pharmaceutical sector has been considerable. These include reduced drug development
time, lower production costs, improved quality in production and a larger choice of
candidate therapeutics and other novel treatments.
The direct impact of past biotechnology performance on R&D can be measured by sales
of biotechnology products. Virtually all of the 27 biotechnology drugs listed on the
world market have been developed by the US specialised sector. Furthermore, many
biotechnology drugs under development are based in US companies. Because the small
biotechnology companies do not have the resources to fully develop and market new
drugs, 17 products have been licensed to large firms. Ten have been licensed to
European companies, the remainder to the US. However, Europe’s pharmaceutical firms
all now have substantial leading in-house R&D programmes with several
biotechnology-derived drugs in development. The threat is that this will encourage more
R&D in the USA (at the expense of the EU), so that the high value added jobs will
increasingly migrate to the USA.
It is much more difficult to measure the impact of biotechnology on production

processes in the pharmaceutical industry. The application of biotechnology to
production often involves incremental improvements in the yield of new and existing
drugs. Although such innovations are often at relatively small scale, they are the subject
nevertheless of patents. However, they are not a subject for publicity. Moreover,
IPTS 1998 15
refinements in production processes are sometimes protected by trade secrets rather

than patents and therefore are not widely discussed.
The first modern large scale application of industrial enzymes for the production of bulk
chemicals occurred in the early 1970s. In the chemical industry it was used to resolve D
and L forms of amino acids while in the pharmaceutical industry the first
biotransformation was for the production of modified (semi-synthetic) penicillin. These
processes all use immobilised enzymes whereby an enzyme (these days usually
produced by GMOs) is attached to a solid matrix. For example, 7000 tonnes of 6-amino-
penicillanic acid (6-APA), the precursor for penicillin, are produced each year. The
amount of resolved amino acids also runs into thousands of tonnes.
Biotransformation adds value in two different ways. In the production of generic

chemicals, biotransformation is economically advantageous and value is added by
converting a heterogeneous mixture of chiral forms of chemicals (two almost identical
compounds -stereoisomers-, the only difference is that they are mirror images of each
other) into a homogeneous form. This is particularly important in the pharmaceutical
industry where in racemic drugs - which contain both isomers -, one isomer has a
specific biological activity with a possibility that the second isomer may well produce
deleterious effects.
The production of chiral compounds (chemicals where a single stereoisomer of a

chemical is produced) is becoming of importance in the pharmaceutical industry. Its
importance stems from the thalidomide disaster. This was a racemic drug where one
stereoisomer had the desired and beneficial pharmacological effect but the other
stereoisomer caused birth deformities in foetuses. Although chirality is not a problem
for most drugs, it can be important as different chiral forms of every new drug have to
be synthesised to study their individual pharmacological effects. In practice, the
resolution of racemic mixtures is performed after synthesis where different
stereoisomers can be separated. Alternatively one stereoisomer may be removed
enzymically. In practice, enantiomeric excess of > 99.9% can only be achieved through
enzyme reactions.
In box 2 examples of using biocatalysts for production of pharmaceuticals are shown.

Most cases are examples of the production of a generic compound in the most cost
effective way. By contrast to the enzymatic processes for penicillin production, the
chemical method of synthesis of 6-APA utilises phosphorus pentachloride which must
be dissolved in organic solvents under strictly anhydrous conditions and at low
temperatures.
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Box 2 Examples of using biocatalysts for the production of pharmaceuticals.
ACE inhibitors ACE (Angiotension-converting enzymes) inhibitors such as Captopril are important
for the treatment for cardiovascular disease. Chirality is resolved by the use of a lipase purified
from Pseudomonas fluorescens.
Ibuprofen Stereoisomers can be resolved by selectively hydrolysing one isomer with an

Aspergillus oryzae protease. Ibuprofen is a major non-steroid anti-inflammatory agent that is now
available across the counter.
L-methionine To resolve chiral mixtures of methionine, the enzyme aminoacylase from

Aspergillus is used in the presence of cobalt, then a yield of 95% can be obtained. Furthermore
the enzyme can be used to resolve other amino acid mixtures such as those of phenylalanine,
tyrosine and leucine.
Penicillins and cephalosporins Almost 90% of penicillins sold for therapeutic use are now semi-
synthetic. These are derived from 6-APA which is made by the hydrolysis of penicillin-V or -G.
Similarly, semi-synthetic cephalosporins are made from 7-ADCA which is in turn made by the
hydrolysis of cephalosporinic acid-G or -V.
Atenolol Many pharmaceutical compounds contain secondary alcohols. There are different routes
to the chiral synthesis of secondary alcohol’s. These involve lipase resolution, whole cell
reduction of ketones and the enzymatic reduction of ketones. Chiral selectivity can also be
achieved by the use of secondary alcohol dehydrogenase.
Insulin Although much of today's human insulin is produced by genetically modified bacteria, a
substantial part is still produced from pancreas. In the latter case, the sequence is not identical to
the human sequence and therefore the pig insulin must be converted to human form by the use of
a carboxypeptidase-Y and threonine amide.
Peptide synthesis To some extent, recombinant bacteria are taking over the role of purified
enzymes in the production of small proteins. However biocatalytic enzymes still have an important
role to play, particularly in the synthesis of small peptides. One of the obvious choices for peptide
synthesis would be proteases and peptidases. It has been found that peptides can also be
synthesised by lipases.
Phenylglycine and Dp-hydroxyphenylglycine These chiral D-amino acids are produced in

quantities of more than 1,000 tonnes per year. The amide can be stereoselectively hydrolysed to
the L-amino acid.
Leukotrienes and lipoxins These molecules play a key role in the control of the cellular
metabolism and signalling. They are produced in the body from arachodonic acid by the enzyme
lipoxygenase. Most lipoxygenase is currently derived from soybean. As well as being of use in the
pharmaceutical industry, the same enzyme is used by bakers to decolorise bread. The addition of
linoleic acid and lipoxygenase results in oxidation of carotenoids that are responsible for much of
the colour of wheatflour. It can also be used to make a range of flavours such as mushroom and
cucumber.
3.3.2 Food and Drinks and Animal Feed

As mentioned before, the use of enzymes in food processing has a long tradition (cheese
and wine making, beer brewing, etc.). Apart from these ‘old’ enzymes (which have been
improved and updated several times to have better performances and higher yields), new
types of enzymes have found applications in food production.
Considering the use of enzymes in food processing and as premixes supplemented to

animal feed, a number of arguments to use enzymes can be identified:
• enhancing processing characteristics (higher yields, more specific conversions, faster
ripening),
IPTS 1998 17
• enhancing product characteristics (flavour, colour, debiting),

• enhancing the use of the product qualities (better digestibility).
The use of added enzymes in food processing is often initiated by the fact that the
endogenous enzymes do not function well enough. The added, exogenous enzymes are
used to correct and supplement the composition of the original, endogenous enzymes in
the plant raw materials. In most cases the use of enzymes is needed to improve product
quality characteristics. This means that application of enzymes may not influence the
product characteristics: thus e.g. the colour, taste, texture, mouth bite have to stay the
same. Only within the boundaries of maintaining the same product characteristics,
process improvements by means of biocatalysts can be made. Consumers are very
influential in the process of product innovation and because consumers of food are
known as very traditional, the use of new food ingredients is limited.
Use of enzymes in the food and feed industries1

The overview of the use of enzymes as presented in this paragraph does not reflect the
dynamic developments in this field, because this implies going too much in detail. We
focus on the most important applications, thereby showing the main driving forces for
the introduction of biocatalysts i.e. exogenous enzymes in this sector2.
Bakery sector
The introduction of industrial enzymes from the early part of this century allowed the
baking industry to use raw materials with a broad range of varieties. Wheat flour
contains both alpha and beta-amylases which can activate the dough to produce more
fermentable sugars for carbon dioxide production. The composition of endogenous
enzymes may be unbalanced, due to a number of reasons (climatological, geographical,
storage, transport etc.). Therefore, it is quite usual to supplement the flour by adding
alpha-amylase (fungal) at the mill.
As there are many different supply sources of this type of enzymes, there can also be
differences in their side effects (side activities) because of small variations in the way
the different amylases break down the starch. That is why also small amounts of a
variety of other enzymes (carbohydrases and proteases, peptidases and lipases) are
added. The selection of specific combination of amylases and additional enzymes can
only be made on the basis of practical and well-interpreted baking tests. At this moment
13 enzyme types are currently used by the baking industry.
The need to find alternatives to strong oxidising agents, such as potassium bromate, has
encouraged the search for enzymes that function in the same way. To create an
oxidising environment and to replace bromate with a similar biochemical step, enzymes
such as glucose oxidase, peroxidase, and catalase are being studied.
Dairy industry
1
Not included in the description are the bioconversion of agricultural raw materials (i.e. bio-alcohol as
fuel), starch conversion, biosensors and diagnostic kits and enzymes for flavour production.
2
The most important information basis for this chapter is Godfrey and West, 1996.
IPTS 1998 18
Cheese is one of the eldest biotechnical products in human food, often produced in local
varieties and made with traditional old recipes and craftsmanship. In the production of
cheese the coagulation of milk catalysed by enzymes is the central process. The group
of milk coagulating enzymes falls into three groups: animal rennet’s extracted primarily
from calves’ stomachs, microbial milk coagulants, derived from production by various
species of fungi and chymosin, identical to the original calf rennet but manufactured by
a cloned bacterium. The type used depends on national regulations, consumers
preferences and differs for each country.
Other enzymes used for cheese production are:

• Lipases which are added to cheese made of pasteurised milk before coagulating in
order to increase the content of free fatty acids during ripening. These fatty acids
probably contribute to the picante flavour of some (Italian) cheeses.
• Lysozyme for decontamination of spore formers in milk used for top quality cheeses.
Lysozyme is the preferred option, as the alternatives have been banned on safety
reasons (nitrates) or/and the equipment is too expensive (bactofugation).
• Endopeptidases for cheese ripening, and reducing storage time and flavour
enhancement in low and reduced fat cheeses.
• Catalase for destroying any excess of hydrogen peroxide remaining after sterilisation
of preservative milk or whey.
The high value of whey protein is well known, both from a nutritional and a technical
point of view. However, lactose in whey is considered to be of less value as it causes
sandiness in ice-cream, has low sweetening power and fermentability, and can cause
intestinal problems for people suffering from lactose intolerance. Value can be added by
hydrolysing the lactose, resulting in hydrolysed whey syrup. This product is highly
valuable in bakery products, confectionery products, deserts and ice-creams, spreadings,
dressings, soft drinks and semi-moist pet foods. The lactose hydrolysis treatment of
whey with immobilised enzymes (lactase or beta-galactosidase) for production of whey
syrups in a continuously operated reactor is another example of using immobilised
enzymes.
Drinks
The major biological reactions which take place in the beer brewing process are
catalysed by naturally produced enzymes from the barley and yeast. The process of
brewing is strongly influenced by the variety of barley, the method of cultivation and
varieties of seasonal weather. As barley is the main raw material, brewers may find
themselves using poorer qualities of malt as they would ideally like, which will require
the addition of exogenous enzymes to supplement the malt enzymes and in some cases
to provide additional activities not inherently present in the malt. Conventionally alpha-
amylase is used as an exogenous enzyme. A heat stable alpha-amylase requires,
however, shorter contact time and lower calcium levels. Thermostable fungal
betaglucanase may also be added. Enzyme suppliers have produced blends of enzymes
to provide the brewer with a single addition, such as a blend of amyloglucosidase and a
pullulanase/beta-amylase for the production of more fermentable sugars, resulting in
low-carbohydrate or ‘diabetic’ beers.
IPTS 1998 19
Addition of enzymes in beer production is limited to the steps before fermentation. The
only exception is the use of a recently introduced maturing enzyme in order to prevent
the forming of undesirable flavour ingredients (a-acetolactate) with a very low
threshold. The use of this enzyme can replace several weeks of maturation.
The use of immobilised brewer’s yeast in beer production is on a full industrial scale,
leading to considerable economic and environmental advantages. Savings in investment
costs on fermentation tanks and facilities, interests on lagered beer and in reduced beer
losses make the process economically feasible. However, also the environmental
impacts of the rapid, continuous process are predominantly positive. Decreased organic
waste water loading is achieved due to lesser need for emptying, draining and cleaning
of tanks and pipes.
Exogenous enzymes are now also widely used in wine making (i.e. one third of the
French wines are treated with enzymes), to obtain a better initial extraction of the must
components, thereby improving the yield. In depectinization - the enzymatic hydrolysis
of pectin -, the high viscosity caused by the pectin in the must is decreased. Moreover,
pectinase increases the juice yield during pressing by decreasing the viscosity.
Additional enzyme mixtures may be used to release the flavour molecules typical in
wine.
Fruit and some vegetables can be processed to produce fruit and vegetable juices or
concentrates. The addition of exogenous enzymes, the most important being pectinases
and glucanases, allows more specific degradation of carbohydrates to give smooth
textures, not found after heat treatments, and at the same time preserving colour and
vitamins. The cell wall is the most important part of the fruit, including the grapes, to be
broken down and to enhance juice recovery. For all the cell wall components, specific
enzymes are commercially available. It is not possible to produce clear concentrates of
fruits juices without adding enzymes. In the fruit juice industry, acid amylases are used
to process fruits containing starch, such as apples at early harvest.
A new trend is to improve the consistency and flavour of vegetable products. New
processes, including enzymes, work on vegetable rheology and develop new blends
from raw vegetables, and even include fruit-vegetable mixtures in which vitamins,
colour, and flavour have been preserved.
Sweeteners
Enzymes used for the conversion of starch to syrups comprise about 25% of all
industrial enzymes. By partial isomerization of glucose to fructose, the sweetness of the
comparably inexpensive glucose can be increased to the same level as the more
expensive sucrose. Glucose can also be prepared from starch by acid hydrolysis which,
however, results in low yields and undesirable by-products. Since there is no convenient
chemical process available for isomerization, syrups containing fructose are produced
by enzymatic processes using the glucose isomerase enzyme.
The isomerization of glucose to fructose is currently the largest-scale technical process
being performed with the aid of an immobilised enzyme. The product is used as liquid
sugar in food processing. The industrial process based on immobilised glucose
isomerase isolated from a Streptomyces species was introduced already in 1972. The
enzyme was adsorbed on DEAE-cellulose and used in a continuous computer controlled
IPTS 1998 20
process. Today, several companies offer a complete technology for production of high
fructore syrups.
Olive and other edible oils

