Biocatalysis: State of the Art in Europe

INSTITUTE FOR PROSPECTIVE TECHNOLOGICAL STUDIESSEVILLEW.T.C., Isla de la Cartuja, s/n,E-41092 Sevilla

IPTS ProjectModern Biotechnology and the Greening of Industry

BIOCATALYSIS:STATE OF THE ART IN EUROPE

Economic and environmental benefitsof a process integrated technology

IPTS, 1998

1.1.1 EditorsSørup, P.(IPTS), Tils, C. (IPTS), Wolf, O. (IPTS)

1.1.2 Project Co-ordinatorEnzing, C. (TNO)

1.1.3 Contributors from the ESTO networkVan 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 COMMISSIONJOINTRESEARCHCENTRE

Biocatalysis: State of the Art in Europe

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Executive Introduction

Process integrated biotechnologies, especially the use of biocatalysts, are expected tohave an important contribution to cleaner production processes in a number ofindustries. However, there are only a few - nowadays very well known - examples ofbiocatalysts in industry; wider implementation seems to meet high barriers.

In order to gain further insight regarding specific factors that hinder or favour theimplementation of process integrated biotechnologies for environmental purposes, IPTShas set up the project “Modern Biotechnology and the Greening of Industry”. Theoverall goal of this framework project, is to generate information for policymakers inthe field of environment, biotechnology and R&D-policy.

In this report the results of one of the studies in this framework project are presented. Inthe study an overview is made of the state of the art of biocatalysis in Europe. Threespecific aspects are addressed: economic and ecological benefits of biocatalysts in fourindustrial sectors, Europe’s scientific and technical potential in biocatalysis, includingpatents, and Europe’s economic potential of biocatalysis. Additionally, an inventory ismade of the future technical developments in biocatalysts.

It is concluded that Europe has a strong basis in biocatalysis and that environmentalbenefits of biocatalysis in industry are in general an outcome, not an input. Methods forthe assessment of economic and environmental cost/benefits of (bio)technologies shouldbe developed, in order to take better informed decisions on process integratedtechnologies 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 INBIOCATALYSIS ........................................................................................................... 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 OFEUROPE’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 toaddress is the greening of industries and more specifically the role of new processintegrated technologies in decreasing environmental pressures. In general, it isrecognised that specific biotechnologies have the potential to be used as integrated cleantechnologies. However, the fact that the introduction of new promising biotechnics,especially biocatalysis, has taken place is somewhat limited. For that reason IPTS, inthe framework of the project Modern Biotechnology and the Greening of Industry,commissioned a number of feasibility studies in order to gain insight in specific issuesrelated to biocatalysis research and its industrial implementation (dynamics ofinnovation, integral conceptual framework, identification of ‘hot spots’ in R&D). Thesestudies were the basis for the analysis and identification of influencing factors thatstimulate or hinder the introduction of biocatalysis as process integrated technology.

In the framework of the contract of IPTS with the ESTO-consortium (the EuropeanScience 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 proposedwhich had as its main goal to make a state of the art of biocatalysis in Europe andcompare it with the USA and Japan.

This rather ambitious project proposal was discussed in the IPTS Advisory BoardModern Biotechnology and the Greening of Industry and with the members of theInterservice Group of the EU in June 1997. The recommendations brought forward inboth discussions, finally resulted in the project proposal ‘Biocatalysis in Europe’. Theresults 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 thequestion 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 mustbe considered as a process integrated biotechnology, as most environmentally soundbiotechnologies are add-on i.e. bioremediation technologies. The main goal of thisproject, as it finally materialised, is to give an overview of the state of the art ofEuropean biocatalysis. On the basis of this overview state further steps can be takentowards initiatives which stimulate the introduction of green process integratedtechnologies, including biotechnology.

The scope of the project is limited to four sectors: the fine chemicals/ pharmaceuticalsindustry, 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 andthe 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 ofthe 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 andinterviews with industry.

Structure of the reportThe structure of this report follows the above mentioned division. Chapters 2, 3 and 4deal with the four aspects mentioned. In addition, we also made an overview of thefuture technological developments in biocatalysis (chapter 5) and finally in chapter 6conclusions are drawn and recommendations for further steps, including EU policy, arepresented.

2.3 Project team

The project was performed by an international ESTO-project team with research groupsfrom 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 wereinput for chapters 2, 3, 4 and 5, which were collected and processed by the chaptermanagers. The fourth group worked on the patent research. The results are included inchapter 3.The preliminary results were presented to IPTS and discussed in the project team. Onthe 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 Netherlandsproject 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 Dalenand ir. Bram de Hoop of TNO-STB. Prof. Dr. Liisa Viikari of VTT Biotechnology and Food Research, Finland, responsiblefor:• 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 BiologicalSciences, 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 arepresented 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 Projectdescription 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 organicmolecules required for life. This diversity is made possible solely due to the widecatalytic scope of enzymes. Enzymes have been used for thousands of years, withoutscientific knowledge, to preserve food. Many of these old processes have survived, andbeen put into efficient technical frames, such as winemaking, beer brewing and theproduction of milk products. However, enzymes can be used for a great number of otherproducts too.

The recent advances in the field of biocatalysts has enabled the biological processes tocompete successfully with conventional chemical processing. Combination of chemicaland biocatalytic systems are being developed thereby utilising the most attractivefeatures of biocatalysts i.e. enzymes, namely high specificity with less side or wasteproducts and higher yields, mild reaction conditions and usually low environmentalimpacts.

Various industrial fields thus already use biotechnical methods in their processes. Inmany cases, a bioprocess is the most straightforward and economical way to producethese products. However, in all biological processes, the synthesis of the desiredproduct is catalysed by enzymes. There are also examples where biocatalysts maycompete with traditional chemical technologies. For instance organic chemicals, such asacids, alcohol’s or acetone are still produced by chemical synthetic routes. Dependingon the raw material’s availability and cost, biological production processes havereplaced the chemical routes.

This chapter describes the basic methodologies of biocatalysts, including enzymetechnology and production methods using living microbial cells, as well as systems toproduce and improve biocatalysts (2.2). Well established examples of biocatalystsapplied 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 andbioremediation are widely used for the degradation of various waste compounds, butthey are not dealt with in this report.Finally in paragraph 2.4 the central question on the most important driving forces forusing biocatalysts in these industries is addressed. Conclusions are drawn on theeconomic and ecological benefits of biocatalysts and whether ecological benefits are aninput 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 frommicrobes, 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 cellbioconversions, living organisms are used to perform the desired reactions, whereas inenzyme technology, isolated or crude enzyme preparations are used as catalysts.

3.2.1 Whole cell bioconversionsBasically 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 woodymaterials).

The technique of growing microbial cells and their biosynthetic reactions are widelyused 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, yeastand fungi, are being used in industrial processes. The use of cultured animal or plantcells differs clearly from microbial cells being generally expensive compared tomicrobes. Most of the development work in this area relates to high value - low volumeproducts 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 growingmicro-organism (first and second type) and bioconversion with resting i.e. dead wholemicrobial cells for even one-enzyme-bioconversions (third type), especially if theenzyme needs a cofactor. An example of the latter is the production of acrylamide byresting cells of Rhodococcus rhodochrous. In this process acrylonitrile is used as asubstrate producing about 20,000 tonnes acrylamide per annum. This is done in a batchprocess with a high substrate concentration and conversion rate. Vitamin C is alsoproduced by whole cell transformation by Acetobacter suboxydans. Finally, a full rangeof steroids are produced by several whole cell transformations including progesteroneand predenisolone.


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3.2.2 Enzyme technologyThe first enzyme produced industrially was the fungal amylase, employed as apharmaceutical agent in the US as early as 1894. The first patent on using enzymes inlaundry detergents was announced in 1915. The utilisation of amylases andamyloglucosidases for the production of glucose from starch was the second major areafor industrial enzymes. Currently, more than 2000 different types of enzymes have beenidentified. They are classified to six major classes (IUPAC). Several hundreds ofenzymes are commercially available as speciality reagents or bulk products.

The industrial bulk enzymes consist mostly of fairly simple enzymes, carrying outmainly hydrolytic reactions, i.e. degrading different natural polymers. These enzymes,such as proteases, lipases, amylases and cellulases, are used on very different industrialareas. The major applications of these enzymes are in food industry and detergentmanufacture. Bulk enzymes are sold as liquid or dried products. Table 2.1 summarisesthe most commonly used commercial bulk enzymes in five industrial areas. In additionto these, several minor applications exist.

Table 2.1. Commercial bulk enzymes for different industrial fields

Industry Type of enzyme Benefits

Food AmylasesProteasesPettinessLipasesGlucose isomerases

Hydrolysis of starchProcessing of cheese and meatClarification of juicesModification of fatsProduction of fructose

Feed HemicellulasesCellulasesPhytases

Digestibility of feedIncreased nutritional valueImproved phosphate uptake

Pharmaceutical Penicillin acylasesLipases, Proteases,Aminoacylase

Production of penicillin derivativesProduction of optically pure compounds (chiralresolution)

Textile AmylasesCellulasesPectinasesProteasesLaccasesCatalases

Starch removal; desizingDenim stone washing, depillingTreatment of flax and other fibresDegumming of silk, detergentsDenim bleachingRemoval of residual hydrogen peroxide

Pulp and paper HemicellulasesCellulasesLipasesCellulases/HemicellulaseLaccase

Improved bleachabilityPaper manufactureRemoval of pitch componentsDeinking of recycled fibresBleaching, fibre treatments

Source: Godfrey and West, 1996

New production technologies have decreased the prices of enzymes and thedevelopment 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 oforganic compounds and the production of commodity chemicals. Speciality enzymes areindustrially 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: bioprocessingConventional biocatalysis is performed by enzymes in aqueous solutions. Two types ofapplications relevant for this project are: enzymes in non-aqueous solutions andimmobilisation of enzymes for continuous conversion processes.

Water is a poor solvent for nearly all applications in chemical industry. Most organiccompounds of commercial interest are very sparingly soluble and are often unstable inaqueous solutions. Over the past decade the application of enzymes in organic mediahas become an alternative to chemical synthesis and analytical applications. There arenumerous potential advantages in employing enzymes in organic as opposed to aqueousmedia. In aqueous solutions, enzymes such as lipases, esterases, proteases andcarbohydrases catalyse hydrolytic reactions. In organic media, however, these enzymescatalyse a variety of synthetic reactions in high yields including esterification,transesterification, interesterification, lactonization, thiotransesterification andaminolysis. All these reactions are possible only in the presence of low-water activities.Various enzymes in non-aqueous media can catalyse reactions formerly limited toexpensive and tedious chemical catalysts.

Continuous operation of biocatalysts can be achieved by immobilisation. Both enzymesand micro-organisms can be immobilised. Immobilisation has several economic andtechnical advantages: e.g. reduction of enzyme’s costs, more efficient reactorperformances and less downstream processing. Furthermore, products are easilyseparated from the biocatalysts, and in some cases the enzyme properties are alteredfavourably by immobilisation. Most enzymes, when immobilised, show a higherstability than the soluble forms. Advanced examples of industrial processes based onimmobilised biocatalysts include isomerization of glucose to fructose, production ofvarious amino acids, and hydrolysis of penicillin’s to 6-aminopenicillanic acid. Morecomplex reactions involving coenzymes have not yet been used to a great extent at anindustrial scale. This technology is in principle applicable to all soluble substrates. Solidsubstrates, however, have to be treated in batch or semicontinuous processes.

3.2.4 Methods to improve biocatalystsEnzymes of commercial interest may be produced by cultivating microbes (bacteria,yeast or filamentous fungi), plant or tissue cells in well controlled conditions in aprocess especially developed for the production of a certain enzyme. Some industrialenzymes are still being extracted from plants (e.g. bromelain, a protease frompineapple) or from tissues (e.g. lysozyme, a carbohydrate hydrolysing enzyme from eggwhite). 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 bacterialgenus Bacillus or species of the filamentous fungal genera Aspergillus andTrichoderma. 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. extracellularenzymes) and the possibility to cultivate these organisms on cheap media. In additionextracellular enzymes are generally very stable, even under more “unnatural”conditions.It is obvious that relatively few species will be used as producers, since the mostefficient enzyme producers will be genetically modified for that purpose. Especially forapplications in the food industry, so called ‘food-grade’ enzymes are produced only byorganisms with the GRAS (Generally Recognised As Safe) approval. The list ofaccepted producers is published by the organisation of enzyme producers AMFEP(Association of Microbial Food Enzyme Producers). The costs for the official approvalof a new organism as a producer of an enzyme for such fields as medical, food or otherconsumer applications are quite often prohibitive.

Enzymes are naturally produced in fairly low quantities and their properties do notnecessarily meet those required in industrial processes. Furthermore, the naturallyproduced enzyme mixtures may contain enzymes, unnecessary for the desiredapplication, impairing the action of the target biocatalyst, or causing even harmful sidereactions. Using methods of modern molecular biology, the commercial productionprocess 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 ofview, justifying the high development costs of new products.

The developments in genetic engineering during the last decade have made it possible tochange the spectrum of the enzymes produced by a microbial species or to make anefficient and approved species to produce an enzyme that by nature it is not able tosynthesise. The degree of purity of commercial enzymes ranges from crude enzymes tohighly purified speciality enzymes and depends on their application.

The development of an economically competitive product, which is optimal with respectto specificity, stability or activity under the sometimes extreme conditions existing inthe process industry, is quite demanding, and various technologies are required for thedevelopment (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 anorganism well adopted for industrial use (the host). Furthermore the catalytic behaviour or otherproperties of enzymes can be changed and even new catalytic features created.

Protein engineering: understanding of the protein function-structure relationship is based on theunderstanding of catalytic mechanisms and on the exact three-dimensional knowledge of proteinstructure. Thus a theoretical route exists from a given enzyme with known catalytic properties toan altered enzyme, the properties of which are changed to give improved characteristics in agiven process. With genetic engineering specific alterations of gene structures and consequentlyin the amino acid sequences of proteins can be made. With knowledge of the chemicalinteractions 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 predeterminedchanges are not easily obtained but require both hard and imaginative experimentation.

Catalytic antibodies: a route to enzymatic catalysis of reactions without any biological functions isobtained through the research in catalytic antibodies. Antibodies are proteins which have evolvedto 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 shownthat antibodies, recognising the transition state form of a reacting molecule (or in practice a stableanalogue thereof) will stabilise this state. Consequently the activation energy is decreasedleading to a specifically catalysed reaction. This is an important fact, and a number of cases haveshown that the concept is true and hold in practice. Catalytic antibodies have, however, not yetentered the industrial scene.

Biochemical engineering: this field deals with unit operations of production and use ofbiocatalysts, describing their scientific and engineering basis, determining their performance andoperating characteristics, studying the factors which influence their performance and aiding intheir integration into complete processes. Today, commercial bulk enzymes can be considered ascheap products, and their production processes with engineered organisms are fairly simple. Theproduct is, however, often present at a low concentration in a mixture of a large number of othercomponents. An essential part of such process is separation and purification of the enzyme atthe extent determined by the application. Separation and purification costs may be 50 to 80 % oftotal production and investment costs. With bulk products their share is 10 to 30 %. Usually a setof subsequent unit operations are used, such as filtration, centrifugation, ultrafiltration,precipitation, or chromatography. Fermentation techniques optimise the production of biocatalystby an organism and separation techniques (down-stream processing) to isolate and, whennecessary, to purify the product from the fermentation broth. The price effect of a biocatalyst in apotential application is often prohibitively high. Knowledge on advanced biochemical engineeringin addition to molecular biology are the keys to improved economy of biotechnical processes. It isclearly desirable to maximise the content of the appropriate enzymes in the biomass thatproduces it. Genetic modifications are important to achieve high product yields or contents ofbiological catalysts.

3.3 Benefits of biocatalysts in industry

There is a abundance of existing knowledge on actual and potential use of biologicalprocesses within a variety of industrial fields. These include areas with strongbiotechnical background such as the food and pharmaceutical industry sectors as well asthe more unconventional areas of textile or forest industries. In this chapter a generaloverview 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: theircatalytic, economic and environmental functions.


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3.3.1 Pharmaceuticals and Fine ChemicalsPharmaceuticals are an European success story. European multinationals in this sectorlead the industry world-wide. All of Europe’s major pharmaceutical companies areusing biotechnology to protect, and in some cases, enhance their global competitiveposition. Although Europe has been the leading producer of pharmaceuticals and hasproduced more NCEs (new chemical entitles) than any other region, it is not clear thatthis position can be sustained. The European industry’s share of NCEs has fallensharply 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 healthspending and a slowdown in the growth rate of the European market have put enormouspressure on the industry, particularly in Europe. Although the European multinationalshave been slow to establish biotechnology in-house as part of their R&D programmesand manufacturing processes, nowadays most kind of biotechnology includingmanufacturing processes - biocatalysis - are entirely integrated into R&D programmes.

Although modern biotechnology has been introduced relatively recently into thepharmaceutical industry, the impacts on the economic performance of thepharmaceutical sector has been considerable. These include reduced drug developmenttime, lower production costs, improved quality in production and a larger choice ofcandidate therapeutics and other novel treatments.

The direct impact of past biotechnology performance on R&D can be measured by salesof biotechnology products. Virtually all of the 27 biotechnology drugs listed on theworld market have been developed by the US specialised sector. Furthermore, manybiotechnology drugs under development are based in US companies. Because the smallbiotechnology companies do not have the resources to fully develop and market newdrugs, 17 products have been licensed to large firms. Ten have been licensed toEuropean companies, the remainder to the US. However, Europe’s pharmaceutical firmsall now have substantial leading in-house R&D programmes with severalbiotechnology-derived drugs in development. The threat is that this will encourage moreR&D in the USA (at the expense of the EU), so that the high value added jobs willincreasingly migrate to the USA.

It is much more difficult to measure the impact of biotechnology on productionprocesses in the pharmaceutical industry. The application of biotechnology toproduction often involves incremental improvements in the yield of new and existingdrugs. Although such innovations are often at relatively small scale, they are the subjectnevertheless of patents. However, they are not a subject for publicity. Moreover,


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refinements in production processes are sometimes protected by trade secrets ratherthan patents and therefore are not widely discussed.

The first modern large scale application of industrial enzymes for the production of bulkchemicals occurred in the early 1970s. In the chemical industry it was used to resolve Dand L forms of amino acids while in the pharmaceutical industry the firstbiotransformation was for the production of modified (semi-synthetic) penicillin. Theseprocesses all use immobilised enzymes whereby an enzyme (these days usuallyproduced 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. Theamount of resolved amino acids also runs into thousands of tonnes.

Biotransformation adds value in two different ways. In the production of genericchemicals, biotransformation is economically advantageous and value is added byconverting a heterogeneous mixture of chiral forms of chemicals (two almost identicalcompounds -stereoisomers-, the only difference is that they are mirror images of eachother) into a homogeneous form. This is particularly important in the pharmaceuticalindustry where in racemic drugs - which contain both isomers -, one isomer has aspecific biological activity with a possibility that the second isomer may well producedeleterious effects.

The production of chiral compounds (chemicals where a single stereoisomer of achemical is produced) is becoming of importance in the pharmaceutical industry. Itsimportance stems from the thalidomide disaster. This was a racemic drug where onestereoisomer had the desired and beneficial pharmacological effect but the otherstereoisomer caused birth deformities in foetuses. Although chirality is not a problemfor most drugs, it can be important as different chiral forms of every new drug have tobe synthesised to study their individual pharmacological effects. In practice, theresolution of racemic mixtures is performed after synthesis where differentstereoisomers can be separated. Alternatively one stereoisomer may be removedenzymically. In practice, enantiomeric excess of > 99.9% can only be achieved throughenzyme 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 costeffective way. By contrast to the enzymatic processes for penicillin production, thechemical method of synthesis of 6-APA utilises phosphorus pentachloride which mustbe dissolved in organic solvents under strictly anhydrous conditions and at lowtemperatures.


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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 importantfor the treatment for cardiovascular disease. Chirality is resolved by the use of a lipase purifiedfrom Pseudomonas fluorescens.

Ibuprofen Stereoisomers can be resolved by selectively hydrolysing one isomer with anAspergillus oryzae protease. Ibuprofen is a major non-steroid anti-inflammatory agent that is nowavailable across the counter.

L-methionine To resolve chiral mixtures of methionine, the enzyme aminoacylase fromAspergillus is used in the presence of cobalt, then a yield of 95% can be obtained. Furthermorethe 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 thehydrolysis of cephalosporinic acid-G or -V.

Atenolol Many pharmaceutical compounds contain secondary alcohols. There are different routesto the chiral synthesis of secondary alcohol’s. These involve lipase resolution, whole cellreduction of ketones and the enzymatic reduction of ketones. Chiral selectivity can also beachieved by the use of secondary alcohol dehydrogenase.

Insulin Although much of today's human insulin is produced by genetically modified bacteria, asubstantial part is still produced from pancreas. In the latter case, the sequence is not identical tothe human sequence and therefore the pig insulin must be converted to human form by the use ofa carboxypeptidase-Y and threonine amide.

Peptide synthesis To some extent, recombinant bacteria are taking over the role of purifiedenzymes in the production of small proteins. However biocatalytic enzymes still have an importantrole to play, particularly in the synthesis of small peptides. One of the obvious choices for peptidesynthesis would be proteases and peptidases. It has been found that peptides can also besynthesised by lipases.

Phenylglycine and Dp-hydroxyphenylglycine These chiral D-amino acids are produced inquantities of more than 1,000 tonnes per year. The amide can be stereoselectively hydrolysed tothe L-amino acid.

Leukotrienes and lipoxins These molecules play a key role in the control of the cellularmetabolism and signalling. They are produced in the body from arachodonic acid by the enzymelipoxygenase. Most lipoxygenase is currently derived from soybean. As well as being of use in thepharmaceutical industry, the same enzyme is used by bakers to decolorise bread. The addition oflinoleic acid and lipoxygenase results in oxidation of carotenoids that are responsible for much ofthe colour of wheatflour. It can also be used to make a range of flavours such as mushroom andcucumber.

3.3.2 Food and Drinks and Animal FeedAs mentioned before, the use of enzymes in food processing has a long tradition (cheeseand wine making, beer brewing, etc.). Apart from these ‘old’ enzymes (which have beenimproved and updated several times to have better performances and higher yields), newtypes of enzymes have found applications in food production.

Considering the use of enzymes in food processing and as premixes supplemented toanimal 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 theendogenous enzymes do not function well enough. The added, exogenous enzymes areused to correct and supplement the composition of the original, endogenous enzymes inthe plant raw materials. In most cases the use of enzymes is needed to improve productquality characteristics. This means that application of enzymes may not influence theproduct characteristics: thus e.g. the colour, taste, texture, mouth bite have to stay thesame. Only within the boundaries of maintaining the same product characteristics,process improvements by means of biocatalysts can be made. Consumers are veryinfluential in the process of product innovation and because consumers of food areknown 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 thedynamic developments in this field, because this implies going too much in detail. Wefocus on the most important applications, thereby showing the main driving forces forthe 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 thebaking industry to use raw materials with a broad range of varieties. Wheat flourcontains both alpha and beta-amylases which can activate the dough to produce morefermentable sugars for carbon dioxide production. The composition of endogenousenzymes may be unbalanced, due to a number of reasons (climatological, geographical,storage, transport etc.). Therefore, it is quite usual to supplement the flour by addingalpha-amylase (fungal) at the mill. As there are many different supply sources of this type of enzymes, there can also bedifferences in their side effects (side activities) because of small variations in the waythe different amylases break down the starch. That is why also small amounts of avariety of other enzymes (carbohydrases and proteases, peptidases and lipases) areadded. The selection of specific combination of amylases and additional enzymes canonly be made on the basis of practical and well-interpreted baking tests. At this moment13 enzyme types are currently used by the baking industry. The need to find alternatives to strong oxidising agents, such as potassium bromate, hasencouraged the search for enzymes that function in the same way. To create anoxidising environment and to replace bromate with a similar biochemical step, enzymessuch 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 localvarieties and made with traditional old recipes and craftsmanship. In the production ofcheese the coagulation of milk catalysed by enzymes is the central process. The groupof milk coagulating enzymes falls into three groups: animal rennet’s extracted primarilyfrom calves’ stomachs, microbial milk coagulants, derived from production by variousspecies of fungi and chymosin, identical to the original calf rennet but manufactured bya cloned bacterium. The type used depends on national regulations, consumerspreferences 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 acidsprobably 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 safetyreasons (nitrates) or/and the equipment is too expensive (bactofugation).