The role of enzymes in edible oil and fat processing is virtually very small, mainly
because the necessary enzymes are not available at low enough prices (phospholipases
for the degumming of oils); the conservative nature of some parts of the oil industry
(e.g. olive oil processing); and technological barriers (enzymatic interesterification
technology). The amount of enzymes being used for oils and fat processing is still tiny
compared with use of enzymes in starch and protein processing in other areas of the
food processing industry. In the season 1993/1994 2% of the olive oil was processed in
Spain using enzymes. Olive oil processing consumes high amounts of water (1 tonne of
olives processed requires 1 tonne of process water). There is a commercially available
enzyme mixture to enhance the oil yield with 8 to 10 kg per tonne and to reduce the
process water use up to 50 percent, but its practical use is still very small.
Meat and fish

Enzymes are also used in meat and fish as a processing aid, e.g. to digest certain tissues,
such as skin or connective tissues. There is also great interest in enzymes from marine
environment and their potential usefulness in food processing: collagenase for crab
hepatopancreas for several applications, such as deskinning of squid, production of
caviar and ripening of salt fish. Lysozyme from clam shell, which is very psychrophilic,
with high activity at 0°C, could be used as bacteriostatic agent specially in food and
feed. The development of enzymes that bind tissues, such as transglutaminase in surimi
(fish product) or meat products in which small parts are bound to new higher added
meat products (Vilhelmsson, 1997) is another new idea.
Animal feed
The use of enzymes to improve animal feed performance is a commercially fast growing
application area. The enzyme additives can be divided in two sections: those dealing
with enhancement of general nutrient availability and phytase which improves the
availability of organic phosphorus (phytic acid) found in cereals and vegetable proteins
for the animal.
The main enzyme application is providing feed with enzymes. capable of degrading
NSP (Non Starch Polysaccharide) found in cereals and vegetable proteins. Use of beta-
glucanases or xylanases in feed increases the availability of dietary energy in feed. A
promising development is the use of microbial enzymes to increase the nutrient
availability of cell-wall carbohydrates.
The intensive rearing of animals in certain regions of Europe asks for a decrease in
nitrogen and phosphorus excretion in manure and their effects on water supplies. Two
enzyme applications can reduce this load of pollutants. First by improving the feed
digestibility, higher amounts of the feed dry matter, particularly nitrogen and soluble
carbohydrates, are retained by the animal, resulting in decreased excretion. Second, the
enzyme phytase can liberate organic phosphorus from feed raw material in order to be
digested by the animal. Herewith the phosphorus excretion of pigs may be reduced with
35% over a growth cycle (Liu and Baidoo, 1997).
IPTS 1998 21
3.3.3 Textile industry

The textile industry is often identified as a key sector where opportunities available
from adapting biotechnology are high but current awareness of biotechnology is still
quite low. However, the textile enzymes have been one of the fastest growing areas in
the enzyme industry. The applications especially in the textile finishing are, however,
easily fluctuated by the changing fashions. As an example, the popularity of stone
washed jeans has somewhat diminished, affecting thus also the enzyme markets.
Fibre preparation
Flax is one the oldest arable crops used by man. Until the 20th century, linen was mass-
produced in Europe. However, due to the reduction in economic subsidies and strong
competition between cotton and man-made fibres, the market share of linen declined
during the 1950’s. Nevertheless, interest in flax has remained and its main market sector
is in the top end of fashion along with the traditional sector such as table linens,
upholstery and others. Currently its total production is, however, less than 2% of the
textile output in the world.
The bast fibres of flax cannot be easily separated from the other plant tissues unless
some decomposition of the stem takes place. This controlled process of decomposition
is called retting. The retting of flax has always been one of the major costs and practical
limitations (environmental pollution) to the more widespread use of this indigenous
source of cellulosic fibre in Northern Europe. In various attempts since the late 1970s it
has been achieved to introduce more rapid and controllable enzyme retting processes.
These types of processes are based on the action of pectinases. To achieve optimum
degree of retting, however, enzymes other than pectinases are also important, such as
cellulases and hemicellulases.
Fabric preparation
In many fabrics production, a coating of starch - size - is used to prevent the threads
from breaking during weaving. After weaving the size has to be removed since sized
fabric is less absorbent. The desizing can be carried out by lengthy cooking or by using
strong chemicals such as acids, bases or oxidising agents. Enzymatic treatment with
amylase enzymes has replaced the harsh processes since the beginning of 1900.
However, there is still considerable scope for improving the speed, economics and
consistency of the process, including the development of more temperature stable
enzymes, as well as a better understanding of how to characterise their activity and
performance with respect to different fabrics, sizes and processing conditions. There are
many commercial alpha amylases available.
Scouring and bleaching of cotton fabrics are attractive targets for enzyme-based
processes. Researchers at several research centres in Europe have shown that pectins,
waxes and colour can all be removed but that residual seed coatings remain a problem.
New enzymes may offer an eventual solution.
The use of catalase enzymes to break down residual hydrogen peroxide after bleaching
process of cotton is an already established application. Reactive dyes are especially
sensitive to peroxides and currently require extended rinsing and/or use of reducing
agents.
Fabric finishing
IPTS 1998 22
Cellulase treatment of cellulose materials such as cotton, viscose, lyocell, cupro or

polynosic fabrics has gained increasing interest with the growing concern about
environmental issues. The best known applications of cellulases are in denim garment
washing - biostoning - as an alternative to stone washing and in surface modification of
cotton fabrics - biopolishing - to improve the surface properties. Cellulase can replace
the pumice stones and result less damage to the clothes, machinery and environment. By
using cellulases, there is no need for the time-consuming and expensive removal of
stone particles from the garments after processing. A small dose of enzyme can
substitute kilograms of stones. The machinery capacity can also be improved by 30-50%
due to reduced processing times. In biopolishing cellulases remove fuzz from the
surface of cellulosic fibres, which eliminates pilling, making the fabrics smoother and
cleaner-looking. A similar process using protease enzymes has recently been developed
for wool. Proteases are also used for the degumming of silk and for producing
sandwashed effects on silk garments.
Textile After-Care (detergents)

In contrast to textile processing, there has been a dramatic increase in the use of
enzymes in detergents since their introduction in the 1960s. The main classes of
enzymes in the detergent industry (proteases, lipases, amylases and cellulases) can
degrade a wide range of stains and their use allows milder washing conditions at lower
temperatures (Hamlyn, 1995).
3.3.4 Pulp and paper industry

The pulp and paper industry has adopted quite few biotechnical methods, so far. The
major reason is that the biocatalysts developed do not generally offer adequate benefits
in terms of their technical performance and costs. Because most commercial products
developed have not found large markets, the enzyme companies are reluctant to
significantly invest in this field. On the other hand, the pulp and paper companies seem
to prefer traditional technologies.
Mechanical pulping
Biotechnical methods aim to overcoming a number of drawbacks in mechanical pulping
processes (generally poorer strength properties, high electrical energy requirements and
the few suitable species of wood). The biotechnical pre-treatment methods of wood
chips - often referred to as biopulping - are based on the ability of white rot fungi to
carry out modifications in the raw material. After the fungal treatment the energy
requirement for the refining of mechanical pulp has been decreased by up to 50%.
Improved strength properties of the fibres have been obtained. Recently, the process,
developed in Wisconsin, has been scaled up to large pilot scale and is being
commercialised. Also in Europe, considerable research activities are carried out in this
field, especially in Austria, Finland and Spain. These efforts are also directed towards
the utilisation of non-woody fibres.
The microbial reduction of pitch (the troublesome extractives that cause negative effects
in the paper making process) can be carried out by two ways; by a microbial method on
wood chips prior to refining or by an enzymatic method on refined fibres before
papermaking. Treatment of wood chips with the fungus decreases both the total resin
IPTS 1998 23
acid and total fatty acid amount by approximately 40 %. By removing the triglycerides
from softwood mechanical pulp by lipases a significant reduction in pitch problems has
been demonstrated. The lipase treatment allows savings in the consumption of white
carbon, surface active chemicals. The cleaning frequency and the number of stops is
decreased.
Bleaching of chemical pulps

The kraft process is the world’s major pulping method. It has evolved over a period of
100 years and has become highly refined. Currently, about 70% of the world’s annual
output of approximately 100 million tonnes is produced by kraft process. The kraft
process results in the degradation and solubilisation of lignin. About 90% of the lignin
is removed; the less than 10% remaining in the pulp is primarily responsible for the
brown colour, characteristic of kraft pulp. In bleaching, the residual lignin is degraded
and dissolved by several chemical compounds, including chlorine. In the search to
produce pulp with non-polluting chemicals, more efficient pulping methods and
alternative bleaching methods have been developed, including enzymatic methods.
Xylanase treatment enables a reduction in chlorine consumption by 15-25%. Today,
enzymes contribute to bleaching sequences where chlorine is completely replaced with
chlorine dioxide or by non-chlorine chemicals. This allows the AOX levels to be
reduced by about 20%. This method is very flexible and applicable to different raw
materials as well as to different bleaching sequences. Investment is very low as the
treatment can be carried out in storage or intermediate tanks. Due to the present low
prices of enzymes, even savings in bleaching chemical costs can be achieved (table 2.3).
Today, long term use of enzymes has been reported by several mills. This rapid
development of xylanase prebleaching was partially due to the availability of reasonably
priced commercial enzymes and the low capital investment required for implementation.
Table 2.3. Chemical and cost savings with bleach boosting enzymes (from Roehm
Enzymes Finland)
Chemical consumption:
Normal ClO2 consumption 65 kg act. Cl/t

Saving of ClO2 with enzyme: 15 10 kg act. Cl/t
%
Savings of chemicals:
4 USD/t
ClO2 price: 0.4 USD/kg act. Cl
Costs:
Enzyme 1.5 USD/t
Acidification 0 - 0.2 USD/t
Net savings: 2.3 - 2.5 USD/t pulp
Source: Roehm Enzymes, Finland
Lignin biodegradation is fundamental in bleaching and other potential applications of

biotechnology in the pulp and paper industry. The commercialisation of efficient lignin-
degrading biocatalyst systems can be expected to take place soon. The future outlook of
these enzymes seems promising.
IPTS 1998 24
Paper manufacture
Enzyme-aided deinking technology has been developed for paper manufacture from
recycled fibres to reduce chemical consumption. There are two principal approaches to
the use of enzymes in waste paper deinking. One employs lipases to hydrolyse soy-
based ink carriers, and the other uses specific carbohydrate hydrolysing enzymes, such
as cellulases, xylanases or pectinases to release ink from fibre surfaces (Welt and Dinus,
1995).
One of the advantages offered by enzymatic deinking is the avoidance of alkaline

deinking chemicals. In an industrial operation, the use of enzymes as deinking aids
could lower the chemical costs and decrease negative environmental impacts. Offset and
letterpress newsprint waste have been enzymatically deinked at low pH in several
laboratory studies. Presently, however, the application of enzymatic deinking in
commercial installations has not yet been reported, although several pilot trials have
been carried out.
The fibrillation and drainage properties of recycled fibres can be improved by using a
mixture of cellulases and hemicellulases (Pergalase A 40 by Genencor Int). This
treatment is at least partly based on the removal of fine cellulose particles that impede
draining. Control of slime deposits in paper mill whitewater systems is another area in
which enzymatic approaches have been investigated. The deposits are mainly microbial
polysaccharides, which can sometimes be solubilised by enzymes. Biotechnical methods
are also used for enzymatic removal of pitch deposits, slimes and solubilised fine
particles. The enzymatic pitch control technology in paper manufacture has been
commercially employed in Japan for several years (Jeffries and Viikari, 1996).
3.4 Economic and ecological benefits of biocatalysts
The general overview of the application of biocatalysts in the four selected industrial
sectors shows that biocatalysts have a number of important benefits: cost efficiency -
(bulk) enzymes are cheaper then chemicals -, saving of energy and water, production of
less waste, shorter processing, ripening and storage time, higher efficiency, more
specific, etc.... In this paragraph we focus on the environmental benefits of the use of
biocatalysts and focus on the question: what are the main driving forces of industry to
use biocatalysts. Table 2.4 lists the main benefits of the major classes of enzymes for
cleaner production.
Table 2.4. Enzymes produced by Novo Nordisk saving energy, chemicals or raw
materials.
Product Application Saving
Proteases Washing detergents Energy, chemicals

Lipases
Amylases
Xylanases Bleaching of paper Chlorine

IPTS 1998 25
Proteases Leather industry Sulphides, COD
Catalases Textile industry Energy, water,

Cellulases acids, alkalis
Amylases
Amylases Starch industry Energy, acids
Source: Marshall and Woodley, 1996
In traditional established applications such as bakery or wine-production,

biotechnological processes clearly represent the technique with the best cost/benefit
ratio. On the contrary, looking at new developing industrial uses, biotechnological
processes are inherently expensive when compared to traditional chemical processes,
due to the present relatively high capital and process development costs. This means
that a biotechnological solution in most of the cases is only suitable for high added
value products with a cost price of 5,000 to 10,000 ECU per tonne or higher i.e. for the
production of fine chemicals and pharmaceuticals.
The fine chemicals industry is one of the industrial segments where the impact of
biotechnology (biocatalysis) is needed for the replacement of traditional, stoichiometric
processes in order to improve the product/waste ratio. The failure to translate
chemocatalytic processes from petrochemicals to fine chemicals makes this more
urgent. Introduction of biocatalysts would meet low entry barriers, i.e. low investments,
in this small scale industry.
The improvements in the environmental efficiency of the fine chemicals industry is due
to the application of biocatalysis, recycling of solvents and (biological) wastewater
treatment. The application of biocatalysis has made the largest contribution in cleaner
production, i.e. about 60%. The introduction of biocatalysis in the 1980’s in the
production of fine chemicals was highly appropriate and timely through which a large
reduction in the production of waste could be achieved (table 2.5). Despite a fourfold
increase in production volume in the fine chemicals sector, the production of waste was
reduced with 20% due to the use of biocatalysis.
Table 2.5. Growth and efficiency in the chemical industry between 1975 and 1995 1)2)
IPTS 1998 26
1975 1995
9% (10) 1 % ( 2 .5 )
Pet r o c hemic al
91 % ( 1 0 0 ) 99 % ( 2 5 0 )
50% (10) 9 % ( 2 .5 )
Bulk c hemic als
50 % ( 1 0 ) 91% (25)
9 % ( 0 .5 ) 33% (2)
Fine c hemic als
91% (5) 67% (4)
2 % ( 0 .1 ) 9 % ( 0 .5 )
Sp ec ialit ie
98% (5) 91% (5)
Wast e Volume
1 ) Product Volume
2 ) Amount i n mi l l i on t onnes i n br acket s
Source: Bruggink, Chemferm, NL.
Though biocatalysis has contributed for 60% to cleaner production in the fine chemicals
sector, also reuse or reduction of solvent demand has contributed to more environment
friendly production processes. As the fine chemicals sector is a small scale industry, the
absolute reduction for each individual production process is small. A list of products
that are now being manufactured using biotechnology is given in table 2.6
Table 2.6. Products and their production volumes as produced by biotechnology in the
fine chemicals sector.
Products Production volume in tonnes

Acrylamid >20,000
6-aminopenicillanic acid 7,000
7-aminocephalosporinic acid > 1,000
Aspartame 600
L-methionine 200
Vitamin B12 12
Vitamin C 70,000
Provitamin D2 5
Vitamin F 1,000
Nicotinamid 3,000
D-p-hydroxyphenylglycine 3,000
Source BUNR, 1996

IPTS 1998 27
This table indicates that biotechnological production of fine chemicals and

pharmaceuticals has been introduced on a large scale. Vitamins are still mainly
produced using traditional organic chemistry, the competition between organic
chemistry and biotechnology is yet strongly in favour of chemistry.
Bearing in mind that biocatalysis has led to a reduction in waste generation from 10 to 2
tonnes per ton of product, the annual reduction in waste generation for the above
examples would be in the order of one million tonnes of waste per annum.
In the pharmaceutical sector a number of biotechnological products, like antibodies,

can only be produced using biotechnology, and by definition cannot be classified as
clean(er) as they neither have zero-discharge nor do they replace or improve existing
technology. However, the volume of these products is usually very low.
Modern advances in biotechnology contribute to cleaner production of semi-synthetic
antibiotics by biocatalysis, optimised fermentation and replacement of organic solvents
by water. For instance, by replacing a chemical reaction in methylene chloride by an
enzymatic step in water, the use of methylene chloride was reduced about 25 ktonnes on
a global scale.
The sales created by clean biotechnology for the chemical sector are very small, as it
does not play a role in either petrochemicals or bulk chemical manufacturing. However,
clean biotechnology is prominent in the production of fine chemicals and
pharmaceuticals. The fine chemicals & pharmaceuticals segment of the chemical
industry produces about 1% of the volume of products. The contribution of clean
biotechnology to the sales value in the fine chemicals and pharmaceuticals segment is
about 60% for fine chemicals and between 5 to 11 % world wide for pharmaceuticals.
(Smith, 1996, Ballantine and Thomas, 1997; Abbott, 1996; Bickerstaff, 1995; OECD,
1996; Godfrey and West, 1996).
Table 2.7. The most important enzymes used in food processing and in feed
Sector Products Enzymes Benefits
Bakery Bread, cakes and alpha-Amylase Flour supplementation
biscuits Proteases Gluten weakening
Amyloglucosidase Improved crust colour
Oxidase Create oxidising environment
Dairy Ice cream beta- Galactosidase Prevention of ‘sandy’ texture caused by
(lactase) lactose crystals
Cheese Chymosin (rennet) Coagulation of milk proteins
Lipases Flavour development
Endopeptidases Accelerated ripening
Lysozymes and Removing spores formers resp. hydrogen
catalases peroxide
Whey syrup Lactases and beta- Remove lactose and produce sweet whey
Galactosidases syrup
Dairy products in Various proteases Modification of milk proteins
general beta-Galactosidase Hydrolysis of lactose for those who are
lactose intolerant
Drinks Beer alpha-Amylases Removal of starch haze
Papain Removal of protein
Amyloglucosidase Saccharification for low-carbohydrate beer
Wine Pectinases Increased yield, clarification
Amyloglucosidase Starch removal
Fruit juices Pectinases Increased yield, clarification
Glucose oxidase Removal of oxygen
IPTS 1998 28
Amyloglucosidase Starch removal

Coffee Pectinases Extraction of the bean
Meat and Meat Proteases Tenderisation and Removal of meat from
fish bones
Caviar Proteases Viscosity reduction of ‘stickwater’ and
increase the yield of roe
Pepsin Ease the riddling process
Tuna Proteases and De-skinning
carbohydrases
Meat and fish Transglutaminases Effect on physical properties of proteins,
thermal stability, gelation capacity, water
holding capacity
Ingredient Sweeteners alpha-Amylase Liquefaction of starch
s Amyloglucosidase Saccharification
Flavourings Lipases Ester synthesis
High fructose Glucose isomerase Conversion of glucose to fructose
syrup
Sweets Soft centred Invertase Liquefaction of sucrose
sweets and Sugar syrups
chocolates
Feed beta-Glucanase, Enhancing metabolizable energy in feed,
amylase and protease better nutrient availability
Phytase Better availability of phytic acid in cereals
and vegetable proteins
Sources: Madden, 1995; Vilhelmsson, 1997; Liu and Baidoo, 1997.
In table 2. 7 an overview of the most important applications of exogenous, microbial

enzymes used in food processing is shown. The table also includes the enzymes added
as ingredients to animal feed to influence digestion processes.
The traditional character of the food industry is one in which innovations only take
place step by step. The most influencing factor in this innovation process is the
consumer. This has consequences for the introduction of new technologies, including
biocatalysis. One important precondition for application of biocatalysts in the food and
drinks industries is that they are not allowed to have any effect on the food: the quality
and safety of the food may not be influenced. This together with the poor
understanding, on molecular level, of sophisticated biological conversions at hand in the
older food production processes (i.e. wine, beer, cheese, bread) results in minor changes
of the production process. Therefore environmental savings related to process changes
will not occur in the food industry. Environmental savings which are to be established
are related to minor process changes, and consequently will be small in effect. We can
also observe that the food industry from its origin and nature, has never used severe
process conditions using chemical compounds as compared with, e.g. textile or
chemical industries. This means that in the food industry there is less need for essential
environmental gains to be obtained by means of biocatalysts. Cleaner in this case refers
to using less energy and less water. The use of immobilised yeast’s in brewing is one of
the few examples of the application of enzymes in the food sector with explicit
environmental impact. Others are enzymes used in olive oil processing leading to water
savings and replacement of traditional technology using hexane, and less water to be
transported in concentrated fruit juices by using enzymes.
The size of clean biotechnology, using genetically modified organisms, in the USA in
food processing, beverages and feeds is relatively large compared to Europe. One of the
reasons for this is that the USA consumer more readily accepts foods manufactured
using modern biotechnology.
IPTS 1998 29
A rough estimate of the impact of process integrated modern biotechnologies in the

food industry - excluding the traditional applications of biotechnological techniques -
shows that the impacts are larger in the USA with a market share (process integrated
biotechnology related sales as part of total sales in this sector) of between 2% and 4%.
In Europe process integrated biotechnology has a market penetration of between 1% and
2 %. In Japan the role of this type of process integrated biotechnologies seems
negligible yet (OECD, 1996; Smith, 1996; Ballantine and Thomas, 1997).
In contrast, in the animal feed industry, things are different. Here a business to
business market exists where product and process changes are more easily accepted.
Enzymes were explicitly developed and used for environmental reasons; the addition of
microbial phytase in animal feed decreases the amount of phosphorus in manure
disposal. The use of enzymes in the feed industry is expected to grow rapidly and so are
the possibilities for environmental savings.
The use of enzymes in textile processing and after-care is one of the best established
examples on the application of biocatalysts to obtain environmental and technical
benefits in process industries. These methods explicitly aim at minimising the
environmental effects as well as improving product quality (reducing the damage caused
to the fibres during processing). The application of biotechnology to textile processes is
facilitated by the use of water solutions and relatively mild process conditions. Actually
biotechnical processing was introduced to textile industry already in the beginning of
1900. Enzymatic treatment replaced the cooking i.e. use of strong chemicals like acids,
alkalis or oxidising agents, previously used in desizing. This sector is likely to continue
to provide some of the most immediate illustrations of its potential also in the near term
future. There has been a dramatic increase in the use of proteases, cellulases and lipases
in after-care detergents since their introduction in the 1960s. The most important
applications are summarized in Table 2.8.
Table 2.8. Benefits of using enzymes in textile processing
Processing stage Benefits

Pre-treatment: Desizing, scouring and bleaching Reduced water, energy and auxiliary
consumption
Finishing : Biopolishing Improved product quality
Finishing : Denim treatment Less damage to machinery
Higher machine capacity
Decreased waste formation
Improved product quality
Improved control and reproducibility
After-care and detergents Energy savings
Reduced use of sodium perborate
Improved product quality
Source: Hamlyn, 1959 and Godfrey and West, 1996
Biotechnical methods have also entered the pulp and paper industry, aiming at
improving process stages to decrease environmental impacts, to save energy or to
improve product quality. Especially interesting today are biotechnical methods
supporting efforts to close the water systems of the mills, leading to minimal waste
release. The most clearly well-established biotechnical process step is the waste water
purification, however, as end-of-pipe technology not being within the scope of this
IPTS 1998 30
study. The functions and potential advantages of biocatalysts at different process stages
are summarized in Table 2.9.
Table 2.9. Benefits of using enzymes in pulp and paper processing.
Process stage Biocatalysts Benefits

Raw material treatment:
Debarking Pectinases Energy and raw material savings
Wood preservation Micro-organisms (fungi) Environmentally benign methods
Mechanical pulping:
Pre-treatment Micro-organisms (fungi) Pitch removal, energy savings
Refining Cellulases Energy savings, improved product quality
Chemical pulping: Micro-organisms (fungi) Savings in chemicals
Pre-treatment Various enzymes
Bleaching: Hemicellulases, Chemical savings, increased capacity,
Laccases reduced AOX-formation
Paper manufacture: Cellulases Increased productivity
Drainage Cellulases, Chemical savings, improved product quality
Deinking hemicellulases Improved products
Chemistry of the wet end Various enzymes Improved runnability
Source: Jeffries and Viikari, 1996
Conclusions
We conclude that the use of biocatalysts is based on their superiority in carrying out the
desired reactions, due to their specificity, economical advantages or improved
environmental impacts. Depending on the field of application, these reasons may vary.
Thus, in the field of food processing, biocatalysts have a history, thousands of years old,
based on first hand empirical findings. Today, these methods represent clearly the best
available technologies. If we observe the pharmaceutical industries, the target reactions
(such as many synthetic reactions) can often be carried out more easily by biocatalysts.
Targeted medicines are often based on knowledge about enzymatic reactions. However,
in both these industries, the minimisation of environmental impacts is not the primary
target, and plays usually no role.
The most important driving force are economic benefits, in some cases in a win-win
situation with environmental savings. However, environmental benefits from the
perspective of companies are, almost by definition, an outcome and not an input.
From several market studies it appears that the major impact in terms of sales value to
these sectors and the resulting sales value of biotechnology in (clean) production can be
found in the food and drinks and fine chemicals/pharmaceuticals sectors. In the process
industries, such as textile or pulp and paper, the use of biocatalysts aims at specific
processes with savings of energy or raw material, simpler processes with lower
investment costs or improved products. These economic driven applications show the
important environmental impact of biotechnology. However the sales to these sectors is
still relatively small (Smiths, 1996; Ballantine and Thomas, 1997; Degenaars en Jansen,
1996).
Often, in the public and political debate, the major advantage of biotechnology is
considered to be its positive environmental impact. The simple rational is that since the
process is biological - and biological is ‘green’ - all side events should also be
environmentally compatible. This is clearly an oversimplification. The simple
substitution of a chemical reaction by a biotransformation will not necessarily alone
IPTS 1998 31
lead to improved environmental impact. The environmental impact and economics of a

process must be considered as a whole, including energy requirements, process
parameters, product and waste streams and recyclability. The scientific, technological,
economic and ecological considerations relevant to a specific process will determine the
best available technology in competition. In practice modern technologies do not
compete but complement each other. The green image of biotechnology can only be
‘proven’ by decent life cycle analysis of competing processes.
Literature
Abbott G., ed., 1996. Biotechnology Industry Study Report 1996., In: In Touch with
Industry: ICAF Industry Studies, Academic Year 1996., Industrial College of the Armed
Forces National Defence University Washington, DC 20319 - 5062.
Ballantine, B. and Thomas, S., 1997. Benchmarking the Competitiveness of

Biotechnology in Europe, The European Association for Bioindustries, Brussels,
Belgium.
Bickerstaff, G.F., 1995. REVIEW: Impact of Genetic Technology on Enzyme

Technology., The Genetic Engineer and Biotechnologist, Vol. 15, No. 1, Journals
Oxford Ltd.
BUNR, 1996. Umweltpolitik; Tagungsband des Fachgesprächs “Beitrag der

Biotechnologie zu einer nachhaltigen, umweltgerechten Entwicklung”,
Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit, Bonn.
Degenaars, G.H. en Janszen, F.H.A., 1996. Modern Biotechnology within the Dutch
Industry: Critical Factors for Success., Erasmus University Rotterdam, Ministry of
Economic Affairs, The Netherlands, pp. 125.
EFPIA, European Federation of Pharmaceutical Industries Association (1995) The

Pharmaceutical Industry in Figures, EFPIA, Brussels
Godfrey T. and West S. (1996) Industrial Enzymology 2 edition., Macmillan/Nature.
Hamlyn, P (1959) The Impact of Biotechnology on the Textile Industry, Textile