• Endopeptidases for cheese ripening, and reducing storage time and flavourenhancement in low and reduced fat cheeses.

• Catalase for destroying any excess of hydrogen peroxide remaining after sterilisationof preservative milk or whey.

The high value of whey protein is well known, both from a nutritional and a technicalpoint of view. However, lactose in whey is considered to be of less value as it causessandiness in ice-cream, has low sweetening power and fermentability, and can causeintestinal problems for people suffering from lactose intolerance. Value can be added byhydrolysing the lactose, resulting in hydrolysed whey syrup. This product is highlyvaluable in bakery products, confectionery products, deserts and ice-creams, spreadings,dressings, soft drinks and semi-moist pet foods. The lactose hydrolysis treatment ofwhey with immobilised enzymes (lactase or beta-galactosidase) for production of wheysyrups in a continuously operated reactor is another example of using immobilisedenzymes.

DrinksThe major biological reactions which take place in the beer brewing process arecatalysed by naturally produced enzymes from the barley and yeast. The process ofbrewing is strongly influenced by the variety of barley, the method of cultivation andvarieties of seasonal weather. As barley is the main raw material, brewers may findthemselves using poorer qualities of malt as they would ideally like, which will requirethe addition of exogenous enzymes to supplement the malt enzymes and in some casesto 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 fungalbetaglucanase may also be added. Enzyme suppliers have produced blends of enzymesto provide the brewer with a single addition, such as a blend of amyloglucosidase and apullulanase/beta-amylase for the production of more fermentable sugars, resulting inlow-carbohydrate or ‘diabetic’ beers.


IPTS 1998 19

Addition of enzymes in beer production is limited to the steps before fermentation. Theonly exception is the use of a recently introduced maturing enzyme in order to preventthe forming of undesirable flavour ingredients (a-acetolactate) with a very lowthreshold. 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 investmentcosts on fermentation tanks and facilities, interests on lagered beer and in reduced beerlosses make the process economically feasible. However, also the environmentalimpacts of the rapid, continuous process are predominantly positive. Decreased organicwaste water loading is achieved due to lesser need for emptying, draining and cleaningof tanks and pipes.

Exogenous enzymes are now also widely used in wine making (i.e. one third of theFrench wines are treated with enzymes), to obtain a better initial extraction of the mustcomponents, thereby improving the yield. In depectinization - the enzymatic hydrolysisof 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 inwine.

Fruit and some vegetables can be processed to produce fruit and vegetable juices orconcentrates. The addition of exogenous enzymes, the most important being pectinasesand glucanases, allows more specific degradation of carbohydrates to give smoothtextures, not found after heat treatments, and at the same time preserving colour andvitamins. The cell wall is the most important part of the fruit, including the grapes, to bebroken down and to enhance juice recovery. For all the cell wall components, specificenzymes are commercially available. It is not possible to produce clear concentrates offruits juices without adding enzymes. In the fruit juice industry, acid amylases are usedto process fruits containing starch, such as apples at early harvest.A new trend is to improve the consistency and flavour of vegetable products. Newprocesses, including enzymes, work on vegetable rheology and develop new blendsfrom raw vegetables, and even include fruit-vegetable mixtures in which vitamins,colour, and flavour have been preserved.

SweetenersEnzymes used for the conversion of starch to syrups comprise about 25% of allindustrial enzymes. By partial isomerization of glucose to fructose, the sweetness of thecomparably inexpensive glucose can be increased to the same level as the moreexpensive 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 convenientchemical process available for isomerization, syrups containing fructose are producedby enzymatic processes using the glucose isomerase enzyme.The isomerization of glucose to fructose is currently the largest-scale technical processbeing performed with the aid of an immobilised enzyme. The product is used as liquidsugar in food processing. The industrial process based on immobilised glucoseisomerase isolated from a Streptomyces species was introduced already in 1972. Theenzyme 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 highfructore syrups.

Olive and other edible oilsThe role of enzymes in edible oil and fat processing is virtually very small, mainlybecause the necessary enzymes are not available at low enough prices (phospholipasesfor the degumming of oils); the conservative nature of some parts of the oil industry(e.g. olive oil processing); and technological barriers (enzymatic interesterificationtechnology). The amount of enzymes being used for oils and fat processing is still tinycompared with use of enzymes in starch and protein processing in other areas of thefood processing industry. In the season 1993/1994 2% of the olive oil was processed inSpain using enzymes. Olive oil processing consumes high amounts of water (1 tonne ofolives processed requires 1 tonne of process water). There is a commercially availableenzyme mixture to enhance the oil yield with 8 to 10 kg per tonne and to reduce theprocess water use up to 50 percent, but its practical use is still very small.

Meat and fishEnzymes 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 marineenvironment and their potential usefulness in food processing: collagenase for crabhepatopancreas for several applications, such as deskinning of squid, production ofcaviar 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 andfeed. 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 addedmeat products (Vilhelmsson, 1997) is another new idea.

Animal feedThe use of enzymes to improve animal feed performance is a commercially fast growingapplication area. The enzyme additives can be divided in two sections: those dealingwith enhancement of general nutrient availability and phytase which improves theavailability of organic phosphorus (phytic acid) found in cereals and vegetable proteinsfor the animal.The main enzyme application is providing feed with enzymes. capable of degradingNSP (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. Apromising development is the use of microbial enzymes to increase the nutrientavailability of cell-wall carbohydrates.

The intensive rearing of animals in certain regions of Europe asks for a decrease innitrogen and phosphorus excretion in manure and their effects on water supplies. Twoenzyme applications can reduce this load of pollutants. First by improving the feeddigestibility, higher amounts of the feed dry matter, particularly nitrogen and solublecarbohydrates, are retained by the animal, resulting in decreased excretion. Second, theenzyme phytase can liberate organic phosphorus from feed raw material in order to bedigested by the animal. Herewith the phosphorus excretion of pigs may be reduced with35% over a growth cycle (Liu and Baidoo, 1997).


IPTS 1998 21

3.3.3 Textile industryThe textile industry is often identified as a key sector where opportunities availablefrom adapting biotechnology are high but current awareness of biotechnology is stillquite low. However, the textile enzymes have been one of the fastest growing areas inthe enzyme industry. The applications especially in the textile finishing are, however,easily fluctuated by the changing fashions. As an example, the popularity of stonewashed jeans has somewhat diminished, affecting thus also the enzyme markets.

Fibre preparationFlax 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 strongcompetition between cotton and man-made fibres, the market share of linen declinedduring the 1950’s. Nevertheless, interest in flax has remained and its main market sectoris 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 thetextile output in the world.The bast fibres of flax cannot be easily separated from the other plant tissues unlesssome decomposition of the stem takes place. This controlled process of decompositionis called retting. The retting of flax has always been one of the major costs and practicallimitations (environmental pollution) to the more widespread use of this indigenoussource of cellulosic fibre in Northern Europe. In various attempts since the late 1970s ithas been achieved to introduce more rapid and controllable enzyme retting processes.These types of processes are based on the action of pectinases. To achieve optimumdegree of retting, however, enzymes other than pectinases are also important, such ascellulases and hemicellulases.

Fabric preparationIn many fabrics production, a coating of starch - size - is used to prevent the threadsfrom breaking during weaving. After weaving the size has to be removed since sizedfabric is less absorbent. The desizing can be carried out by lengthy cooking or by usingstrong chemicals such as acids, bases or oxidising agents. Enzymatic treatment withamylase enzymes has replaced the harsh processes since the beginning of 1900.However, there is still considerable scope for improving the speed, economics andconsistency of the process, including the development of more temperature stableenzymes, as well as a better understanding of how to characterise their activity andperformance with respect to different fabrics, sizes and processing conditions. There aremany commercial alpha amylases available.Scouring and bleaching of cotton fabrics are attractive targets for enzyme-basedprocesses. 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 bleachingprocess of cotton is an already established application. Reactive dyes are especiallysensitive to peroxides and currently require extended rinsing and/or use of reducingagents.

Fabric finishing


IPTS 1998 22

Cellulase treatment of cellulose materials such as cotton, viscose, lyocell, cupro orpolynosic fabrics has gained increasing interest with the growing concern aboutenvironmental issues. The best known applications of cellulases are in denim garmentwashing - biostoning - as an alternative to stone washing and in surface modification ofcotton fabrics - biopolishing - to improve the surface properties. Cellulase can replacethe pumice stones and result less damage to the clothes, machinery and environment. Byusing cellulases, there is no need for the time-consuming and expensive removal ofstone particles from the garments after processing. A small dose of enzyme cansubstitute kilograms of stones. The machinery capacity can also be improved by 30-50%due to reduced processing times. In biopolishing cellulases remove fuzz from thesurface of cellulosic fibres, which eliminates pilling, making the fabrics smoother andcleaner-looking. A similar process using protease enzymes has recently been developedfor wool. Proteases are also used for the degumming of silk and for producingsandwashed effects on silk garments.

Textile After-Care (detergents)In contrast to textile processing, there has been a dramatic increase in the use ofenzymes in detergents since their introduction in the 1960s. The main classes ofenzymes in the detergent industry (proteases, lipases, amylases and cellulases) candegrade a wide range of stains and their use allows milder washing conditions at lowertemperatures (Hamlyn, 1995).

3.3.4 Pulp and paper industryThe pulp and paper industry has adopted quite few biotechnical methods, so far. Themajor reason is that the biocatalysts developed do not generally offer adequate benefitsin terms of their technical performance and costs. Because most commercial productsdeveloped have not found large markets, the enzyme companies are reluctant tosignificantly invest in this field. On the other hand, the pulp and paper companies seemto prefer traditional technologies.

Mechanical pulpingBiotechnical methods aim to overcoming a number of drawbacks in mechanical pulpingprocesses (generally poorer strength properties, high electrical energy requirements andthe few suitable species of wood). The biotechnical pre-treatment methods of woodchips - often referred to as biopulping - are based on the ability of white rot fungi tocarry out modifications in the raw material. After the fungal treatment the energyrequirement 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 beingcommercialised. Also in Europe, considerable research activities are carried out in thisfield, especially in Austria, Finland and Spain. These efforts are also directed towardsthe utilisation of non-woody fibres.

The microbial reduction of pitch (the troublesome extractives that cause negative effectsin the paper making process) can be carried out by two ways; by a microbial method onwood chips prior to refining or by an enzymatic method on refined fibres beforepapermaking. 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 triglyceridesfrom softwood mechanical pulp by lipases a significant reduction in pitch problems hasbeen demonstrated. The lipase treatment allows savings in the consumption of whitecarbon, surface active chemicals. The cleaning frequency and the number of stops isdecreased.

Bleaching of chemical pulpsThe kraft process is the world’s major pulping method. It has evolved over a period of100 years and has become highly refined. Currently, about 70% of the world’s annualoutput of approximately 100 million tonnes is produced by kraft process. The kraftprocess results in the degradation and solubilisation of lignin. About 90% of the ligninis removed; the less than 10% remaining in the pulp is primarily responsible for thebrown colour, characteristic of kraft pulp. In bleaching, the residual lignin is degradedand dissolved by several chemical compounds, including chlorine. In the search toproduce pulp with non-polluting chemicals, more efficient pulping methods andalternative 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 withchlorine dioxide or by non-chlorine chemicals. This allows the AOX levels to bereduced by about 20%. This method is very flexible and applicable to different rawmaterials as well as to different bleaching sequences. Investment is very low as thetreatment can be carried out in storage or intermediate tanks. Due to the present lowprices 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 rapiddevelopment of xylanase prebleaching was partially due to the availability of reasonablypriced 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

Saving of ClO2 with enzyme: 15%

Savings of chemicals:

ClO2 price: 0.4 USD/kg act. Cl

65 kg act. Cl/t

10 kg act. Cl/t

4 USD/t

Costs:

Enzyme

Acidification

1.5 USD/t

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 ofbiotechnology in the pulp and paper industry. The commercialisation of efficient lignin-degrading biocatalyst systems can be expected to take place soon. The future outlook ofthese enzymes seems promising.