Magazine, 3, pp. 6-10.
Jeffries and L. Viikari eds. (1996) Enzymes for pulp and paper processing, ACS
Symposium Series 655.
Link, Matt (1990) Enzymes in the forefront of food and feed industries, Keynote lecture
on the First international Symposium on ‘Enzymes in the Forefront of Food and Feed
industries’, Food Research Foundation, ELINTARVIKKEIDEN TUTKIMUSSAATIO.
IPTS 1998 32
Liu, Yonggang and Baidoo Samual K. (1997) Exogenous enzymes for pig diets: an
overview, in: Enzymes in Poultry and Swine Nutrition, R.R. Marquardt and Zhengkang
Han eds. International Development Research Centre, Canada.
Marshall, C.T. and Woodley, J.M. (1996), Process Synthesis for Multi-step Microbial
Conversions, BIO/TECHNOLOGY, Vol. 13, October, pp. 1072 - 1078.
Madden (1995), Food Biotechnology: an introduction, ISLI Europe, Brussels.
OECD, 1996 The OECD STAN Database for Industrial Analysis: 1975 - 1994., OECD,
Paris, France, pp. 362.
OTA, 1991. U.S. Congress, Office of Technology Assessment Biotechnology in a

Global Economy, OTA-BA-494, Washington DC, U.S. Government Printing Office.
Smith, J. ed., 1996. the Future of Biotechnology in Europe: From Research &
Development to Industrial Competitiveness., Club de Bruxelles, contribution for the
Conference organised by the Club de Bruxelles on Sept. 26 and 27, 1996, Bruxelles,
Belgium.
Vilhelmsson Oddur (1997) The state of Enzyme Biotechnology in the Fish processing
industry, in: Trends in Food Science and Technology, August 1997, vol. 8, p. 266.
IPTS 1998 33
4. Europe’s scientific-technical competence in

biocatalysis
4.1 Science base in life sciences and biotechnology: EU vs. US
The science base is critically important in biotechnology because it produces new

knowledge that provides the foundations for R&D and ultimately, new products or
processes. The science base, as well as producing new knowledge, gives rise to new
instrumentation and methodologies, new skills and creates spin-off companies. The
science base is particularly important in biotechnology in general because
biotechnology is closely linked to developments in basic research. Realising the
potential of biotechnology depends on a strong science base and good links between the
science base, specialised biotechnology companies and large or user companies.
In general, there is a perception that the science base in the US is stronger than in
Europe and that this factor has contributed to the US lead in biotechnology. This
evidence comes from a range of previous studies which generally support this notion. A
recent survey of over 50 biotech companies with operations in both Europe and the
USA revealed that these companies have the perception that the science base in the US
is superior to that in Europe. The scale and quality of public investment in R&D, the
relevance of public investment in R&D, the overall scale and quality of academic-
industry collaboration and the effectiveness of the technology transfer mechanisms were
all factors felt to be significant in achieving the strengths in the US. However, the same
group of companies also consider that the quality of public sector research in Europe is
as good in the USA (Ballantine and Thomas, 1997).
What are the main reasons behind these differences? In the first place, the USA spends
probably 50% more than Europe on life sciences; thus, expenditure on life sciences in
Europe is estimated at approximately ECU 15 per capita compared to ECU 22 per capita
in the US. However, the reality is that this spending gap between the USA and Europe is
actually greater than that given by these figures because the cumulative impact of high
US spending over a longer period and the relatively fragmented nature of spending in
the 15 EU member states enforce the differences. It has also been estimated that
biotechnology-specific investment in the US science base is at least three times higher
than that in Europe. Concerning links between universities and companies, in-depth
interviews also revealed that scientists in Europe often find it difficult to identify
themselves with commercial opportunities (IMD, 1996).
Finally, bibliometric indicators also revealed that Europe’s scientific research has less
impact than that of the US (table 3.1). In general, European scientific papers in life
sciences have markedly less citations per paper in disciplines central to biotechnology.
IPTS 1998 34
Table 3.1. Bibliometric indicators: impact (average citations per paper, 1982-1992)
Europe USA Other

All sciences 3.77 5.26 3.71
Biology 7.24 9.83 7.51
Biochemistry and molecular biology 3.26 4.07 3.14
Biotechnology and applied 3.90 5.55 4.00
biotechnology
Chemistry 3.19 5.54 2.83
Plant science 2.83 3.19 2.63
Based on articles, notes and reviews only (three year citation window).
Source: J.S. Katz, BESST Project, SPRU, University of Sussex in: Ballantine and Thomas, 1997,
p58.
4.2 European competence in biocatalysis
What are Europe’s relative strengths in biocatalysis in relation to its science base as
outlined above? In Europe there is relatively little academic research on biocatalysts
which has a bearing on the pharmaceutical industry. Simply by its nature most of this
research is highly applied, often focused on the improvement of production processes,
and carried out by companies. This kind of research is generally either confidential and
protected by trade secrets or patented. Despite which of these two modes of intellectual
property are used, the outcome, in terms of publications, is broadly the same. In other
words the amount of published material on biocatalysis by European academics is at a
very low level.
A further problem is that it is difficult to allocate published papers on basic research as

being specifically relevant to different sectors. For example, about half of the published
papers in table 3.1 cannot be attributed to a specific industrial sector, thus a basic
research paper on, for example, proteases may ultimately have relevance to both the
food industry and the pharmaceutical industry.
However, several indicators reveal that Europe is internationally competitive in

biomedicine and molecular biology. For example, nearly half of the top molecular
biology institutes ranked by citation impact in 1991 were European (Sciencewatch,
1992).
An indication of the scientific competence of Europe in the field of biocatalysis can be

obtained by an analysis of published papers. By assessing the authorship of papers
published in ISI cited journals it is possible to obtain a crude estimate of productivity in
a particular area. A search was performed using the keyword "Biocatalysis" in both the
title and the abstract of the paper. Between 1994 and 1997, 303 "hits" were made and
the country of origin of the paper was determined. This is shown in table 3.2.
IPTS 1998 35
Figure 3.1 Origin of papers with a keyword on “Biocatalysis” between 1994-1997.

Papers (%)
South Korea 2
Country Papers (%)
Italy USA 2 30
UK 10
Austria Canada3 7
France 4 6
Spain
Netherlands 5
Sweden Japan 4 5
Sweden 4
Japan Spain 5 4
Austria 5 3
Netherlands
Italy 2
France South Korea 6 2
Canada 7
UK 10
USA 30
0 5 10 15 20 25 30
As might be expected, the leading country is the USA with 30% of the papers
containing the keyword "Biocatalysis" either in the title or the abstract. Next is the UK
followed by Canada. The columns in the above table do not add up to 100% as countries
with less than 3 papers are not included in the above analysis. However, if the papers
from all EU countries are added together, these make up 42% of the total. Included in
this are countries such as Portugal (1.5%), Ireland (1.5%), Finland (1.5%) and Germany
(2.5%).
This analysis, however, is very broad in the sense that it covers everything from site-
directing mutagenesis, chemical engineering and the isolation of novel enzymes. On the
other hand, the number of hits does apparently not correspond to the numbers of papers
published within this area (of biocatalysts). However, we believe it reasonably and
accurately reflects the competence of particular countries. There do however seem to be
some surprises in this list, which may turn out to be some artefact of the data. Firstly,
Japan (5%) is under-represented and secondly, Denmark (with just a single publication)
may also be underestimated.
4.3 Scientific competence in the industrial sectors
Big parts of research capabilities in biocatalysts is concentrated in the industrial

laboratories of enzyme producers. The research needed for industrial production of
enzymes needs development of production host, of their excretion systems, of
fermentation technology, of protein chemistry, of molecular modelling, identification of
new enzymes and applications and also development of the application. This means that
together with the enzyme specialists there is a big number of specialists for each of the
application areas. Besides the actual enzyme producers, extensive development work is
being performed by the enzyme suppliers (who do not necessarily have their own
production).
IPTS 1998 36
Pharmaceutical industry
Biocatalysis research in relation to pharmaceuticals, such as it is, is spread across
departments of chemical engineering and microbiology in public sector institutes. A
tradition of collaboration between these two types of departments is largely absent. This
contrasts with the situation in the pharmaceutical companies undertaking this type of
research where the chemical engineering and microbiological aspects of biocatalysis
need to be thoroughly integrated to be incorporated within the production processes.
Academic research in this area tends to be highly focused with groups perhaps working
on a single enzyme over a long period of time3.
Food and feed

Food processing is only partly based on scientific knowledge of the molecular basis of
these processes. Craftsmanship is still a very important compound of expertise in
enzyme research; the fundamental processes underlying important food processes like
the baking of bread are not yet understood on a molecular level. For the other food
sectors things may be ‘better’, but a total understanding of the processes is not present.
This of course is due to the complex biological processes in food processing. Every
enzyme product shows additional activities or side effects when added to a substrate.
Consequently, improvements in the processes are related to extensive testing and
research in specific applications. So, one of the largest test-baking lab’s in the world is
Quest Research Lab in Naarden. As this craftsman application knowledge on the
conduct of enzymes is not patentable, the enzyme industries have to keep their research
in-house to protect it.
All this results in a rather big, heterogeneous and diffuse structure of the public research
community in the field of biocatalysts used in the food and feed industry. At many
universities biocatalys research is taking place but it is scattered in small subgroups and
mostly related to part of other subjects (bio-processing, organic chemistry, biological
chemistry, biotechnology, food chemistry, food technology, chemical engineering, fine
chemicals etc.).
Textile, pulp and paper

Both areas involve traditional technologies which have been developed under a long
period, and which have undergone extensive modifications and improvements already.
This is especially true in the pulp and paper industry where the environmental loads
have significantly decreased, especially in Northern Europe, during the last decade.
Excluding the waste water purification technology, this has mainly been carried out
without the use of biocatalysts. The present applications of biocatalysts involve
processes where enzymes act rather as enhancers or additives, and these process stages
are not based on biotechnology, solely. Thus, these can be considered as applications of
the first generation. Extensive modification of the traditional processes (into
“biotechnical” unit operations) would require more efficient biocatalysts and
application technologies.
The European research has many strong areas in this field. Research on traditional
cotton production areas, such as US, is however strong. The textile research tradition is
3
For example, research at the University of Exeter focuses on thermophilic enzymes.
IPTS 1998 37
practically absent in Europe. The European know how is especially good in the fields of
cellulases and cellulose structure studies. Northern Europe has traditionally excellent
research and know how in the pulp and paper area, but there are also other high-quality
research units in Central Europe. Research on biocatalysts in the pulp and paper area is
widespread, but there are only few centres of excellence.
4.4 Patent analysis of biocatalysis
4.4.1 Relevance of patent statistics

Patent statistics have long been used as indicators of technological activities by
academics as well as policy-makers. As with other technology indicators, such as R&D
expenditure, they have their own relative advantages and disadvantages and these have
been reviewed in detail elsewhere (Pavitt, 1988; Griliches, 1990; Patel and Pavitt,
1995). Does counting the number of patents in industrial sectors and companies provide
a useful indicator of competitive success? As Sharp et al (1996) have shown, there is
no consistent relationship between R&D expenditure, number of patents and number of
products in the Top 50 pharmaceutical companies. Thus Rôche, which is first in the set
in terms of the percentage of sales devoted to R&D, and is amongst the top group in
patenting activity and new drug development, has only one of the top selling drugs.
Similarly Glaxo's patenting activity and the number of new drugs under development
were relatively low in comparison with companies with much less R&D expenditure.
The development and wide application of biotechnology has posed major challenges for
the patent system in the US, Europe and Japan. Although patents for new chemicals
have been granted over a century, the patent system of many countries has explicitly
excluded the patenting of any naturally occurring substance or life forms. While the
strengths and limitations of existing patent protection has been effectively adapted to
inventions in classical microbiology, the specific exclusions laid down long before the
development of biotechnology continue to cause major problems of interpretation. As
commercial exploitation of biotechnology is now gathering momentum in the
pharmaceutical and agrochemical sectors, strong patent protection for biotechnology-
related inventions is assuming an increasingly critical part of corporate strategy.
Biotechnology has been widely integrated within the pharmaceutical industry which is
already known to be a high user of patents (Levin, 1987). Patenting as a means of
protecting inventions is also fairly well established in the food industry.
The main advantage of patent statistics as indicators of technological activity is that

such datasets are available over long periods of time and can be grouped into technical
fields and by organisation. However, there is considerable empirical evidence for the
limitations of patents as indicators and biocatalysis as a field is no exception. It is
important to bear in mind that only about 7% of patents will ever actually be used. The
remainder will not be commercially exploited. Patents are an important way to protect
hard-earned R&D results, but they are equally significant as a source of technical
information. An often-quoted figure is that around 80 per cent of all publicly-available
technical information is published in patent documentation - and often nowhere else.
Around 90 per cent of this information can be directly used by people other than the
respective patent holders; only the remaining 10 per cent is protected by valid patents.
IPTS 1998 38
The steady growth shown in some of the patent databases in biotechnology patents
reflects the fact that biotechnology has become increasingly integrated within the
pharmaceutical sector which habitually patents heavily. These patents will, for both
products and processes, contain a wide range of claims. In some cases where products
cannot be patented because of existing prior art or lack of novelty or inventiveness, a
process patent can be used instead. For biotechnology companies, particularly those in
the US, patents have become a very important asset with regard to the raising of funds
from venture capitalists and shareholders. Several studies have shown that small
companies preferentially patent over large companies in terms of numbers of
biotechnology patents per company (Thomas et. al., 1997).
4.4.2 Biocatalysis patents

From 1990 onwards there has been an increase in the number of biocatalysis patents and
general biotechnology patents in the physics and engineering fields. Whether this is due
to strategic registration policies or the result of knowledge and technology transfer from
classic biochemistry/biology is not clear from the patent data.
Most patents are in the following sectors of industry.
• Medicine È biochemical therapies, diagnosis and cancer medication;

• Pharmaceutics È large-scale production;
• Chemicals È environmentally compatible production processes;
• Food È producing, processing and preserving foodstuffs.
A wide variety of sectors are involved in the field. As the processes are used in a wide
range of industrial sectors more than one allocation is not possible. The main user of
enzyme processes however is clearly the pharmaceutical industry.
There are two distinct areas:
• Biocatalysis as a production process, and
• Biocatalytic products themselves as ingredients in medication, chemicals
(preservatives) or food (60% of all hits).
The figures show the patent database analysis of biocatalysis patents in the most
relevant EU member States (figure 3.1) and for EU, USA and Japan (figure 3.2).
Figure 3.2. Patent registrations in the biocatalysis sector (enzyme classes) in different
EU-countries, 1960-96
IPTS 1998 39
1000
815
800
600 562
400 339
200 70 72 30 38 6
0
DE GB NL IT FR ES BE NO
Figure 3.3. Patent registrations in Europe, USA and Japan in the biocatalysis sector
from 1960 to 1996
600
500
400
300
200
100
0
96 95 94 93 92 91 90 89 88 85 80 75 70 65 60
US JP EU
Patent analysis for the industrial sectors

’Biocatalysis’ is only used as a search word in a small number of patents. Therefore
additional searches were made with the word ’enzyme’. A search in the World Patent
Index revealed 4,763 patents in total. Figure 3.3 shows the distribution of patents across
the fields "pulp and paper", "pharmaceutics" ,"fine chemicals" and "food". All search
terms were truncated so as to include plurals and genitives. The search for individual
enzyme classes together with biocatalysis will take place at a later date.
Figure 3.4. Thematic overview of patents in the field of biocatalysis
IPTS 1998 40
61%
Pulp & Paper

Pharmaceutics
3%
Fine Chemicals
36% Food
0,5%
Pharmaceutical sector
An analysis was made of the overall situation of the "initial registering country" of the
pharmaceutics patents in terms of which country registered them first. The results from
the "enzyme and biocat" search were combined with the "pharmaceutics" (figure 3.4).
The analysis shows that in countries where one could expect higher values on account
of their scientific/technological situation (e.g. Denmark, DK) few patents were
registered in their own countries.
Figure 3.5. Patent registration in pharmaceutics world wide (1960 to today)
Number of patent registrations

888
900
800 745
700
600
500
400
300 260
186 182
200
79
100 23 4 11 30 40 20
6 19
0
US JP DE DK NO GB ES FR NL BE IT SE CH AU Country
Therefore, the next analysis carried out was for the ’pharmaceutical biocatalysis’
thematic complex (search for "biocat and enzyme " and "pharmaceutics") according to
the registering country (originating country of the patent). Denmark was selected
because NOVO has its home base in Denmark. The search for Danish patents resulted
in a total of 327 compared to 23 patents registered in Denmark. The patents in the
countries in figure 3.4 were then examined to see which were Danish. The results are
presented in figure 3.5.
Figure 3.6. Danish biocatalysis patents in the pharmaceutics sector by country (1960 to
1996)
Number of patents
IPTS 1998 41
140 136
120
100
80
60 48
41
40 25
20 2 5
0
US JP DE NO GB DK
Country
The distribution shows an interesting phenomenon. Danish developments are registered
predominantly in countries with a high level of industrial application for biotechnology,
and not in Denmark itself. The patents are registered to gain strategic benefits. This
explains the registration of Danish research results mainly in the USA which is the
leader in biotechnology.
The results of registration within Europe is also interesting. Whilst no Danish patents
can be found for Spain or Italy, Germany and Britain have the most registrations after
the USA within these countries - a phenomenon which can be explained by the overall
economic and technological situation. Both countries have a high R&D expenditure in
the pharmaceuticals and general biochemistry sectors and a large number of industrial
production companies.
Food
A similar analysis was also made for the food sector. The results show similarities with
that of the pharmaceutical sector. Interesting is the Japanese patent situation in the
sector of food. 1919 patents are applied at first in Japan (figure 3.6).
IPTS 1998 42
Figure 3.7. Patent registration in "food" world wide (1960 to today)
Patent registration
2000
1919
1800
1600
1400
1200
1000
780
800
600
400 246 182 191

200 47 2 16 47 20
12 29 33 40
0
US JP DE DK NO GB ES FR NL BE IT SE CH AU
Country
A possible cause for this situation may be the high export rate of Japanese food to other
Asian countries. For example the fish industry in Japan is one of the most important in
the world. There is high R&D budget for research on fish based food and ingredients.
Paper and pulp

In a third step the pulp and paper sector has been analysed for patent situation in a
number of countries. Figure 3.7 shows the result.
Figure 3.8. Patent registration in "pulp and paper" world wide (1960 to today)
Patent registration
80
71
70
58
60
50
41
40
30
20 16
10
6 4 7
10 2
0 0 0 0 0
0
US JP DE DK NO GB ES FR NL BE IT SE CH AU
Country
IPTS 1998 43
EU patenting system
The fact that in 1996 less patents have been registered by the EPO as to 1994 and 1995
might indicate that patenting is becoming more and more a strategic competition
instrument which is highly influenced by the national patent system. It seems that the
high costs and the lack of awareness mean that European companies are failing to make
best use of the patent system. The relatively cheap US patent registration system serves
as a strategic competition tool, which helps controlling market(s) / shares. The present
system of patents in the EU is complex and expensive, and does not provide a unitary
patent for all the Member States.
However, innovations should enjoy the same protection throughout the single market, in
order to develop the role played by innovation in competitiveness and job creation.
Probably the most apparent problem with the current system is costs. Patent costs are
not always easy to compare, because they are made up of several different components
payable at different stages during the lifetime of the patent. Nevertheless, there is no
doubt that European patents are far more expensive than those in America or Japan.
A typical European patent to be protected in eight Member States costs around ECU
20,000 and this does not include mandatory translation costs. A US patent, by
comparison, costs around ECU 1,500. Japanese patents cost only ECU 1,100 each,
though this figure is somewhat misleading because Japanese patents tend to be smaller
in scope than their western counterparts. The total cost includes the fees charged by the
EPO and national patent offices, patent attorneys’ charges and translation costs. The
EPO is currently in the process of cutting its fees by around 20 per cent, but many users
think this is still too expensive. These and other problems all stem from today’s ’dual
system’ arrangements. While inventors can apply for a European patent through either
the EPO or a national patent office, in reality, patent applications are split equally
between these two routes. In fact, more than 90% of applications filed by EU nationals
are based on a previous national application.
Patenting of enzymes has historically not been a major issue: many companies produce
the same or equivalent enzymes. For example, there are over 12 companies world-wide
which produce amylase. In future patenting of enzymes will become of great importance
as companies realize that this is the way to protect their investment in research and
development and also give them leverage to obtain market share.
Since there is no single European patent, fragmentation of the internal market, with
separate patents needed for each Member State, is a realistic option. The cost of
securing patent protection in every Member State will discourage companies from
exploiting their innovative potential, while the lack of legal mechanisms and legal
infrastructure at European level means that interpretations and applications of patent
legislation are different. The USA and Japan both have a single patent mechanism and
legal framework allowing protection in the whole territory.
The existing structure in Europe consists of the European Patent Convention (EPC),
which includes non-EU Member States, and the Agreement relating to Community
patents, which has not yet entered into force. These two instruments represent a
complex legal framework for those seeking patent protection, and are subject to
different legal jurisdictions. Alongside national patents, which continue to exist, there is
IPTS 1998 44
a European patent, though only an embryonic one: once granted by the European Patent
Office in Munich, the European patent operates to all intents and purposes like a
national patent. There is no provision for a court of law with jurisdiction at European
level to decide disputes in patent cases, such as actions for infringement or revocation
of a patent, so that there is always the danger that the courts that hear such actions in the
Member States may deliver conflicting judgements. The present system consequently
places serious difficulties in the openness and smooth operation of the single market.
The given market situation in the biocatalysis sector clearly calls for the adoption of a
"truly operational Community patent system", which would be comparable to that of its
two main competitors, Japan and the USA. This system should also provide adequate
and non-discriminatory treatment for non-EU Member States. Under this system, the
costs for patent protection within Europe would be reduced to a level comparable to the
USA and Japan (although the costs of translation have to be considered in setting the
fees). In the USA, SMEs benefit from a 50% reduction in the costs of patenting,
whereas, in Europe, such reduction does not exist under the EPC.
A unitary Community patent would have the advantage that its effects would be the
same throughout the Union; it could be granted, transferred, revoked or allowed to lapse
only in respect of the whole of the Union.
4.5 Conclusions
Biocatalyst research is in the first place concentrated in the industrial laboratories of

enzyme producers and enzyme suppliers which market and sell enzyme products for
specialised fields. There are several groups in the public funded research organisations -
universities and research institutes - in Europe, specialised in different types of
biocatalysts and there are of course the basic scientific disciplines which are the pole of
knowledge, dedicated or not, for the applied biocatalysis research. Taking into account
the different area’s of expertise involved in the biocatalysis research, in theory it is
possible to give an overview of Europe’s scientific and technological potential in the
field of biocatalysis. However, identifying the scientist in public and private
laboratories, describing their activities and their products and finding reports with an
assessment of their products (audits, visiting committees, referee’s etc.) would take
several years. Hence, we had to conclude that in the practical context of this project it
was impossible to make such an overview, especially if in addition specific attention
should be given to the environmental benefits of biocatalysis.
Nevertheless, on the basis of recently published reports with more qualitative data we
were able to make a number of observations on Europe’s scientific and technological
potential in the field of biocatalysis. In industry the perception is that the science base in
the field of biotechnology, which includes biocatalysis, is stronger in the USA than in
Europe. The quality of public sector research in Europe is evaluated as good as in the
USA. An indication of the strength of biocatalysis research in Europe compared with
other regions in the basis of the productivity of papers reveals that Europe as a whole
(42%) is somewhat stronger then the USA (30%) or Japan (5%). One last indication of
the present state of Europe’s research base in biocatalysis, is that it is well organised.
The European Federation of Biotechnology (professional organisation of biotechnology
IPTS 1998 45
researchers) has a working party on Applied Biocatalysis. The members of this party are
from 23 countries from Western and Eastern Europe.
Patent data base analysis shows that the number of EU biocatalysis patents is lagging
behind those of the US and Japan. However the results have to be interpreted with care.
First the key word ‘biocatalysis’ is too general, and may give references to especially
organic chemistry since biocatalysis is not necessarily given as a key word in works
related to enzymes. It is also important to realize that because patent strategies differ
considerably between regions, it is not allowed to estimate the scientific-technological
potential of European biocatalysis research on the basis of these patent analysis.
Literature
Ballantine, B and S.M. Thomas, (1997) Benchmarking the Competitiveness of

Biotechnology in Europe, report prepared for EuropaBio, Brussels.
Griliches, Z. (1990): 'Patent statistics as economic indicators', Journal of Economic

Literature, p.28.
IMD (1996). The World Competitiveness Yearbook, Geneva.
Levin, R.C. et al (1987): 'Appropriating the returns from industrial research and
development', The Brookings Papers on Economic Activity, Vol. 3, pp 783-831.
Patel, P. and Pavitt, K. (1995): 'Patents of technological activity: Their measurement

and interpretation' in Handbook of Economics of Innovation and Technical Change,
Stoneman, P. (ed), Blackwells, Oxford.
Pavitt, K. (1988): 'Uses and abuses of patent statistics' in Handbook of Quantitative

Studies of Science and Technology, van Raan, A. (ed), Amsterdam, North-Holland.
Sharp, M. and Patel, P. (1996) Europe’s pharmaceutical industry - an innovation

profile, STEEP Discussion Paper No. 34, SPRU, University of Sussex.
Thomas, S. et al., 'DNA sequence patents in the public sector', Nature, 7 August 1997)
IPTS 1998 46
5. An overview of the economic potential of Europe’s

biocatalysis industry
5.1 Introduction
In this chapter the economic potential of Europe’s biocatalysis industry will be

discussed. First the size and structure of the biocatalyst industry and the biocatalyst
market is addressed. In addition, an estimate and description is made for each sector of
the working processes in the sectors in which biocatalysis is used. The chapter will
finish with the general bottlenecks (technical, economic, socio/cultural) met by industry
in applying biocatalysis.
5.2 The economic capabilities in Europe
5.2.1 Structure of the industry

In general, the development of new enzymes is performed by the enzyme producers.
However new enzyme applications can also be developed by enzyme providers or by the
customers, in most cases the bigger food and drink companies and the pharmaceutical
industry. The latter screen commercially available enzymes and develop new
applications, sometimes with the assistance of the enzyme providers.
The enzyme providers develop enzyme mixtures for specific applications on the basis of
their knowledge of enzymes and specific applications and market niches. This is of
course also an activity of the enzyme producers themselves - all producers in the
enzyme industry buy and sell enzymes from each other - but these provider companies
have an important position in the market. The enzyme providers are especially active in
the bakery ingredients sector. It is estimated that in this sector an enzyme is sold 2.5
times before being used in a baking process.
Godfrey and West (1996) made an inventory of the enzyme supplier companies. They
found that 400 companies world-wide offer enzymes in their sales programmes. A
considerable number of these companies are agents and distributors and if these are
excluded a list of 137 companies is left.
Table 4.1 shows the geographical distribution of these companies. Even though the
market as a whole seems to be dominated by three major producers, the number of
companies selling enzymes (this includes the providers) doubled during the 1980s.
IPTS 1998 47
Table 4.1. Distribution of enzyme providers world-wide
Region/Country No. of companies Region/Country No. of companies

Europe 70 Australia 2
USA 30 South America 2
(Mexico and Brazil)
Japan 24 Republic of South Africa 1
Canada 4 Russia 1
Southeast Asia 3
(Thailand, India and Taiwan) Total 137
Source: Godfrey and West, 1996.
Table 4.2 shows the distribution of the 70 European enzyme providers and producers
within Europe and their activities in the sectors relevant for this project.
Table 4.2. Number of companies in a country and their range of enzyme products
Country # Food Feed Textiles Pulp and Pharma Others*

paper
Diary Baking Others
Austria 4 x
Belgium 4 x x
Denmark 4 x x x x x
Spain 2 x
Finland 3 x x x x x
France 10 x x x x x x
Germany 13 x x x x x
Italy 2 x x
Netherland 5 x x x x x x
s.
Switzerland 4 x x x x
UK 19 x x x x
Source: Godfrey and West, 1996.