IPTS 1998 24

Paper manufactureEnzyme-aided deinking technology has been developed for paper manufacture fromrecycled fibres to reduce chemical consumption. There are two principal approaches tothe use of enzymes in waste paper deinking. One employs lipases to hydrolyse soy-based ink carriers, and the other uses specific carbohydrate hydrolysing enzymes, suchas 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 alkalinedeinking chemicals. In an industrial operation, the use of enzymes as deinking aidscould lower the chemical costs and decrease negative environmental impacts. Offset andletterpress newsprint waste have been enzymatically deinked at low pH in severallaboratory studies. Presently, however, the application of enzymatic deinking incommercial installations has not yet been reported, although several pilot trials havebeen carried out.

The fibrillation and drainage properties of recycled fibres can be improved by using amixture of cellulases and hemicellulases (Pergalase A 40 by Genencor Int). Thistreatment is at least partly based on the removal of fine cellulose particles that impededraining. Control of slime deposits in paper mill whitewater systems is another area inwhich enzymatic approaches have been investigated. The deposits are mainly microbialpolysaccharides, which can sometimes be solubilised by enzymes. Biotechnical methodsare also used for enzymatic removal of pitch deposits, slimes and solubilised fineparticles. The enzymatic pitch control technology in paper manufacture has beencommercially 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 industrialsectors shows that biocatalysts have a number of important benefits: cost efficiency -(bulk) enzymes are cheaper then chemicals -, saving of energy and water, production ofless waste, shorter processing, ripening and storage time, higher efficiency, morespecific, etc.... In this paragraph we focus on the environmental benefits of the use ofbiocatalysts and focus on the question: what are the main driving forces of industry touse biocatalysts. Table 2.4 lists the main benefits of the major classes of enzymes forcleaner production.

Table 2.4. Enzymes produced by Novo Nordisk saving energy, chemicals or raw materials.

Product Application Saving

ProteasesLipasesAmylases

Washing detergents Energy, chemicals

Xylanases Bleaching of paper Chlorine


IPTS 1998 25

Proteases Leather industry Sulphides, COD

CatalasesCellulasesAmylases

Textile industry Energy, water,acids, alkalis

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/benefitratio. On the contrary, looking at new developing industrial uses, biotechnologicalprocesses are inherently expensive when compared to traditional chemical processes,due to the present relatively high capital and process development costs. This meansthat a biotechnological solution in most of the cases is only suitable for high addedvalue products with a cost price of 5,000 to 10,000 ECU per tonne or higher i.e. for theproduction of fine chemicals and pharmaceuticals.

The fine chemicals industry is one of the industrial segments where the impact ofbiotechnology (biocatalysis) is needed for the replacement of traditional, stoichiometricprocesses in order to improve the product/waste ratio. The failure to translatechemocatalytic processes from petrochemicals to fine chemicals makes this moreurgent. 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 dueto the application of biocatalysis, recycling of solvents and (biological) wastewatertreatment. The application of biocatalysis has made the largest contribution in cleanerproduction, i.e. about 60%. The introduction of biocatalysis in the 1980’s in theproduction of fine chemicals was highly appropriate and timely through which a largereduction in the production of waste could be achieved (table 2.5). Despite a fourfoldincrease in production volume in the fine chemicals sector, the production of waste wasreduced with 20% due to the use of biocatalysis.

Table 2.5. Growth and efficiency in the chemical industry between 1975 and 19951)2)


IPTS 1998 26

Pet ro chemical

Bulk chemicals

Fine chemicals

Sp ec ialit ie

1 9 7 5 1 99 5

91% (100)

9% (10)

99% (250)

1% (2 .5)

50% (10)

50% (10)

91% (25)

9% (2.5)

9% (0 .5)

91% (5)

33% (2)

67% (4)

2% (0 .1)

98% (5)

9% (0.5)

91% (5)

Product Volume

Wast e Volume

Amount i n mi l l i on tonnes i n br ackets2 )

1 )

Source: Bruggink, Chemferm, NL.

Though biocatalysis has contributed for 60% to cleaner production in the fine chemicalssector, also reuse or reduction of solvent demand has contributed to more environmentfriendly production processes. As the fine chemicals sector is a small scale industry, theabsolute reduction for each individual production process is small. A list of productsthat 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 andpharmaceuticals has been introduced on a large scale. Vitamins are still mainlyproduced using traditional organic chemistry, the competition between organicchemistry 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 2tonnes per ton of product, the annual reduction in waste generation for the aboveexamples 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 asclean(er) as they neither have zero-discharge nor do they replace or improve existingtechnology. However, the volume of these products is usually very low.Modern advances in biotechnology contribute to cleaner production of semi-syntheticantibiotics by biocatalysis, optimised fermentation and replacement of organic solventsby water. For instance, by replacing a chemical reaction in methylene chloride by anenzymatic step in water, the use of methylene chloride was reduced about 25 ktonnes ona global scale.

The sales created by clean biotechnology for the chemical sector are very small, as itdoes not play a role in either petrochemicals or bulk chemical manufacturing. However,clean biotechnology is prominent in the production of fine chemicals andpharmaceuticals. The fine chemicals & pharmaceuticals segment of the chemicalindustry produces about 1% of the volume of products. The contribution of cleanbiotechnology to the sales value in the fine chemicals and pharmaceuticals segment isabout 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 BenefitsBakery Bread, cakes and

biscuitsalpha-AmylaseProteases

Flour supplementationGluten weakening

AmyloglucosidaseOxidase

Improved crust colourCreate oxidising environment

Dairy Ice cream beta- Galactosidase(lactase)

Prevention of ‘sandy’ texture caused bylactose crystals

Cheese Chymosin (rennet)LipasesEndopeptidasesLysozymes andcatalases

Coagulation of milk proteinsFlavour developmentAccelerated ripeningRemoving spores formers resp. hydrogenperoxide

Whey syrup Lactases and beta-Galactosidases

Remove lactose and produce sweet wheysyrup

Dairy products ingeneral

Various proteasesbeta-Galactosidase

Modification of milk proteinsHydrolysis of lactose for those who arelactose intolerant

Drinks Beer alpha-AmylasesPapainAmyloglucosidase

Removal of starch hazeRemoval of proteinSaccharification for low-carbohydrate beer

Wine PectinasesAmyloglucosidase

Increased yield, clarificationStarch removal

Fruit juices PectinasesGlucose oxidase

Increased yield, clarificationRemoval of oxygen


IPTS 1998 28

Amyloglucosidase Starch removalCoffee Pectinases Extraction of the bean

Meat andfish

Meat Proteases Tenderisation and Removal of meat frombones

Caviar Proteases

Pepsin

Viscosity reduction of ‘stickwater’ andincrease the yield of roeEase the riddling process

Tuna Proteases andcarbohydrases

De-skinning

Meat and fish Transglutaminases Effect on physical properties of proteins,thermal stability, gelation capacity, waterholding capacity

Ingredients

Sweeteners alpha-AmylaseAmyloglucosidase

Liquefaction of starchSaccharification

Flavourings Lipases Ester synthesisHigh fructosesyrup

Glucose isomerase Conversion of glucose to fructose

Sweets Soft centredsweets andchocolates

Invertase Liquefaction of sucroseSugar syrups

Feed beta-Glucanase,amylase and proteasePhytase

Enhancing metabolizable energy in feed,better nutrient availabilityBetter availability of phytic acid in cerealsand vegetable proteins

Sources: Madden, 1995; Vilhelmsson, 1997; Liu and Baidoo, 1997.

In table 2. 7 an overview of the most important applications of exogenous, microbialenzymes used in food processing is shown. The table also includes the enzymes addedas ingredients to animal feed to influence digestion processes.

The traditional character of the food industry is one in which innovations only takeplace step by step. The most influencing factor in this innovation process is theconsumer. This has consequences for the introduction of new technologies, includingbiocatalysis. One important precondition for application of biocatalysts in the food anddrinks industries is that they are not allowed to have any effect on the food: the qualityand safety of the food may not be influenced. This together with the poorunderstanding, on molecular level, of sophisticated biological conversions at hand in theolder food production processes (i.e. wine, beer, cheese, bread) results in minor changesof the production process. Therefore environmental savings related to process changeswill not occur in the food industry. Environmental savings which are to be establishedare related to minor process changes, and consequently will be small in effect. We canalso observe that the food industry from its origin and nature, has never used severeprocess conditions using chemical compounds as compared with, e.g. textile orchemical industries. This means that in the food industry there is less need for essentialenvironmental gains to be obtained by means of biocatalysts. Cleaner in this case refersto using less energy and less water. The use of immobilised yeast’s in brewing is one ofthe few examples of the application of enzymes in the food sector with explicitenvironmental impact. Others are enzymes used in olive oil processing leading to watersavings and replacement of traditional technology using hexane, and less water to betransported in concentrated fruit juices by using enzymes.The size of clean biotechnology, using genetically modified organisms, in the USA infood processing, beverages and feeds is relatively large compared to Europe. One of thereasons for this is that the USA consumer more readily accepts foods manufacturedusing modern biotechnology.


IPTS 1998 29

A rough estimate of the impact of process integrated modern biotechnologies in thefood industry - excluding the traditional applications of biotechnological techniques -shows that the impacts are larger in the USA with a market share (process integratedbiotechnology 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% and2 %. In Japan the role of this type of process integrated biotechnologies seemsnegligible yet (OECD, 1996; Smith, 1996; Ballantine and Thomas, 1997).

In contrast, in the animal feed industry, things are different. Here a business tobusiness market exists where product and process changes are more easily accepted.Enzymes were explicitly developed and used for environmental reasons; the addition ofmicrobial phytase in animal feed decreases the amount of phosphorus in manuredisposal. The use of enzymes in the feed industry is expected to grow rapidly and so arethe possibilities for environmental savings.

The use of enzymes in textile processing and after-care is one of the best establishedexamples on the application of biocatalysts to obtain environmental and technicalbenefits in process industries. These methods explicitly aim at minimising theenvironmental effects as well as improving product quality (reducing the damage causedto the fibres during processing). The application of biotechnology to textile processes isfacilitated by the use of water solutions and relatively mild process conditions. Actuallybiotechnical processing was introduced to textile industry already in the beginning of1900. 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 continueto provide some of the most immediate illustrations of its potential also in the near termfuture. There has been a dramatic increase in the use of proteases, cellulases and lipasesin after-care detergents since their introduction in the 1960s. The most importantapplications 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 auxiliaryconsumption

Finishing : Biopolishing Improved product qualityFinishing : Denim treatment Less damage to machinery

Higher machine capacityDecreased waste formationImproved product qualityImproved control and reproducibility

After-care and detergents Energy savingsReduced use of sodium perborateImproved product quality

Source: Hamlyn, 1959 and Godfrey and West, 1996

Biotechnical methods have also entered the pulp and paper industry, aiming atimproving process stages to decrease environmental impacts, to save energy or toimprove product quality. Especially interesting today are biotechnical methodssupporting efforts to close the water systems of the mills, leading to minimal wasterelease. The most clearly well-established biotechnical process step is the waste waterpurification, however, as end-of-pipe technology not being within the scope of this


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study. The functions and potential advantages of biocatalysts at different process stagesare summarized in Table 2.9.

Table 2.9. Benefits of using enzymes in pulp and paper processing.

Process stage Biocatalysts Benefits

Raw material treatment:DebarkingWood preservation

PectinasesMicro-organisms (fungi)

Energy and raw material savingsEnvironmentally benign methods

Mechanical pulping:Pre-treatmentRefining

Micro-organisms (fungi)Cellulases

Pitch removal, energy savingsEnergy savings, improved product quality

Chemical pulping:Pre-treatment

Micro-organisms (fungi)Various enzymes

Savings in chemicals

Bleaching: Hemicellulases,Laccases

Chemical savings, increased capacity,reduced AOX-formation

Paper manufacture:DrainageDeinkingChemistry of the wet end

CellulasesCellulases,hemicellulasesVarious enzymes

Increased productivityChemical savings, improved product qualityImproved productsImproved runnability

Source: Jeffries and Viikari, 1996

ConclusionsWe conclude that the use of biocatalysts is based on their superiority in carrying out thedesired reactions, due to their specificity, economical advantages or improvedenvironmental 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 bestavailable 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 primarytarget, and plays usually no role.

The most important driving force are economic benefits, in some cases in a win-winsituation with environmental savings. However, environmental benefits from theperspective 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 tothese sectors and the resulting sales value of biotechnology in (clean) production can befound in the food and drinks and fine chemicals/pharmaceuticals sectors. In the processindustries, such as textile or pulp and paper, the use of biocatalysts aims at specificprocesses with savings of energy or raw material, simpler processes with lowerinvestment costs or improved products. These economic driven applications show theimportant environmental impact of biotechnology. However the sales to these sectors isstill relatively small (Smiths, 1996; Ballantine and Thomas, 1997; Degenaars en Jansen,1996).

Often, in the public and political debate, the major advantage of biotechnology isconsidered to be its positive environmental impact. The simple rational is that since theprocess is biological - and biological is ‘green’ - all side events should also beenvironmentally compatible. This is clearly an oversimplification. The simplesubstitution of a chemical reaction by a biotransformation will not necessarily alone


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lead to improved environmental impact. The environmental impact and economics of aprocess must be considered as a whole, including energy requirements, processparameters, product and waste streams and recyclability. The scientific, technological,economic and ecological considerations relevant to a specific process will determine thebest available technology in competition. In practice modern technologies do notcompete 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 withIndustry: ICAF Industry Studies, Academic Year 1996., Industrial College of the ArmedForces National Defence University Washington, DC 20319 - 5062.

Ballantine, B. and Thomas, S., 1997. Benchmarking the Competitiveness ofBiotechnology in Europe, The European Association for Bioindustries, Brussels,Belgium.

Bickerstaff, G.F., 1995. REVIEW: Impact of Genetic Technology on EnzymeTechnology., The Genetic Engineer and Biotechnologist, Vol. 15, No. 1, JournalsOxford Ltd.

BUNR, 1996. Umweltpolitik; Tagungsband des Fachgesprächs “Beitrag derBiotechnologie 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 DutchIndustry: Critical Factors for Success., Erasmus University Rotterdam, Ministry ofEconomic Affairs, The Netherlands, pp. 125.

EFPIA, European Federation of Pharmaceutical Industries Association (1995) ThePharmaceutical 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, TextileMagazine, 3, pp. 6-10.

Jeffries and L. Viikari eds. (1996) Enzymes for pulp and paper processing, ACSSymposium Series 655.

Link, Matt (1990) Enzymes in the forefront of food and feed industries, Keynote lectureon the First international Symposium on ‘Enzymes in the Forefront of Food and Feedindustries’, Food Research Foundation, ELINTARVIKKEIDEN TUTKIMUSSAATIO.


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Liu, Yonggang and Baidoo Samual K. (1997) Exogenous enzymes for pig diets: anoverview, in: Enzymes in Poultry and Swine Nutrition, R.R. Marquardt and ZhengkangHan eds. International Development Research Centre, Canada.

Marshall, C.T. and Woodley, J.M. (1996), Process Synthesis for Multi-step MicrobialConversions, 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 aGlobal 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 theConference 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 processingindustry, in: Trends in Food Science and Technology, August 1997, vol. 8, p. 266.


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4. Europe’s scientific-technical competence inbiocatalysis

4.1 Science base in life sciences and biotechnology: EU vs. US

The science base is critically important in biotechnology because it produces newknowledge that provides the foundations for R&D and ultimately, new products orprocesses. The science base, as well as producing new knowledge, gives rise to newinstrumentation and methodologies, new skills and creates spin-off companies. Thescience base is particularly important in biotechnology in general becausebiotechnology is closely linked to developments in basic research. Realising thepotential of biotechnology depends on a strong science base and good links between thescience 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 inEurope and that this factor has contributed to the US lead in biotechnology. Thisevidence comes from a range of previous studies which generally support this notion. Arecent survey of over 50 biotech companies with operations in both Europe and theUSA revealed that these companies have the perception that the science base in the USis superior to that in Europe. The scale and quality of public investment in R&D, therelevance of public investment in R&D, the overall scale and quality of academic-industry collaboration and the effectiveness of the technology transfer mechanisms wereall factors felt to be significant in achieving the strengths in the US. However, the samegroup of companies also consider that the quality of public sector research in Europe isas good in the USA (Ballantine and Thomas, 1997).

What are the main reasons behind these differences? In the first place, the USA spendsprobably 50% more than Europe on life sciences; thus, expenditure on life sciences inEurope is estimated at approximately ECU 15 per capita compared to ECU 22 per capitain the US. However, the reality is that this spending gap between the USA and Europe isactually greater than that given by these figures because the cumulative impact of highUS spending over a longer period and the relatively fragmented nature of spending inthe 15 EU member states enforce the differences. It has also been estimated thatbiotechnology-specific investment in the US science base is at least three times higherthan that in Europe. Concerning links between universities and companies, in-depthinterviews also revealed that scientists in Europe often find it difficult to identifythemselves with commercial opportunities (IMD, 1996).

Finally, bibliometric indicators also revealed that Europe’s scientific research has lessimpact than that of the US (table 3.1). In general, European scientific papers in lifesciences have markedly less citations per paper in disciplines central to biotechnology.


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Table 3.1. Bibliometric indicators: impact (average citations per paper, 1982-1992)

Europe USA Other

All sciences 3.77 5.26 3.71Biology 7.24 9.83 7.51Biochemistry and molecular biology 3.26 4.07 3.14Biotechnology and appliedbiotechnology

3.90 5.55 4.00

Chemistry 3.19 5.54 2.83Plant 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 asoutlined above? In Europe there is relatively little academic research on biocatalystswhich has a bearing on the pharmaceutical industry. Simply by its nature most of thisresearch is highly applied, often focused on the improvement of production processes,and carried out by companies. This kind of research is generally either confidential andprotected by trade secrets or patented. Despite which of these two modes of intellectualproperty are used, the outcome, in terms of publications, is broadly the same. In otherwords the amount of published material on biocatalysis by European academics is at avery low level.

A further problem is that it is difficult to allocate published papers on basic research asbeing specifically relevant to different sectors. For example, about half of the publishedpapers in table 3.1 cannot be attributed to a specific industrial sector, thus a basicresearch paper on, for example, proteases may ultimately have relevance to both thefood industry and the pharmaceutical industry.

However, several indicators reveal that Europe is internationally competitive inbiomedicine and molecular biology. For example, nearly half of the top molecularbiology 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 beobtained by an analysis of published papers. By assessing the authorship of paperspublished in ISI cited journals it is possible to obtain a crude estimate of productivity ina particular area. A search was performed using the keyword "Biocatalysis" in both thetitle and the abstract of the paper. Between 1994 and 1997, 303 "hits" were made andthe country of origin of the paper was determined. This is shown in table 3.2.


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Figure 3.1 Origin of papers with a keyword on “Biocatalysis” between 1994-1997.

Country Papers (%)USA 30UK 10Canada 7France 6Netherlands 5Japan 5Sweden 4Spain 4Austria 3Italy 2South Korea 2

USA

UK

Canada

France

Netherlands

Japan

Sweden

Spain

Austria

Italy

South Korea

30

10

7

6

5

5

4

4

3

2

2

0 5 10 15 20 25 30

USA

UK

Canada

France

Netherlands

Japan

Sweden

Spain

Austria

Italy

South Korea

Papers (%)

As might be expected, the leading country is the USA with 30% of the paperscontaining the keyword "Biocatalysis" either in the title or the abstract. Next is the UKfollowed by Canada. The columns in the above table do not add up to 100% as countrieswith less than 3 papers are not included in the above analysis. However, if the papersfrom all EU countries are added together, these make up 42% of the total. Included inthis 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 theother hand, the number of hits does apparently not correspond to the numbers of paperspublished within this area (of biocatalysts). However, we believe it reasonably andaccurately reflects the competence of particular countries. There do however seem to besome 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 industriallaboratories of enzyme producers. The research needed for industrial production ofenzymes needs development of production host, of their excretion systems, offermentation technology, of protein chemistry, of molecular modelling, identification ofnew enzymes and applications and also development of the application. This means thattogether with the enzyme specialists there is a big number of specialists for each of theapplication areas. Besides the actual enzyme producers, extensive development work isbeing performed by the enzyme suppliers (who do not necessarily have their ownproduction).


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Pharmaceutical industryBiocatalysis research in relation to pharmaceuticals, such as it is, is spread acrossdepartments of chemical engineering and microbiology in public sector institutes. Atradition of collaboration between these two types of departments is largely absent. Thiscontrasts with the situation in the pharmaceutical companies undertaking this type ofresearch where the chemical engineering and microbiological aspects of biocatalysisneed to be thoroughly integrated to be incorporated within the production processes.Academic research in this area tends to be highly focused with groups perhaps workingon a single enzyme over a long period of time3.

Food and feedFood processing is only partly based on scientific knowledge of the molecular basis ofthese processes. Craftsmanship is still a very important compound of expertise inenzyme research; the fundamental processes underlying important food processes likethe baking of bread are not yet understood on a molecular level. For the other foodsectors 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. Everyenzyme product shows additional activities or side effects when added to a substrate.Consequently, improvements in the processes are related to extensive testing andresearch in specific applications. So, one of the largest test-baking lab’s in the world isQuest Research Lab in Naarden. As this craftsman application knowledge on theconduct of enzymes is not patentable, the enzyme industries have to keep their researchin-house to protect it.

All this results in a rather big, heterogeneous and diffuse structure of the public researchcommunity in the field of biocatalysts used in the food and feed industry. At manyuniversities biocatalys research is taking place but it is scattered in small subgroups andmostly related to part of other subjects (bio-processing, organic chemistry, biologicalchemistry, biotechnology, food chemistry, food technology, chemical engineering, finechemicals etc.).

Textile, pulp and paperBoth areas involve traditional technologies which have been developed under a longperiod, and which have undergone extensive modifications and improvements already.This is especially true in the pulp and paper industry where the environmental loadshave significantly decreased, especially in Northern Europe, during the last decade.Excluding the waste water purification technology, this has mainly been carried outwithout the use of biocatalysts. The present applications of biocatalysts involveprocesses where enzymes act rather as enhancers or additives, and these process stagesare not based on biotechnology, solely. Thus, these can be considered as applications ofthe first generation. Extensive modification of the traditional processes (into“biotechnical” unit operations) would require more efficient biocatalysts andapplication technologies.

The European research has many strong areas in this field. Research on traditionalcotton 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.


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practically absent in Europe. The European know how is especially good in the fields ofcellulases and cellulose structure studies. Northern Europe has traditionally excellentresearch and know how in the pulp and paper area, but there are also other high-qualityresearch units in Central Europe. Research on biocatalysts in the pulp and paper area iswidespread, but there are only few centres of excellence.

4.4 Patent analysis of biocatalysis

4.4.1 Relevance of patent statisticsPatent statistics have long been used as indicators of technological activities byacademics as well as policy-makers. As with other technology indicators, such as R&Dexpenditure, they have their own relative advantages and disadvantages and these havebeen reviewed in detail elsewhere (Pavitt, 1988; Griliches, 1990; Patel and Pavitt,1995). Does counting the number of patents in industrial sectors and companies providea useful indicator of competitive success? As Sharp et al (1996) have shown, there isno consistent relationship between R&D expenditure, number of patents and number ofproducts in the Top 50 pharmaceutical companies. Thus Rôche, which is first in the setin terms of the percentage of sales devoted to R&D, and is amongst the top group inpatenting 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 developmentwere relatively low in comparison with companies with much less R&D expenditure.