* This category includes industrial enzymes and enzyme products of companies who have not
specified their products or which have a wide range of products.
Novo Nordisk and Genencor are active world-wide in the market for industrial enzymes:
detergents, starch conversion, textiles and pulp and paper. Gist Brocades recently sold a
part of its activities in industrial enzymes to Genencor and now focuses on food and
feed enzymes and ingredients.
It remains obvious that Novo Nordisk is the absolute market leader, especially
concerning its research activities linked to promising new enzyme products. Each year it
introduces approximately ten new enzyme products. This is quite a high figure for an
established market such as the enzyme one, and is equivalent to all the market
introductions of competing companies put together. Novo is based in Europe but has, as
almost all world-wide operating companies, its research facilities based in other regions,
especially in the USA.
IPTS 1998 48
Table 4.3. Total turnover of the most important producers in the enzymes sector 1995
(in US $ millions)
Company Turnover
Novo Nordisk (DK) 580
Genencor (USA) 235
Total Japanese companies (J) 90
Gist Brocades (NL) 65
Quest International (NL) 32
Hansen (DK) 25
Grintsted (DK) 25
Rohm (D) 25
Others 53
5.2.2 The structure of the market

In 1980 the total value of enzyme sales world-wide was estimated at around US $ 300
million. Between 1991 and 1996 the global market for industrial enzymes more than
doubled from US $ 650 million to US $ 1.4 billion (Novo Nordisk, 1997; OTA, 1991).
In 1995, Germany and France were the biggest European markets for industrial
enzymes, resp. 23,0% and 18,3%.
Table 4.4. Total sales in industrial enzyme markets (US and Europe) and estimates for
the year 2000 (in US $ millions)
Area 1985 1990 1995 2000 Total

Detergents 110 170 280 400 960
Food 100 160 240 370 870
Beverages 100 160 200 320 780
Other 60 100 160 320 640
Total 370 590 880 1.410 3.250
Source: Bickerstaff (1995)
Although the enzyme producing industry has less companies and investments than the
pharmaceutical industry, it is recognised as the most profitable area today (Abbott,
1996). Detergents for the home laundry market, proteases and in the future lipases, are
the leading area (Smith, 1996). Within the total market volume of food, drinks and feed
enzymes, carboxyhydrases and enzymes for starch hydrolysis and fructose syrup
production make up for more then 50%. The enzymes for baking (including the bulk
amylases and the number of special enzymes for structure and freshness) are in second
position (14%), followed by brewing and fruit processes (10% and 8% respectively).
The enzymes for wine production and feed/fodder account for approx. 6% and the
cheese enzymes occupy the last position with 1.6%.
Of the some 18 enzymes commercially available in bulk in 1991, five were the most
important. These were amylases, bacterial proteases, papain, glucosidases, rennin and
chymosin.
Data on the actual market size for food enzymes in Europe and the USA is conflicting.
However, several sources suggest the food enzyme markets to be similar in size in the
USA and Europe with total sales of US $ 100 - 200 million for each region in 1995
IPTS 1998 49
(Marrs, 1997; Bickerstaff, 1996; Novo Nordisk, 1997; Godfrey and West, 1996). Table
4.5 shows the estimated world sales of food enzymes in 1990.
Table 4.5. Estimated World Sales Value of Food Enzymes by Product Type, 1990
Enzyme Consumption (US $ millions)

Rennet (animal and microbial) 75
Glucose isomerase 40
Glucoamylase 75
alpha-Amylase 50
Papain 8
Trypsin 8
Other food proteases 8
Invertase 8
Pectinase 7
Others (beta-Glucanase, cellulase, dextranase, glucose oxidase, 20
lipase, pullulanase)
Source: Sahm et al., 1991-1997.
Enzymes are relatively new additives in the feed industry. Before 1985 the use of
enzyme additives was ignorably small. In 1995 it represented US $100 million of the
total $5.8 billion market of feed additives. It is the fastest growing market for industrial
enzymes and will continue to be so in the near future. Other feed markets are expected
to follow the example of the poultry market which is now more or less saturated. It is
expected that the enzyme market for the feed industry will grow at 25% per year
(Godfrey and West, 1996). About 18% of the world production of animal feed takes
place in the EU. Production is concentrated: 30% of the world’s feed mills produce 80%
of the total feed production.
Table 4.6. Total turnover in 1995 and main market suppliers (US $ millions)
Enzyme market Turnover Main Supplier

Detergents 448 Novo Nordisk and Genencor
Starch conversion 138 Novo Nordisk and Genencor
Textiles and pulp and paper 164 Novo Nordisk and Genencor
Dairy 130 Gist Brocades
Bakery 80 All
Animal feed 75 Gist brocades and Novo Nordisk
Brewing and distillery 50 Quest
Fruit juice and wine 25 Gist brocades
Other 40
Future markets
The commercial outlook for industrial enzymes is very good. According to a report of
Frost & Sullivan, a market research company, the European turnover for industrial
enzymes will double from $ 450 million in 1995 to more then $ 900 million in 2003.
However the prices of enzymes dropped between 1993 and 1996 and will continue to do
so in the year 2000. Frost & Sullivan report this is mainly due to modern
biotechnology’s which allow for a more efficient production of enzymes. The detergents
sector was between 1993 and 1996 the fastest growing market for industrial enzymes.
Frost and Sullivan expect this will change: the less traditional applications will grow
faster. Examples are the treatment of waste, paper and pulp followed by chemical and
pharmaceutical processes (NIABA, 1997).
IPTS 1998 50
5.3 Integration of biocatalysts in industry
5.3.1 The pharmaceutical industry

The use of enzymes in the pharmaceutical industry has two primary functions. First, to
separate different chiral forms of drugs and second, to play a major role in the synthesis
of drug precursors. The use of enzymes has grown steadily in the pharmaceutical
industry having been initiated in the above two areas about 20 years ago. Prior to this
the separation of different chiral forms was largely achieved using silica columns which
are a relatively inefficient and lengthy process.
Today, enzymes are used in a minority of drug production processes. Excepting that
about 10% of all drugs are biotechnology-based, a small proportion of the remaining
90% uses enzymes for the separation of chiral forms or the synthesis of drug precursors.
About 10% of these non-recombinant drugs (i.e. 90% of the total number of drugs) are
in the chiral form that requires separation. Thus the importance of enzymes in the
pharmaceutical industry as a whole is limited by this use in only a minority of cases.
Similarly, in the use of enzymes for the synthesis of drug precursors, only about 6-7%
of all drugs are involved in this set of processes. This small group of drugs uses a range
of enzymes such as lipases to synthesise precursors to the final drug product. The
introduction of enzymes has become established in this process because they offer a
more economic production of precursors.
The majority of drugs do not require chiral separation. This may be for two reasons.
Firstly, the efficacy of the drug is not affected by the different stereoisomers, i.e. the L
and D forms. Secondly, the final drug products exist in one form only. This means that
the applications for enzymes in this area are relatively limited and not likely to increase.
In the case of drug precursors, the use of enzymes to synthesis precursors is not likely to
change significantly in the short to medium term. Finally, it should be noted that the
early biotechnology protein-derived drugs such as tissue plasminogen activator or t-PA
and erythropoietin or EPO are produced directly by genes cloned in bacteria or yeast
cells. These products are relatively pure and are not produced in the form of
stereoisomers.
At present the number of biotechnology-based drugs on the market is relatively small

(~10%) but this percentage is likely to increase as there are over 200 such drugs in
clinical trials. However these drugs do not require the use of enzymes either to separate
different chiral forms or to synthesise precursors on a significant level.
5.3.2 The food and drink and the feed industries

The food and drink is an extremely heterogeneous industry and enzymes are used in a
considerable number of its subsectors. The brewing of beer and the baking of bread are
two totally different and separate industries. Not only are there many differences
between subsectors, there are also considerable differences between countries within
each subsector: the production and marketing of beer in Denmark differs in terms of
production technology and consumer preferences markedly from countries like
Germany. These two factors (differing markets, differing products) result in a highly
IPTS 1998 51
diversified market for biocatalysts in the food and drink sector in contrast to more
homogenous markets such as the textile or detergents markets.
It is clear that Europe’s major food and drinks companies have the ability now to apply
and exploit the potential of biotechnology. This is particularly so in the area of
enzymes. They have tended to rely on developing in-house capabilities as opposed to
building complex relationships with the specialised biotechnology sector. Competencies
of Europe’s major food and drink companies in biotechnology generally, have recently
been considered at least equal to that of their US competitors (Ballantine and Thomas,
1997). However it is now apparent that biotechnology in manufacturing is more
advanced in the US than in Europe. On the whole, enzymes produced by biotechnology
in the food and drink industry are used more extensively in the USA and manufacturers
of food ingredients are generally more willing to use biotechnology to improve their
manufacturing processes in the US when compared to Europe.
When we consider the development of the US and European markets for novel foods
and beverages, we can conclude that they are largely at a similar stage. In both regions it
is clear that few such products have yet reached the market. This situation is likely to
change shortly however, as several new genetically engineered crops, with improved
processing characteristics, are being grown in the US and are now being exported to
Europe. At the same time, approvals for genetically engineered crops to be grown in
Europe is also being initiated. These products with improved processing characteristics
will be used by the US food and drink industry very shortly to enhance productivity in
manufacturing processes whereas Europe has yet to grow commercially any transgenic
plants. In general, the way in which European companies are able to exploit the
potential of biotechnology in the food and drink sector, depends largely on consumer
attitudes rather than their technology competencies. We should note that Europe’s small
food and drink companies may face difficulties in the future as product innovation and
improvements in operating efficiency, which have been achieved through the
application of biocatalysis, come to depend more and more on the application of such
technologies.
5.3.3 The textile sector

The textile industry has widely and generally accepted the use of enzymes in its
processes, especially in fairly simple large-scale applications, such as stone washing.
The market share of industrial enzymes in the textile field stood at some US $160
million in 1996 (about 10% of the total market), and is estimated to grow by
approximately 50% before 2005.
In textile industries, technologies based on biocatalysts are already established. The

stone washing of jeans for instance, clearly offers an example of a Best Available
Technology (BAT). The process based on biocatalysts is both economically and
technically competitive and brings environmental benefits. The enzyme markets are,
however, at least to some extent dependent on the yearly changing fashion, and
fluctuations in the market volumes may be caused by this. The consumers can be
expected to have increasingly positive attitudes towards textiles produced by greener
methods.
IPTS 1998 52
5.3.4 The pulp and paper sector

The large scale application of enzymes in the pulp and paper industry has grown fairly
slowly but steadily since 1990. This is mainly due to the decreased prices of enzymes,
but also due to improved commercial preparations which have less negative side-effects
and are more suited to industrial processing conditions. Bleach-boosting using
xylanases and dewatering fibres using cellulases are the most important commercial
applications today. The share of the pulp and paper sector is, however, still fairly small,
estimated to be only around US $ 6 million in 1996.
A major obstacle to applying biocatalysts in the pulp and paper industry seems to be the
fairly slow adoption of new techniques in the sector. It is generally considered that
attitudes in the sector (and in many research organisations of the p&p sector too) are
conservative.
The technical solutions based on biocatalysts must offer clear advantages over existing
ones. For the pulp and paper industry to accept biocatalysts, economic savings must be
possible. Biotechnology can offer competing technologies only in very limited cases.
Thus, the adoption of biocatalysts as BAT cannot be generalised. Instead, biocatalysts
may be beneficial in certain mill-specific cases, e.g. when savings in new investments
can be avoided (as in the case of bleach-boosting enzymes). The introduction of new
biocatalysts in unconventional areas, such as pulp and paper, requires experienced and
careful technical research from the biotech companies to find the optimum conditions
for the use of biocatalysts.
5.4 Bottlenecks
There are several factors of the external business environment which influence
investment in the application of biotechnology (Ballantine and Thomas, 1997). These
include:
• Market conditions: in particular, consumer attitudes, competitive intensity and the

nature of demand. These conditions may encourage or inhibit the use of
biotechnologys in companies and the entrepreneurial investment in specialised
ventures.
• The regulations which cover the use of biotechnology and specific marketing
approvals also influence the scope of markets. The availability of experienced
entrepreneurial managers and high quality staff are also important.
• Entrepreneurship and particularly attitudes to entrepreneurship and risk-taking will
influence development of biotechnology.
• Attitudes influence the level of enthusiasm for setting-up specialised companies and
the willingness of managers within large companies to explore new products and
processes.
• The science base influences the entire innovation system and particularly the
development of the specialised biotechnology sector.
• The availability of appropriate capital for specialised companies is a fundamental
precondition for their growth. At the same time, fiscal policies, in particular tax
regimes on capital gains, share options and tax credits for R&D affect the cost of
investment in research. A basic obstacle is also seen in the capital structure in
IPTS 1998 53
Europe; the availability of risk capital from the stock market differs from that in the
US. Thus, there are too few start-ups with a strong financial basis.
• Finally, supplier factors are important to both specialised companies and also the
pharmaceutical sector which is increasingly outsourcing R&D.
All of these factors identified in the recent EuropaBio study will have some influence
on the successful growth and development of biocatalysis in Europe across the different
sectors. The importance of these external factors to the biotechnology industry as a
whole has been emphasised in several recent studies. Focusing on other areas of
biotechnology and biocatalysis in this respect is no exception.
Europe has a leading position in the global enzymes market. The European companies,
particularly Novo Nordisk, Cultor (through its Genencor venture with Eastman
Chemicals of the USA) and Gist Brocade dominate the market world-wide. However,
European strengths, although impressive, are specific to be challenged in the first two
areas mentioned. The public acceptance of enzymes in food processing and especially
the acceptance of the use of enzymes produced with genetically modified organisms,
constitutes a very serious bottleneck for the diffusion of new technologies in a number
of EU member states. In 1994 Gist Brocades had marketing approval in the Netherlands
and France for their bacterial chymosine produced with rDNA technology. The German
government denied marketing approval due to the lack of public acceptance. Because
Germany is such an important market Gist Brocades did not sell in France and the
Netherlands, fearing a German boycott of their products. In the meantime the product
has been approved but it is still not sold in Germany or the Netherlands.
In the case of pharmaceuticals, enzymes used for the separation of chiral forms of drug
compounds and for the synthesis of precursors are increasingly likely to be produced by
recombinant micro-organisms. However it is unlikely that there will be a significant
problem in consumer attitudes in relation to the use of biocatalysis in healthcare
products. Even in Europe, the general public has shown support for the use of
biotechnology in the context of healthcare which obviously contrasts the attitudes
towards the food and drink sector. The use of enzymes, therefore, in the pharmaceutical
industry is largely related to economic efficiency and need. The market for enzymes
here is largely limited by the need of separative chiral compounds and, as we have seen,
this is absent in the case of biotechnology-derived drugs.
Although EU regulations should lead to uniform legal systems, the legal situation in the
EU member states varies widely. Whether legislation is seen as a hindrance or stimulus
to developments depends on local factors and actors. The German ‘Reinheitsgebot’
(purity laws) for beer prohibits the use of novel ingredients in the brewing process. This
prompted the German beer industry to develop sophisticated brewing technologies
which had a positive impact on developing new products and processes.
At the same time, Europe lacks specialised biotechnology companies which are working
on advanced areas of enzymatic technologies. Overall, the producers of industrial
enzymes have, for some years, consisted of small numbers of large firms and several
smaller, largely local, suppliers. However, strong pricing pressures have recently
IPTS 1998 54
resulted in the consolidation of the industry with two of the larger firms recently
withdrawing from the market. This has added to European strength.
Another bottleneck is the (still) high cost of many enzymes, their low efficacy in certain
cases and their limited applicability. Investment costs in R&D necessary for the
development of new applications of biocatalysts are high. This is not always understood
by the companies using them which may consider the applied development work
carried out by the enzyme producer companies insufficient. In some sectors, the
applying companies participate readily in the development costs of biocatalysts, in
others not. Although public basic research work is often carried out at universities
funded by national governments or at EU level, the additional development work which
needs to be carried out by the companies on production strains, enzyme properties and
applications, including product approval, is substantial.
Literature
Abbott G. (ed.) (1996) Biotechnology Industry Study Report 1996. In: In Touch with
Industry: ICAF Industry Studies, Academic Year 1996. Industrial College of the Armed
Forces National Defence University Washington, DC 20319 - 5062.
Ballantine, B and S.M. Thomas, (1997) Benchmarking the Competitiveness of