The development and wide application of biotechnology has posed major challenges forthe patent system in the US, Europe and Japan. Although patents for new chemicalshave been granted over a century, the patent system of many countries has explicitlyexcluded the patenting of any naturally occurring substance or life forms. While thestrengths and limitations of existing patent protection has been effectively adapted toinventions in classical microbiology, the specific exclusions laid down long before thedevelopment of biotechnology continue to cause major problems of interpretation. Ascommercial exploitation of biotechnology is now gathering momentum in thepharmaceutical 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 isalready known to be a high user of patents (Levin, 1987). Patenting as a means ofprotecting inventions is also fairly well established in the food industry.

The main advantage of patent statistics as indicators of technological activity is thatsuch datasets are available over long periods of time and can be grouped into technicalfields and by organisation. However, there is considerable empirical evidence for thelimitations of patents as indicators and biocatalysis as a field is no exception. It isimportant to bear in mind that only about 7% of patents will ever actually be used. Theremainder will not be commercially exploited. Patents are an important way to protecthard-earned R&D results, but they are equally significant as a source of technicalinformation. An often-quoted figure is that around 80 per cent of all publicly-availabletechnical 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 therespective patent holders; only the remaining 10 per cent is protected by valid patents.


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The steady growth shown in some of the patent databases in biotechnology patentsreflects the fact that biotechnology has become increasingly integrated within thepharmaceutical sector which habitually patents heavily. These patents will, for bothproducts and processes, contain a wide range of claims. In some cases where productscannot be patented because of existing prior art or lack of novelty or inventiveness, aprocess patent can be used instead. For biotechnology companies, particularly those inthe US, patents have become a very important asset with regard to the raising of fundsfrom venture capitalists and shareholders. Several studies have shown that smallcompanies preferentially patent over large companies in terms of numbers ofbiotechnology patents per company (Thomas et. al., 1997).

4.4.2 Biocatalysis patentsFrom 1990 onwards there has been an increase in the number of biocatalysis patents andgeneral biotechnology patents in the physics and engineering fields. Whether this is dueto strategic registration policies or the result of knowledge and technology transfer fromclassic 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 widerange of industrial sectors more than one allocation is not possible. The main user ofenzyme 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 mostrelevant 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


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DE GB NL IT FR ES BE NO

815

562

70 72

339

30 38 60

200

400

600

800

1000

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

0

100

200

300

400

500

600

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. Thereforeadditional searches were made with the word ’enzyme’. A search in the World PatentIndex revealed 4,763 patents in total. Figure 3.3 shows the distribution of patents acrossthe fields "pulp and paper", "pharmaceutics" ,"fine chemicals" and "food". All searchterms were truncated so as to include plurals and genitives. The search for individualenzyme classes together with biocatalysis will take place at a later date.

Figure 3.4. Thematic overview of patents in the field of biocatalysis


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3%

61%

36%

0,5%

Pulp & PaperPharmaceuticsFine ChemicalsFood

Pharmaceutical sectorAn analysis was made of the overall situation of the "initial registering country" of thepharmaceutics patents in terms of which country registered them first. The results fromthe "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 accountof their scientific/technological situation (e.g. Denmark, DK) few patents wereregistered in their own countries.

Figure 3.5. Patent registration in pharmaceutics world wide (1960 to today)

US JP DE DK NO GB ES FR NL BE IT SE CH AU

888

745

260

23 4

186

6

182

19 1179

30 40 20

0

100

200

300

400

500

600

700

800

900


Therefore, the next analysis carried out was for the ’pharmaceutical biocatalysis’thematic complex (search for "biocat and enzyme " and "pharmaceutics") according tothe registering country (originating country of the patent). Denmark was selectedbecause NOVO has its home base in Denmark. The search for Danish patents resultedin a total of 327 compared to 23 patents registered in Denmark. The patents in thecountries in figure 3.4 were then examined to see which were Danish. The results arepresented in figure 3.5.

Figure 3.6. Danish biocatalysis patents in the pharmaceutics sector by country (1960 to 1996)

Country

Number of patents

Number of patent registrations


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US JP DE NO GB DK

136

25

41

2

48

5

0

20

40

60

80

100

120

140

US JP DE NO GB DK

The distribution shows an interesting phenomenon. Danish developments are registeredpredominantly in countries with a high level of industrial application for biotechnology,and not in Denmark itself. The patents are registered to gain strategic benefits. Thisexplains the registration of Danish research results mainly in the USA which is theleader in biotechnology.

The results of registration within Europe is also interesting. Whilst no Danish patentscan be found for Spain or Italy, Germany and Britain have the most registrations afterthe USA within these countries - a phenomenon which can be explained by the overalleconomic and technological situation. Both countries have a high R&D expenditure inthe pharmaceuticals and general biochemistry sectors and a large number of industrialproduction companies.

FoodA similar analysis was also made for the food sector. The results show similarities withthat of the pharmaceutical sector. Interesting is the Japanese patent situation in thesector of food. 1919 patents are applied at first in Japan (figure 3.6).

Country


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Figure 3.7. Patent registration in "food" world wide (1960 to today)


780

1919

246

47 2

182

12

191

29 16 33 47 40 20

0

200

400

600

800

1000

1200

1400

1600

1800

2000


A possible cause for this situation may be the high export rate of Japanese food to otherAsian countries. For example the fish industry in Japan is one of the most important inthe world. There is high R&D budget for research on fish based food and ingredients.

Paper and pulpIn a third step the pulp and paper sector has been analysed for patent situation in anumber of countries. Figure 3.7 shows the result.

Figure 3.8. Patent registration in "pulp and paper" world wide (1960 to today)


58

71

16

41

06

0

10

0

4

0

7

02

0

10

20

30

40

50

60

70

80


Patent registration

Country

Patent registration

Country


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EU patenting systemThe fact that in 1996 less patents have been registered by the EPO as to 1994 and 1995might indicate that patenting is becoming more and more a strategic competitioninstrument which is highly influenced by the national patent system. It seems that thehigh costs and the lack of awareness mean that European companies are failing to makebest use of the patent system. The relatively cheap US patent registration system servesas a strategic competition tool, which helps controlling market(s) / shares. The presentsystem of patents in the EU is complex and expensive, and does not provide a unitarypatent for all the Member States.However, innovations should enjoy the same protection throughout the single market, inorder 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 arenot always easy to compare, because they are made up of several different componentspayable at different stages during the lifetime of the patent. Nevertheless, there is nodoubt 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 ECU20,000 and this does not include mandatory translation costs. A US patent, bycomparison, 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 smallerin scope than their western counterparts. The total cost includes the fees charged by theEPO and national patent offices, patent attorneys’ charges and translation costs. TheEPO is currently in the process of cutting its fees by around 20 per cent, but many usersthink this is still too expensive. These and other problems all stem from today’s ’dualsystem’ arrangements. While inventors can apply for a European patent through eitherthe EPO or a national patent office, in reality, patent applications are split equallybetween these two routes. In fact, more than 90% of applications filed by EU nationalsare based on a previous national application.

Patenting of enzymes has historically not been a major issue: many companies producethe same or equivalent enzymes. For example, there are over 12 companies world-widewhich produce amylase. In future patenting of enzymes will become of great importanceas companies realize that this is the way to protect their investment in research anddevelopment and also give them leverage to obtain market share.

Since there is no single European patent, fragmentation of the internal market, withseparate patents needed for each Member State, is a realistic option. The cost ofsecuring patent protection in every Member State will discourage companies fromexploiting their innovative potential, while the lack of legal mechanisms and legalinfrastructure at European level means that interpretations and applications of patentlegislation are different. The USA and Japan both have a single patent mechanism andlegal 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 Communitypatents, which has not yet entered into force. These two instruments represent acomplex legal framework for those seeking patent protection, and are subject todifferent legal jurisdictions. Alongside national patents, which continue to exist, there is


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a European patent, though only an embryonic one: once granted by the European PatentOffice in Munich, the European patent operates to all intents and purposes like anational patent. There is no provision for a court of law with jurisdiction at Europeanlevel to decide disputes in patent cases, such as actions for infringement or revocationof a patent, so that there is always the danger that the courts that hear such actions in theMember States may deliver conflicting judgements. The present system consequentlyplaces 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 itstwo main competitors, Japan and the USA. This system should also provide adequateand non-discriminatory treatment for non-EU Member States. Under this system, thecosts for patent protection within Europe would be reduced to a level comparable to theUSA and Japan (although the costs of translation have to be considered in setting thefees). 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 thesame throughout the Union; it could be granted, transferred, revoked or allowed to lapseonly in respect of the whole of the Union.

4.5 Conclusions

Biocatalyst research is in the first place concentrated in the industrial laboratories ofenzyme producers and enzyme suppliers which market and sell enzyme products forspecialised fields. There are several groups in the public funded research organisations -universities and research institutes - in Europe, specialised in different types ofbiocatalysts and there are of course the basic scientific disciplines which are the pole ofknowledge, dedicated or not, for the applied biocatalysis research. Taking into accountthe different area’s of expertise involved in the biocatalysis research, in theory it ispossible to give an overview of Europe’s scientific and technological potential in thefield of biocatalysis. However, identifying the scientist in public and privatelaboratories, describing their activities and their products and finding reports with anassessment of their products (audits, visiting committees, referee’s etc.) would takeseveral years. Hence, we had to conclude that in the practical context of this project itwas impossible to make such an overview, especially if in addition specific attentionshould be given to the environmental benefits of biocatalysis.

Nevertheless, on the basis of recently published reports with more qualitative data wewere able to make a number of observations on Europe’s scientific and technologicalpotential in the field of biocatalysis. In industry the perception is that the science base inthe field of biotechnology, which includes biocatalysis, is stronger in the USA than inEurope. The quality of public sector research in Europe is evaluated as good as in theUSA. An indication of the strength of biocatalysis research in Europe compared withother 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 ofthe present state of Europe’s research base in biocatalysis, is that it is well organised.The European Federation of Biotechnology (professional organisation of biotechnology


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researchers) has a working party on Applied Biocatalysis. The members of this party arefrom 23 countries from Western and Eastern Europe.

Patent data base analysis shows that the number of EU biocatalysis patents is laggingbehind 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 especiallyorganic chemistry since biocatalysis is not necessarily given as a key word in worksrelated to enzymes. It is also important to realize that because patent strategies differconsiderably between regions, it is not allowed to estimate the scientific-technologicalpotential of European biocatalysis research on the basis of these patent analysis.

Literature

Ballantine, B and S.M. Thomas, (1997) Benchmarking the Competitiveness ofBiotechnology in Europe, report prepared for EuropaBio, Brussels.

Griliches, Z. (1990): 'Patent statistics as economic indicators', Journal of EconomicLiterature, p.28.

IMD (1996). The World Competitiveness Yearbook, Geneva.

Levin, R.C. et al (1987): 'Appropriating the returns from industrial research anddevelopment', The Brookings Papers on Economic Activity, Vol. 3, pp 783-831.

Patel, P. and Pavitt, K. (1995): 'Patents of technological activity: Their measurementand 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 QuantitativeStudies of Science and Technology, van Raan, A. (ed), Amsterdam, North-Holland.

Sharp, M. and Patel, P. (1996) Europe’s pharmaceutical industry - an innovationprofile, STEEP Discussion Paper No. 34, SPRU, University of Sussex.

Thomas, S. et al., 'DNA sequence patents in the public sector', Nature, 7 August 1997)


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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 bediscussed. First the size and structure of the biocatalyst industry and the biocatalystmarket is addressed. In addition, an estimate and description is made for each sector ofthe working processes in the sectors in which biocatalysis is used. The chapter willfinish with the general bottlenecks (technical, economic, socio/cultural) met by industryin applying biocatalysis.

5.2 The economic capabilities in Europe

5.2.1 Structure of the industryIn 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 thecustomers, in most cases the bigger food and drink companies and the pharmaceuticalindustry. The latter screen commercially available enzymes and develop newapplications, sometimes with the assistance of the enzyme providers.The enzyme providers develop enzyme mixtures for specific applications on the basis oftheir knowledge of enzymes and specific applications and market niches. This is ofcourse also an activity of the enzyme producers themselves - all producers in theenzyme industry buy and sell enzymes from each other - but these provider companieshave an important position in the market. The enzyme providers are especially active inthe bakery ingredients sector. It is estimated that in this sector an enzyme is sold 2.5times before being used in a baking process.

Godfrey and West (1996) made an inventory of the enzyme supplier companies. Theyfound that 400 companies world-wide offer enzymes in their sales programmes. Aconsiderable number of these companies are agents and distributors and if these areexcluded a list of 137 companies is left.Table 4.1 shows the geographical distribution of these companies. Even though themarket as a whole seems to be dominated by three major producers, the number ofcompanies selling enzymes (this includes the providers) doubled during the 1980s.


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Table 4.1. Distribution of enzyme providers world-wide

Region/Country No. of companies Region/Country No. of companies

Europe 70 Australia 2USA 30 South America

(Mexico and Brazil)2

Japan 24 Republic of South Africa 1Canada 4 Russia 1Southeast Asia(Thailand, India and Taiwan)

3Total 137

Source: Godfrey and West, 1996.

Table 4.2 shows the distribution of the 70 European enzyme providers and producerswithin 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 andpaper

Pharma Others*

Diary Baking Others

Austria 4 xBelgium 4 x xDenmark 4 x x x x xSpain 2 xFinland 3 x x x x xFrance 10 x x x x x xGermany 13 x x x x xItaly 2 x xNetherlands.

5 x x x x x x

Switzerland 4 x x x xUK 19 x x x x

Source: Godfrey and West, 1996.* This category includes industrial enzymes and enzyme products of companies who have notspecified 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 apart of its activities in industrial enzymes to Genencor and now focuses on food andfeed enzymes and ingredients.

It remains obvious that Novo Nordisk is the absolute market leader, especiallyconcerning its research activities linked to promising new enzyme products. Each year itintroduces approximately ten new enzyme products. This is quite a high figure for anestablished market such as the enzyme one, and is equivalent to all the marketintroductions of competing companies put together. Novo is based in Europe but has, asalmost all world-wide operating companies, its research facilities based in other regions,especially in the USA.


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Table 4.3. Total turnover of the most important producers in the enzymes sector 1995(in US $ millions)

Company Turnover

Novo Nordisk (DK) 580Genencor (USA) 235Total Japanese companies (J) 90Gist Brocades (NL) 65Quest International (NL) 32Hansen (DK) 25Grintsted (DK) 25Rohm (D) 25Others 53


5.2.2 The structure of the marketIn 1980 the total value of enzyme sales world-wide was estimated at around US $ 300million. Between 1991 and 1996 the global market for industrial enzymes more thandoubled 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 industrialenzymes, 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 960Food 100 160 240 370 870Beverages 100 160 200 320 780Other 60 100 160 320 640Total 370 590 880 1.410 3.250

Source: Bickerstaff (1995)

Although the enzyme producing industry has less companies and investments than thepharmaceutical industry, it is recognised as the most profitable area today (Abbott,1996). Detergents for the home laundry market, proteases and in the future lipases, arethe leading area (Smith, 1996). Within the total market volume of food, drinks and feedenzymes, carboxyhydrases and enzymes for starch hydrolysis and fructose syrupproduction make up for more then 50%. The enzymes for baking (including the bulkamylases and the number of special enzymes for structure and freshness) are in secondposition (14%), followed by brewing and fruit processes (10% and 8% respectively).The enzymes for wine production and feed/fodder account for approx. 6% and thecheese enzymes occupy the last position with 1.6%.

Of the some 18 enzymes commercially available in bulk in 1991, five were the mostimportant. These were amylases, bacterial proteases, papain, glucosidases, rennin andchymosin.

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 theUSA and Europe with total sales of US $ 100 - 200 million for each region in 1995


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(Marrs, 1997; Bickerstaff, 1996; Novo Nordisk, 1997; Godfrey and West, 1996). Table4.5 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) 75Glucose isomerase 40Glucoamylase 75alpha-Amylase 50Papain 8Trypsin 8Other food proteases 8Invertase 8Pectinase 7Others (beta-Glucanase, cellulase, dextranase, glucose oxidase,lipase, pullulanase)

20

Source: Sahm et al., 1991-1997.

Enzymes are relatively new additives in the feed industry. Before 1985 the use ofenzyme additives was ignorably small. In 1995 it represented US $100 million of thetotal $5.8 billion market of feed additives. It is the fastest growing market for industrialenzymes and will continue to be so in the near future. Other feed markets are expectedto follow the example of the poultry market which is now more or less saturated. It isexpected 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 takesplace 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 GenencorStarch conversion 138 Novo Nordisk and GenencorTextiles and pulp and paper 164 Novo Nordisk and GenencorDairy 130 Gist BrocadesBakery 80 AllAnimal feed 75 Gist brocades and Novo NordiskBrewing and distillery 50 QuestFruit juice and wine 25 Gist brocadesOther 40


Future marketsThe commercial outlook for industrial enzymes is very good. According to a report ofFrost & Sullivan, a market research company, the European turnover for industrialenzymes 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 doso in the year 2000. Frost & Sullivan report this is mainly due to modernbiotechnology’s which allow for a more efficient production of enzymes. The detergentssector was between 1993 and 1996 the fastest growing market for industrial enzymes.Frost and Sullivan expect this will change: the less traditional applications will growfaster. Examples are the treatment of waste, paper and pulp followed by chemical andpharmaceutical processes (NIABA, 1997).


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5.3 Integration of biocatalysts in industry

5.3.1 The pharmaceutical industryThe use of enzymes in the pharmaceutical industry has two primary functions. First, toseparate different chiral forms of drugs and second, to play a major role in the synthesisof drug precursors. The use of enzymes has grown steadily in the pharmaceuticalindustry having been initiated in the above two areas about 20 years ago. Prior to thisthe separation of different chiral forms was largely achieved using silica columns whichare a relatively inefficient and lengthy process.

Today, enzymes are used in a minority of drug production processes. Excepting thatabout 10% of all drugs are biotechnology-based, a small proportion of the remaining90% 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) arein the chiral form that requires separation. Thus the importance of enzymes in thepharmaceutical 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 rangeof enzymes such as lipases to synthesise precursors to the final drug product. Theintroduction of enzymes has become established in this process because they offer amore 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 Land D forms. Secondly, the final drug products exist in one form only. This means thatthe 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 tochange significantly in the short to medium term. Finally, it should be noted that theearly biotechnology protein-derived drugs such as tissue plasminogen activator or t-PAand erythropoietin or EPO are produced directly by genes cloned in bacteria or yeastcells. These products are relatively pure and are not produced in the form ofstereoisomers.

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 inclinical trials. However these drugs do not require the use of enzymes either to separatedifferent chiral forms or to synthesise precursors on a significant level.

5.3.2 The food and drink and the feed industriesThe food and drink is an extremely heterogeneous industry and enzymes are used in aconsiderable number of its subsectors. The brewing of beer and the baking of bread aretwo totally different and separate industries. Not only are there many differencesbetween subsectors, there are also considerable differences between countries withineach subsector: the production and marketing of beer in Denmark differs in terms ofproduction technology and consumer preferences markedly from countries likeGermany. These two factors (differing markets, differing products) result in a highly


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diversified market for biocatalysts in the food and drink sector in contrast to morehomogenous markets such as the textile or detergents markets.

It is clear that Europe’s major food and drinks companies have the ability now to applyand exploit the potential of biotechnology. This is particularly so in the area ofenzymes. They have tended to rely on developing in-house capabilities as opposed tobuilding complex relationships with the specialised biotechnology sector. Competenciesof Europe’s major food and drink companies in biotechnology generally, have recentlybeen considered at least equal to that of their US competitors (Ballantine and Thomas,1997). However it is now apparent that biotechnology in manufacturing is moreadvanced in the US than in Europe. On the whole, enzymes produced by biotechnologyin the food and drink industry are used more extensively in the USA and manufacturersof food ingredients are generally more willing to use biotechnology to improve theirmanufacturing processes in the US when compared to Europe.

When we consider the development of the US and European markets for novel foodsand beverages, we can conclude that they are largely at a similar stage. In both regions itis clear that few such products have yet reached the market. This situation is likely tochange shortly however, as several new genetically engineered crops, with improvedprocessing characteristics, are being grown in the US and are now being exported toEurope. At the same time, approvals for genetically engineered crops to be grown inEurope is also being initiated. These products with improved processing characteristicswill be used by the US food and drink industry very shortly to enhance productivity inmanufacturing processes whereas Europe has yet to grow commercially any transgenicplants. In general, the way in which European companies are able to exploit thepotential of biotechnology in the food and drink sector, depends largely on consumerattitudes rather than their technology competencies. We should note that Europe’s smallfood and drink companies may face difficulties in the future as product innovation andimprovements in operating efficiency, which have been achieved through theapplication of biocatalysis, come to depend more and more on the application of suchtechnologies.

5.3.3 The textile sectorThe textile industry has widely and generally accepted the use of enzymes in itsprocesses, 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 $160million in 1996 (about 10% of the total market), and is estimated to grow byapproximately 50% before 2005.

In textile industries, technologies based on biocatalysts are already established. Thestone washing of jeans for instance, clearly offers an example of a Best AvailableTechnology (BAT). The process based on biocatalysts is both economically andtechnically competitive and brings environmental benefits. The enzyme markets are,however, at least to some extent dependent on the yearly changing fashion, andfluctuations in the market volumes may be caused by this. The consumers can beexpected to have increasingly positive attitudes towards textiles produced by greenermethods.


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5.3.4 The pulp and paper sectorThe large scale application of enzymes in the pulp and paper industry has grown fairlyslowly 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-effectsand are more suited to industrial processing conditions. Bleach-boosting usingxylanases and dewatering fibres using cellulases are the most important commercialapplications 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 thefairly slow adoption of new techniques in the sector. It is generally considered thatattitudes in the sector (and in many research organisations of the p&p sector too) areconservative.

The technical solutions based on biocatalysts must offer clear advantages over existingones. For the pulp and paper industry to accept biocatalysts, economic savings must bepossible. Biotechnology can offer competing technologies only in very limited cases.Thus, the adoption of biocatalysts as BAT cannot be generalised. Instead, biocatalystsmay be beneficial in certain mill-specific cases, e.g. when savings in new investmentscan be avoided (as in the case of bleach-boosting enzymes). The introduction of newbiocatalysts in unconventional areas, such as pulp and paper, requires experienced andcareful technical research from the biotech companies to find the optimum conditionsfor the use of biocatalysts.

5.4 Bottlenecks

There are several factors of the external business environment which influenceinvestment in the application of biotechnology (Ballantine and Thomas, 1997). Theseinclude:

• Market conditions: in particular, consumer attitudes, competitive intensity and thenature of demand. These conditions may encourage or inhibit the use ofbiotechnologys in companies and the entrepreneurial investment in specialisedventures.

• The regulations which cover the use of biotechnology and specific marketingapprovals also influence the scope of markets. The availability of experiencedentrepreneurial managers and high quality staff are also important.

• Entrepreneurship and particularly attitudes to entrepreneurship and risk-taking willinfluence development of biotechnology.

• Attitudes influence the level of enthusiasm for setting-up specialised companies andthe willingness of managers within large companies to explore new products andprocesses.

• The science base influences the entire innovation system and particularly thedevelopment of the specialised biotechnology sector.

• The availability of appropriate capital for specialised companies is a fundamentalprecondition for their growth. At the same time, fiscal policies, in particular taxregimes on capital gains, share options and tax credits for R&D affect the cost ofinvestment in research. A basic obstacle is also seen in the capital structure in


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Europe; the availability of risk capital from the stock market differs from that in theUS. Thus, there are too few start-ups with a strong financial basis.

• Finally, supplier factors are important to both specialised companies and also thepharmaceutical sector which is increasingly outsourcing R&D.