Biotechnology in Europe, report prepared for EuropaBio, Brussels.
Bickerstaff, G.F. (1995) REVIEW: Impact of Genetic Technology on Enzyme

Technology., The Genetic Engineer and Biotechnologist, Vol. 15, No. 1, Journals
Oxford Ltd.
Godfrey T. and West S. (1996) Industrial Enzymology 2 edition, Macmillan/Nature,

UK.
Marrs, B. (1997) Chapter 3: The Industrial Sectors., Draft OECD study, 8-6-1997,
Facsimile message.
NIABA (1997). Enzymen vervangen chemische processen. Article on the results of a

study of Frost and Sullivan in the future market of industrial enzymes published in
Biotechnologies Nieuws, a publication of the Dutch Industrial and Agro Biotech
Association NIABA.
Novo Nordisk (1997) Annual Report 96., Denmark
OTA (1991). Biotechnology in a Global Economy, OTA-BA-494, Washington DC, U.S.

Congress, Office of Technology Assessment, U.S. Government Printing Office.
Smith, J., ed. (1996) Conference Proceedings: The Future of Biotechnologies in Europe:
From Research & Development to Industrial Competitiveness, Club de Bruxelles,
contribution for the Conference organised by the Club de Bruxelles on Sept. 26 and 27
1996, Bruxelles, Belgium.
IPTS 1998 55
IPTS 1998 56
6. Future technical developments in biocatalysis
6.1 Introduction
Without doubt there will be an increase in the use of biocatalysis over the next 10 years.
Biocatalysis will be used to add value to food and drink, increase the number of chiral
and chemical intermediates used in the synthesis of pharmaceuticals and biochemicals
and thirdly, biocatalysis will be used to replace processes that require high energy
inputs and are environmentally challenging.
Two technical factors will be responsible for such progress. Firstly, the use of site-
directed mutagenesis to modify the activity and/or substrate specificity of enzymes that
are in current use in the biocatalysis industry. Secondly, the discovery of new enzymes
as a result of genomic sequencing projects currently underway and proposed. A further
driver in the move to biocatalysis will be the importance of apparently ’natural’ products
that are at the moment produced by very ’unnatural’ processes. For example, the
production of margarine from oils requires high temperatures and inorganic catalysts
whereas it is potentially possible to synthesise margarine’s from oils using enzymes to
have the same effect. As is noted in this report, biocatalysis comes into its own when
commodity chemicals or intermediates are considered and the cost of production is of
crucial importance (e.g. penicillin intermediates).
In this chapter we present the future technical developments in the field of biocatalysis.
The character of these developments is very diverse and shows the innovativeness of
this field of research. We can observe that the traditional innovation trajectory is
followed in order to develop improved enzymes, which already are used on a broad
scale. But also more intelligent ways are found to mimic the most essential mechanisms
of biocatalysis or find totally environments for biocatalysts to work in (in vivo and in
vitro biocatalysis).
6.2 Modern enzymes and other new types of biocatalysts
Modern methods of creating new biocatalytic processes are to modify existing enzymes,
ribozymes or antibodies. Use of enzymes has been discussed widely in this report.
However ribozymes are derived from the observation that the catalytic activity of
several enzymes is dependent on an RNA molecule. As RNA molecules can be
synthesised and selected for on a vast scale (selection of one molecule in 100 million to
perform a particular reaction is not uncommon) then such molecules have a potential to
catalyse a large number of reactions although none are yet in industrial use. Antibodies
offer another way of performing novel catalytic reactions in that antibodies can be
raised against transition intermediates. Antibodies stabilize the products of a
biochemical reaction which avoids a backward reaction. As antibodies can be made
against a wide range of molecules then it is theoretically possible to produce
biocatalytic molecules to run processes that cannot be done naturally.
IPTS 1998 57
There are still a vast number of organisms that have not been exploited or screened for
their ability to catalyse specific reactions and this, together with their ever increasing
size of genomic databases, stressed that there is a huge potential for novel biocatalytic
enzymes. The limitation in exploiting these two areas is the lack of high throughput
screens commonly found in the biopharmaceutical industry but less so in the area of
biocatalysis.
Extremozymes
Further expansion is expected from the use of extremozymes, or enzymes which stem
from micro-organisms capable of surviving in extreme environments: at pH’s,
temperatures, pressures, ionoc and solvent environments long thought to be destructive
to biomolecules (Adams et. al., 1995). One specific area is the development of
thermophiles. Obviously of great interest, a new thermostable glucosedehydrogenase
(GDH) has been isolated from a soil bacteria near a hot spring and has optimal activity
of 75° C. There is some evidence, particularly from the Russian Academy of Sciences,
that suggests that marine invertebrates may be a source of unique enzymes with some
novel properties.
Engineered enzymes
Genetic engineering of enzymes holds considerable promise and in particular the
recognition of proteins can often be divided into discrete domains and creates the
possibility that swapping these domains will create novel catalysts which change
substrate or reaction characteristics. Indeed it would be possible to make catalysts that
involve composites between naturally occurring amino acids (in the form of proteins)
and other organic reagents may be introduced by synthetic or recombinant methods. The
semi-synthetic enzymes can be made by chemical modification with fatty acids or
polyethyleneglycol. One problem with the use of biocatalytic enzymes is the need for
co-factors. However it has now been shown that enzymes can be re-engineered so as to
utilise a different form of co-factor. For example, NADP rather than NAD. Subtilisin, a
protease from bacillus can now be engineered so that it can cut and splice specific
proteins. Such activities will become extremely important and valuable in the area of
biocatalysis.
Ribozymes
The use of synthetic RNA as a factor for gene inactivation is now well demonstrated.
Several US biotechnology companies are in a position to exploit this commercial
technology and clinical trials have been initiated involving gene therapy for AIDS.
IPTS 1998 58
Microbial catalysts
More recent advances are related to carrying out a series of sequential organic chemical
reactions using a single microbial catalyst (Marshall and Woodley, 1996). This is
achieved by fluctuating carbon sources for the microbial catalyst, triggering a complete
or partial metabolic pathway to catalyse a sequential series of organic reactions. The use
of microbial catalysts for mediating multiple organic reactions as part of a longer
chemical synthesis may prove completely new routes to chemical products.
Changing chirality
One of the main uses of enzymes in biocatalysis is to produce a single chiral from a
racemic mixture. What would be of considerable use would be to select one form or
another from a mixture. This has recently been achieved and is likely to be of major
consequence, not only for secondary alcohol dehydrogenases but for other enzymes as
well. The stereochemistry is temperature dependent, one form being favoured over
another depending on the temperature. Therefore use of enzymes with increased
stability may well result in the ability to purify different chiral forms from the same
mixture. Developments will occur particularly in the production of generic compounds
whereby enzymes will be used in various solvents at various temperatures in order to
increase the yield of the desired product. From an intellectual property point of view it
remains to be seen whether or not such processes and applications are patentable or will
be the subject of trade secrets (Benkovic and Ballesteros, 1997; May, 1997).
6.3 Modification of plant components
One aspect of the genetic manipulation of plants and enzymology is that in the future
biocatalysis will be seen to move freely between the use of purified enzymes in the
pharmaceutical industry through fermentation using either natural or modified bacteria
or yeasts into plants which have been genetically modified to produce either a particular
biocatalytic enzyme. For example, genetically engineered feed crops containing phytase
or plants that have been modified by the production of enzymes so that secondary
metabolites can be either produced in greater amounts or in altered properties. This
form of ’molecular farming’ is well on the way to fruition.
What may be of particular importance is the ability to produce artificial membranes in

transgenic plants. This allows the possibility that membrane proteins can be
overexpressed and furthermore the products of such reactions could be stored in
independent compartments of the plant cell. This can allow the production of otherwise
toxic compounds and furthermore may allow novel plant products to be processed in
conventional ways through oil mills etc. Several laboratories are pioneering the concept
of engineering value added components into crop. These include increasing the amount
of sulphur rich amino acids in transgenic alfalfa and subterranean clover with the
intention of improving wool growth in grazing sheep.
Another aspect is to incorporate enzymes so as to make the endosperm cell wall of
barley easier to degrade during malting. This process involves incorporating a gene for a
thermostable glucanase.
Encouraging progress has also been achieved in the production of biodegradable
plastics in plants. The product quality and yield could be increased continuously. The
IPTS 1998 59
still existing obstacle to market entry is the non-competitive price in comparison to oil-
based plastics.
In vivo bio-production
Biocatalysis can lead to more efficient ways of producing raw material for production
processes. Similar to this, it is the goal of some companies to use plants as an in vivo
bio-factory for the production of specific molecules (eicosapentaenoic acid,
docosahexaenoic acid and linolenic acid). It is thought that these compounds may be
useful in the prevention and treatment of heart disease, asthma, arthritis and possibly
some cancers. The synthetic pathways of these polyunsaturated fatty acids (PUFAs)
have been identified in certain marine unicellular algae and some fungi. At present the
largest source is fish oil but this is relatively expensive and the product is subject to
oxidation. It is therefore tempting to produce PUFAs in edible plant oils as an attractive
(green, environmentally sensitive) and cost effective alternative. In vivo biocatalysis
will allow the production of custom made oils with different long chain fatty acids.
In vitro bio-production
Additional to the in vivo bio-production described above cellulose has now been
synthesised in vitro using the enzyme cellulase. In vitro synthesis of cellulose will allow
variants of the ‘plant material’ to be produced. One area where in vitro bio-production
is likely to be of importance is in the synthesis of polymers. This can be accomplished
by the use of stereoselective lipase catalysed polymerisation reactions. Remarkably the
lipase enzyme works well in toluene, the organic solvent. What is different from the
chemical synthesis of these polymers is that they are synthesised as one stereoisomer. It
is likely that such polymers will have interesting, particularly biological, properties.
Lipase can also be used to synthesise polyester, in this case in ether. Yields range from
6-8%. Recently horseradish peroxidase has been used to synthesise a fluorescent
polymer of 2-napthol. Perhaps one of the most exciting applications of future
biocatalysis in the production of polymers is the synthesis of mosaic nucleic acids
composed of 50% DNA and 50% RNA. These can be produced in milligram quantities
and have obvious applications as pharmaceuticals and genotherapeutic agents. Direct
evolution is a real possibility of the development of biocatalysis whereby enzymes are
subject to random mutagenesis and screening.
Although these results are impressive, they arise from a tremendous amount of hard
work and in future experiments are going to rely heavily upon genetics and
biochemistry.
IPTS 1998 60
Literature
Adams, M.W.W. et. al. (1995) Extromozymes: Expanding the Limits of Biocatalysis,
BIO/TECHNOLOGY Vol. 13, July, pp. 662-668.
Benkovic, S.J. and A. Ballesteros (1997) Biocatalysts the next generation, TIBTECH
October 1997, Vol. 15, pp. 385-386.
Marshall, C.T. and J.M. Woodley (1996) Process Synthesis for Multiple-Step Microbial
Conversions, BIO/TECHNOLOGY Vol. 13, October, pp. 1072-1078.
May, S.W. (1997) New applications of biocatalysis, Current Opinion in Biotechnology

1997, 8, pp. 181 - 186.
IPTS 1998 61
7. Summary, conclusions and recommendations
Economic and ecological benefits of biocatalysts: input or outcome
The use of biocatalysts is based on their superiority in carrying out the desired
reactions, due to their specificity, economical advantages or improved environmental
impacts. Depending on the field of application, these reasons may vary. Thus, in the
field of food processing, biocatalysts have a history, thousands of years old, based on
first hand empirical findings. Today, these methods represent clearly the best available
technologies.
In general, environmental savings by enzymes can be reached in three ways:

• Enzymes work best at mild temperatures and in mild conditions. They can be used to
replace harsh conditions and harsh chemicals, thus saving energy and preventing
pollution. They are also highly specific, which means fewer unwanted side_effects
and by-products in the production process.
• Enzymes can also be used to treat waste consisting of biological material.
• Finally, enzymes themselves are biodegradable, so they are readily absorbed back
into nature.
If we observe the fine chemicals/pharmaceutical industries, the target reactions (such as

many synthetic reactions) can often be carried out most easily by biocatalysts. Targeted
medicines are often based on knowledge about enzymatic reactions. Environmental
savings in the food and drinks sector will be related to minor process changes, and
consequently will be small in effect. However, there are interesting future
implementations to be expected, for instance in the oil industry for the extraction of
vegetable oil from seeds by enzymes in water, replacing the traditional technology using
hexane (toxic and explosive). However, in both these industries, the minimisation of
environmental impacts is not the primary target, and has usually no role. The
environmental savings in the food and drinks and the feed sector is and will stay very
low as compared to the potential savings in the detergent and pulp and paper industry.
The use of biocatalysts leads, especially in the paper-and-pulp and the textile sector, to
important environmental savings.
The most important driving force for applying biocatalysts are economic benefits, in
some cases in a win-win situation with environmental savings. However environmental
benefits from the perspective of companies are, almost by definition, only attractive if
they combine with low costs. Costs and higher added value are the main driving forces
in companies to use biocatalysts. For industry, greening is no driving force for using
biocatalysts; greening is an output.
IPTS 1998 62
Europe’s scientific-technological potential in biocatalysis
From a scientific and technological perspective, biocatalysis is an interdisciplinary field

of research including basic and applied research. A considerable percentage of
biocatalysis research is being performed in industry and difficult to trace. This makes it
very hard to make a clear cut assessment of the quantity and quality of its potential. The
general perception, based on interviews with companies in Europe, is that the quality of
public sector research in biotechnology, including biocatalysis research, in Europe is as
good as in the USA. An indication of the strength of biocatalysis research in Europe
compared with other regions on the basis of the productivity of papers reveals that
Europe as a whole (42%) is somewhat stronger then the USA (30%) or Japan (5%).
However, the perception is also that the total quantitative science base in Europe is
weaker than in the USA.
Patent database analysis shows that in general European patents in all sectors is lagging
behind if compared with the US and Japan. However the analysis is no basis for
conclusions on Europe’s scientific potential because patenting strategies and patenting
costs differ between these regions and what is also important to note: the keyword
biocatalysis is too general.
Economic potential of Europe’s biocatalysis industry
The economic potential of Europe’s enzyme producing industry is rather strong. Novo
Nordisk, a Danish company, is clearly world-wide the biggest, with about 50 % of the
total market and is for all applications the number 1 supplier. Novo is followed by
Genencor - half owned by the Cultor Company in Finland and half by Eastman Kodak
in the USA. Genencor has less then 20% of the total market and ‘only’ produces
technical (non food grade) enzymes. Gist Brocades, based in the Netherlands is the third
followed by a number of other European companies.
Europe has a major presence in the production of enzymes for many different processes.
However, it is often the case, with one or two exceptions, that these companies are very
small and only produce one or two enzyme products.
Despite the size of the enzyme market (more than ECU 500 million world-wide), the
industry itself is not very profitable. This is primarily because enzymes make up such a
small component of a product (e.g. enzymes in washing powder) and enzymes are often
produced by more than one manufacturer - patenting of enzymes has historically not
been a major issue - , hence the competition is intense.
It is likely that in the future there will be considerable consolidation of the enzyme
companies in an attempt to achieve greater profitability. Food manufacturers, feed
suppliers and the generic pharmaceutical industry will attempt to vertically integrate
their production processes. This will result in the acquisition of a number of
independent producers. The lack of profitability and relatively small sales of
independent enzyme producers is largely due to the nature of catalytic reactions
themselves, for example, the amount of enzyme added in a typical biocatalytic
conversion is of the order of 0.5 kg/10 tonnes of material to be converted. As each
IPTS 1998 63
enzyme molecule is capable of cycling with a turnover approaching 10,000 molecules

per second, then it is easy to see why such small amounts are required. In the case of
immobilised enzymes (which is the way the industry is moving) enzyme input is not a
significant cost in the production process. It is expected that an annual growth rate of
10% within the enzyme business is realistic within the next 3-5 years.
Fine chemical/ pharmaceutical industry

In the fine chemical/ pharmaceutical industry the impact of biocatalysis is explicitly
used in order to replace traditional, stoichiometric processes in order to improve the
product/waste ratio. Another argument is the low entry barrier, i.e. low investments, for
new technologies in this small scale industry. About 10% of all drugs are
biotechnology-based and a small proportion of the remaining 90% uses enzymes for the
separation of chiral forms or the synthesis of drug precursors. Similarly, when using
enzymes for the synthesis of drug precursors, only about 6-7% of all drugs are involved
in this set of processes. This small group of drugs use a range of enzymes such as
lipases to synthesise precursors to the final drug product. The introduction of enzymes
has become established in this process because they offer a more economic production
of precursors.
Food and drinks and animal feed industries

In the food and drink industry the use of enzymes is fully integrated. However in the
exploitation of the results of new biotechnique fruits which are mostly genetically
engineered enzymes in this sector depend largely on consumers attitude rather than on
technological competences. The animal feed market is the fastest growing market for
enzymes, and the expectations are high for the future.
Textiles and pulp-and-paper industries

In the textiles industry the production of fibres from less valuable raw materials, i.e.
upgrading the quality of fibres, is an area of increased interest to biocatalysts.
Bioscouring, denim and garment washing, biological dyeing and bleaching, enzymatic
fibre modification and biofinishing of cellulosics are considered the most promising
new applications.
The expected growth rate of biocatalysts in the pulp-and-paper is highly dependent on

break-throughs within the field. According to inquiries made, the growth may be fast
(when expecting successful R&D and commercialisation) multiplying the present
market figures by several decades within 10 years - or not. Especially in the past, there
have been expectations of rapid growth, but at present, the attitudes are fairly realistic.
It is a general observation that the applying companies are not aware of the R&D costs
for developing new enzymes. However, the biocatalysts developed by the academia or
enzyme companies must offer clear technical or economic improvements. The most
relevant application areas are still bleaching, modification of fibres for e.g. improving
the deinking process and applications of new oxidative enzymes.
Bottlenecks
The potential consumer resistance to the use of enzymes that are essentially produced
by biotechnology in food, drink and animal feeds may have the effect of limiting the
market growth. The public acceptance of using enzymes produced with genetically
IPTS 1998 64
modified organisms, constitutes a very serious bottleneck for the diffusion of new
technologies in a number of EU member states.
The regulations which cover the use of biotechnology and specific marketing approvals
also influence the scope of markets. The availability of experienced entrepreneurial
managers and high quality staff are also important.
Barriers to Biocatalysis
Notwithstanding the high promises of the use of biocatalysts in industry for

environmentally savings, the factual implementation is still rather limited. One very
important barrier are the sunk investments in existing processes. This is a general
problem which is met by policymakers when they try to introduce clean technologies in
mature industries. In these industries far reaching optimisation of existing processes is
the main cost-saving driving force for innovation.
Another barrier are the other competing technologies. However, does biocatalysis
already have such a fixed position in the setting of competing technologies that a fair
competition can be made? In this respect it can be observed that not only sunk
investments, but sunk experiences and cultures can make it more difficult to integrate
new ‘scientific cultures’, like the biological sciences, into the existing culture in
industry4. It must be concluded that the integration of modern biotechnologies not only
meets difficulties by the public, but for other reasons, it also seems a not very
approachable and attractive issue to policymakers in companies and government5.
Nevertheless there are companies where barriers between chemical and biological
cultures are broken down and biotechnologies are part of the research tool kit6.
Although the conclusion might be correct that biocatalysis has until now not been in a
very fair and favourable position to compete with other process integrated technologies,
a plea for an increase of the use of biocatalysts in industry as such because “Bio is
green, Bio should, because Bio is good” is not correct. However it is quite clear that
biocatalysis has a number of advantages for environmental savings and that biocatalysis
should be in an equal position to other technologies in the race for the most competitive
technology. As we concluded, this position has not been reached yet and this legitimates
organisations including government bodies, who give a high priority to environmental
issues, to stimulate the use of these technologies.
4
C.M. Enzing, (1993), Midterm evaluation of the Innovation Research Programme Katalyse, report for
the Dutch Ministry of Economic Affairs, TNO-STB, Apeldoorn.
5
As Floris Maljers, a former CEO of Unilever, put forward in an interview in a Dutch newspaper:
biotechnology is, compared to information technology, not a sexy issue that is talked about by man in
bars. Biotechnology has a soft image and it has no such gadgets as mobile telephones, powerfull
computers, etc. Also in the Advisory Board of this project, it was acknowledged by one of the members
from industry that knowledge and perception of a technology play a major role in the uptake of new
technologies.
6
The breaking down has been done by non-traditional persons, as for instance in DSM where the self-
made man and excentric personality of Wilie Boesten has done some very extremenly innovative
research on the borderline of biocatalysis and organic-chemical synthesis (Chemisch Weekblad, 1 Maart
1997). ‘Better be bio’ is the slogan for DSM, a Dutch chemical company that already practiced
biocatalysis, but now also starts to work on fermentation (Chemisch Weekblad, 11 Oktober 1997).
IPTS 1998 65
Recommendations
In order to develop this stimulus, one should bear in mind that the decisions taken in
industries as to which technologies should be used are based on the knowledge of
existing technologies and on economic arguments.
This leads to the recommendations that stimulation instruments should:

1. Make knowledge of biocatalysis and the benefits more available to the users, i.e. the
European industries. One way of doing this is by setting up an easily accessible
database with the economic and environmental benefits of biocatalysts in specific
applications.
2. Stimulate innovative entrepreneurs inside and outside companies to build up
practices with these new process integrated biotechnologies and use these as
illustration projects for other companies.
3. Stimulate the development and use of methods for the evaluation of the
environmental costs/benefits of competing (bio)technologies from an economical
perspective.
4. In those cases were the cost/benefits evaluation shows that from an environmental
perspective a specific technology, i.e. biocatalysis, is more favourable than other
technologies, regulation can be tuned into direct the economic driving forces to
green production processes.
Regulations promoting the industrial application of biocatalysts must be clearly based

on the acknowledged facts on the properties of biocatalysts and their customary use.
Regulations should however, not unfavourably prohibit or hamper the use of
biocatalysts. At present this trend may be foreseen, especially in the food and feed
sectors.
One last recommendation is based on the fact that the enzyme producing companies
consider their participation difficult in public, e.g. EU projects due to confidentiality
aspects, and due to participation of their competitors in the projects. This is especially
problematic in applied areas as in most biocatalysis research.
However, it is seen desirable to support more applied oriented work within the sector. It
is generally considered that the applying industry should be more efficiently informed
about the projects, and consequently be more involved in the development phase. It is
also considered of utmost importance to promote further the co-operation of industry
and research institutes. Within research programmes, however, the fundamental and
applied research should be more clearly separated.

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State of Art in Europe

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INSTITUTE FOR PROSPECTIVE TECHNOLOGICAL STUDIES

1.1.2 Project Co-ordinator

1.1.3 Contributors from the ESTO network

Process integrated biotechnologies, especially the use of biocatalysts, are expected to

2. TYPES AND FUNCTIONS OF BIOCATALYSTS...................................................... 8

3. EUROPES SCIENTIFIC-TECHNICAL COMPETENCE IN

4. AN OVERVIEW OF THE ECONOMIC POTENTIAL OF

5. FUTURE TECHNICAL DEVELOPMENTS IN BIOCATALYSIS ......................... 56

5.1 INTRODUCTION ............................................................................................................... 56

6. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS................................ 61

2.1 Background of the project: Greening of Industry

2.2 Content of the project: Biocatalysis in Europe

This project focuses on a specific part of biotechnology: biocatalysis. Biocatalysts must

Structure of the report

2.3 Project team

The participants and their part in the project are:

• chapter manager of chapter 2.

3. Types and functions of biocatalysts

This chapter describes the basic methodologies of biocatalysts, including enzyme

3.2.1 Whole cell bioconversions

3.2.2 Enzyme technology

Industry Type of enzyme Benefits

Food Amylases Hydrolysis of starch

Feed Hemicellulases Digestibility of feed

Pharmaceutical Penicillin acylases Production of penicillin derivatives

Textile Amylases Starch removal; desizing

Pulp and paper Hemicellulases Improved bleachability

Source: Godfrey and West, 1996

New application areas are, in healthcare, diagnostics, synthesis and purification of

3.2.3 Industrial application of enzymes: bioprocessing

Continuous operation of biocatalysts can be achieved by immobilisation. Both enzymes

3.2.4 Methods to improve biocatalysts

The development of an economically competitive product, which is optimal with respect

Box 1 Modern biotechnologies for the improvement of biocatalysts

Protein engineering: understanding of the protein function-structure relationship is based on the

3.3 Benefits of biocatalysts in industry

There is a abundance of existing knowledge on actual and potential use of biological

3.3.1 Pharmaceuticals and Fine Chemicals

Table 2.2. Share of new chemical entitles (NCEs, %)

Source: EFPIA, 1995.

It is much more difficult to measure the impact of biotechnology on production

refinements in production processes are sometimes protected by trade secrets rather

Biotransformation adds value in two different ways. In the production of generic

The production of chiral compounds (chemicals where a single stereoisomer of a

In box 2 examples of using biocatalysts for production of pharmaceuticals are shown.

Box 2 Examples of using biocatalysts for the production of pharmaceuticals.

Ibuprofen Stereoisomers can be resolved by selectively hydrolysing one isomer with an

L-methionine To resolve chiral mixtures of methionine, the enzyme aminoacylase from

Phenylglycine and Dp-hydroxyphenylglycine These chiral D-amino acids are produced in

3.3.2 Food and Drinks and Animal Feed

Considering the use of enzymes in food processing and as premixes supplemented to

• enhancing product characteristics (flavour, colour, debiting),

Use of enzymes in the food and feed industries1

Other enzymes used for cheese production are:

Olive and other edible oils

Meat and fish

3.3.3 Textile industry

Cellulase treatment of cellulose materials such as cotton, viscose, lyocell, cupro or

Textile After-Care (detergents)

3.3.4 Pulp and paper industry

Bleaching of chemical pulps

Normal ClO2 consumption 65 kg act. Cl/t

Acidification 0 - 0.2 USD/t