All of these factors identified in the recent EuropaBio study will have some influenceon the successful growth and development of biocatalysis in Europe across the differentsectors. The importance of these external factors to the biotechnology industry as awhole has been emphasised in several recent studies. Focusing on other areas ofbiotechnology 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 EastmanChemicals of the USA) and Gist Brocade dominate the market world-wide. However,European strengths, although impressive, are specific to be challenged in the first twoareas mentioned. The public acceptance of enzymes in food processing and especiallythe acceptance of the use of enzymes produced with genetically modified organisms,constitutes a very serious bottleneck for the diffusion of new technologies in a numberof EU member states. In 1994 Gist Brocades had marketing approval in the Netherlandsand France for their bacterial chymosine produced with rDNA technology. The Germangovernment denied marketing approval due to the lack of public acceptance. BecauseGermany is such an important market Gist Brocades did not sell in France and theNetherlands, fearing a German boycott of their products. In the meantime the producthas 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 drugcompounds and for the synthesis of precursors are increasingly likely to be produced byrecombinant micro-organisms. However it is unlikely that there will be a significantproblem in consumer attitudes in relation to the use of biocatalysis in healthcareproducts. Even in Europe, the general public has shown support for the use ofbiotechnology in the context of healthcare which obviously contrasts the attitudestowards the food and drink sector. The use of enzymes, therefore, in the pharmaceuticalindustry is largely related to economic efficiency and need. The market for enzymeshere 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 theEU member states varies widely. Whether legislation is seen as a hindrance or stimulusto developments depends on local factors and actors. The German ‘Reinheitsgebot’(purity laws) for beer prohibits the use of novel ingredients in the brewing process. Thisprompted the German beer industry to develop sophisticated brewing technologieswhich had a positive impact on developing new products and processes.

At the same time, Europe lacks specialised biotechnology companies which are workingon advanced areas of enzymatic technologies. Overall, the producers of industrialenzymes have, for some years, consisted of small numbers of large firms and severalsmaller, largely local, suppliers. However, strong pricing pressures have recently


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resulted in the consolidation of the industry with two of the larger firms recentlywithdrawing from the market. This has added to European strength.

Another bottleneck is the (still) high cost of many enzymes, their low efficacy in certaincases and their limited applicability. Investment costs in R&D necessary for thedevelopment of new applications of biocatalysts are high. This is not always understoodby the companies using them which may consider the applied development workcarried out by the enzyme producer companies insufficient. In some sectors, theapplying companies participate readily in the development costs of biocatalysts, inothers not. Although public basic research work is often carried out at universitiesfunded by national governments or at EU level, the additional development work whichneeds to be carried out by the companies on production strains, enzyme properties andapplications, including product approval, is substantial.

Literature

Abbott G. (ed.) (1996) Biotechnology Industry Study Report 1996. In: In Touch withIndustry: ICAF Industry Studies, Academic Year 1996. Industrial College of the ArmedForces National Defence University Washington, DC 20319 - 5062.

Ballantine, B and S.M. Thomas, (1997) Benchmarking the Competitiveness ofBiotechnology in Europe, report prepared for EuropaBio, Brussels.

Bickerstaff, G.F. (1995) REVIEW: Impact of Genetic Technology on EnzymeTechnology., The Genetic Engineer and Biotechnologist, Vol. 15, No. 1, JournalsOxford 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 astudy of Frost and Sullivan in the future market of industrial enzymes published inBiotechnologies Nieuws, a publication of the Dutch Industrial and Agro BiotechAssociation 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 271996, Bruxelles, Belgium.


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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 chiraland chemical intermediates used in the synthesis of pharmaceuticals and biochemicalsand thirdly, biocatalysis will be used to replace processes that require high energyinputs 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 thatare in current use in the biocatalysis industry. Secondly, the discovery of new enzymesas a result of genomic sequencing projects currently underway and proposed. A furtherdriver in the move to biocatalysis will be the importance of apparently ’natural’ productsthat are at the moment produced by very ’unnatural’ processes. For example, theproduction of margarine from oils requires high temperatures and inorganic catalystswhereas it is potentially possible to synthesise margarine’s from oils using enzymes tohave the same effect. As is noted in this report, biocatalysis comes into its own whencommodity chemicals or intermediates are considered and the cost of production is ofcrucial 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 ofthis field of research. We can observe that the traditional innovation trajectory isfollowed in order to develop improved enzymes, which already are used on a broadscale. But also more intelligent ways are found to mimic the most essential mechanismsof biocatalysis or find totally environments for biocatalysts to work in (in vivo and invitro 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 ofseveral enzymes is dependent on an RNA molecule. As RNA molecules can besynthesised and selected for on a vast scale (selection of one molecule in 100 million toperform a particular reaction is not uncommon) then such molecules have a potential tocatalyse a large number of reactions although none are yet in industrial use. Antibodiesoffer another way of performing novel catalytic reactions in that antibodies can beraised against transition intermediates. Antibodies stabilize the products of abiochemical reaction which avoids a backward reaction. As antibodies can be madeagainst a wide range of molecules then it is theoretically possible to producebiocatalytic molecules to run processes that cannot be done naturally.


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There are still a vast number of organisms that have not been exploited or screened fortheir ability to catalyse specific reactions and this, together with their ever increasingsize of genomic databases, stressed that there is a huge potential for novel biocatalyticenzymes. The limitation in exploiting these two areas is the lack of high throughputscreens commonly found in the biopharmaceutical industry but less so in the area ofbiocatalysis.

ExtremozymesFurther expansion is expected from the use of extremozymes, or enzymes which stemfrom micro-organisms capable of surviving in extreme environments: at pH’s,temperatures, pressures, ionoc and solvent environments long thought to be destructiveto biomolecules (Adams et. al., 1995). One specific area is the development ofthermophiles. Obviously of great interest, a new thermostable glucosedehydrogenase(GDH) has been isolated from a soil bacteria near a hot spring and has optimal activityof 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 somenovel properties.

Engineered enzymesGenetic engineering of enzymes holds considerable promise and in particular therecognition of proteins can often be divided into discrete domains and creates thepossibility that swapping these domains will create novel catalysts which changesubstrate or reaction characteristics. Indeed it would be possible to make catalysts thatinvolve composites between naturally occurring amino acids (in the form of proteins)and other organic reagents may be introduced by synthetic or recombinant methods. Thesemi-synthetic enzymes can be made by chemical modification with fatty acids orpolyethyleneglycol. One problem with the use of biocatalytic enzymes is the need forco-factors. However it has now been shown that enzymes can be re-engineered so as toutilise a different form of co-factor. For example, NADP rather than NAD. Subtilisin, aprotease from bacillus can now be engineered so that it can cut and splice specificproteins. Such activities will become extremely important and valuable in the area ofbiocatalysis.

RibozymesThe 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 commercialtechnology and clinical trials have been initiated involving gene therapy for AIDS.


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Microbial catalystsMore recent advances are related to carrying out a series of sequential organic chemicalreactions using a single microbial catalyst (Marshall and Woodley, 1996). This isachieved by fluctuating carbon sources for the microbial catalyst, triggering a completeor partial metabolic pathway to catalyse a sequential series of organic reactions. The useof microbial catalysts for mediating multiple organic reactions as part of a longerchemical synthesis may prove completely new routes to chemical products.

Changing chiralityOne of the main uses of enzymes in biocatalysis is to produce a single chiral from aracemic mixture. What would be of considerable use would be to select one form oranother from a mixture. This has recently been achieved and is likely to be of majorconsequence, not only for secondary alcohol dehydrogenases but for other enzymes aswell. The stereochemistry is temperature dependent, one form being favoured overanother depending on the temperature. Therefore use of enzymes with increasedstability may well result in the ability to purify different chiral forms from the samemixture. Developments will occur particularly in the production of generic compoundswhereby enzymes will be used in various solvents at various temperatures in order toincrease the yield of the desired product. From an intellectual property point of view itremains to be seen whether or not such processes and applications are patentable or willbe 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 futurebiocatalysis will be seen to move freely between the use of purified enzymes in thepharmaceutical industry through fermentation using either natural or modified bacteriaor yeasts into plants which have been genetically modified to produce either a particularbiocatalytic enzyme. For example, genetically engineered feed crops containing phytaseor plants that have been modified by the production of enzymes so that secondarymetabolites can be either produced in greater amounts or in altered properties. Thisform of ’molecular farming’ is well on the way to fruition.

What may be of particular importance is the ability to produce artificial membranes intransgenic plants. This allows the possibility that membrane proteins can beoverexpressed and furthermore the products of such reactions could be stored inindependent compartments of the plant cell. This can allow the production of otherwisetoxic compounds and furthermore may allow novel plant products to be processed inconventional ways through oil mills etc. Several laboratories are pioneering the conceptof engineering value added components into crop. These include increasing the amountof sulphur rich amino acids in transgenic alfalfa and subterranean clover with theintention of improving wool growth in grazing sheep.Another aspect is to incorporate enzymes so as to make the endosperm cell wall ofbarley easier to degrade during malting. This process involves incorporating a gene for athermostable glucanase.Encouraging progress has also been achieved in the production of biodegradableplastics in plants. The product quality and yield could be increased continuously. The


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still existing obstacle to market entry is the non-competitive price in comparison to oil-based plastics.

In vivo bio-productionBiocatalysis can lead to more efficient ways of producing raw material for productionprocesses. Similar to this, it is the goal of some companies to use plants as an in vivobio-factory for the production of specific molecules (eicosapentaenoic acid,docosahexaenoic acid and linolenic acid). It is thought that these compounds may beuseful in the prevention and treatment of heart disease, asthma, arthritis and possiblysome cancers. The synthetic pathways of these polyunsaturated fatty acids (PUFAs)have been identified in certain marine unicellular algae and some fungi. At present thelargest source is fish oil but this is relatively expensive and the product is subject tooxidation. It is therefore tempting to produce PUFAs in edible plant oils as an attractive(green, environmentally sensitive) and cost effective alternative. In vivo biocatalysiswill allow the production of custom made oils with different long chain fatty acids.

In vitro bio-productionAdditional to the in vivo bio-production described above cellulose has now beensynthesised in vitro using the enzyme cellulase. In vitro synthesis of cellulose will allowvariants of the ‘plant material’ to be produced. One area where in vitro bio-productionis likely to be of importance is in the synthesis of polymers. This can be accomplishedby the use of stereoselective lipase catalysed polymerisation reactions. Remarkably thelipase enzyme works well in toluene, the organic solvent. What is different from thechemical synthesis of these polymers is that they are synthesised as one stereoisomer. Itis 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 from6-8%. Recently horseradish peroxidase has been used to synthesise a fluorescentpolymer of 2-napthol. Perhaps one of the most exciting applications of futurebiocatalysis in the production of polymers is the synthesis of mosaic nucleic acidscomposed of 50% DNA and 50% RNA. These can be produced in milligram quantitiesand have obvious applications as pharmaceuticals and genotherapeutic agents. Directevolution is a real possibility of the development of biocatalysis whereby enzymes aresubject to random mutagenesis and screening.Although these results are impressive, they arise from a tremendous amount of hardwork and in future experiments are going to rely heavily upon genetics andbiochemistry.


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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, TIBTECHOctober 1997, Vol. 15, pp. 385-386.

Marshall, C.T. and J.M. Woodley (1996) Process Synthesis for Multiple-Step MicrobialConversions, BIO/TECHNOLOGY Vol. 13, October, pp. 1072-1078.

May, S.W. (1997) New applications of biocatalysis, Current Opinion in Biotechnology1997, 8, pp. 181 - 186.


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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 desiredreactions, due to their specificity, economical advantages or improved environmentalimpacts. Depending on the field of application, these reasons may vary. Thus, in thefield of food processing, biocatalysts have a history, thousands of years old, based onfirst hand empirical findings. Today, these methods represent clearly the best availabletechnologies.

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 preventingpollution. They are also highly specific, which means fewer unwanted side_effectsand 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 asmany synthetic reactions) can often be carried out most easily by biocatalysts. Targetedmedicines are often based on knowledge about enzymatic reactions. Environmentalsavings in the food and drinks sector will be related to minor process changes, andconsequently will be small in effect. However, there are interesting futureimplementations to be expected, for instance in the oil industry for the extraction ofvegetable oil from seeds by enzymes in water, replacing the traditional technology usinghexane (toxic and explosive). However, in both these industries, the minimisation ofenvironmental impacts is not the primary target, and has usually no role. Theenvironmental savings in the food and drinks and the feed sector is and will stay verylow 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, toimportant environmental savings.

The most important driving force for applying biocatalysts are economic benefits, insome cases in a win-win situation with environmental savings. However environmentalbenefits from the perspective of companies are, almost by definition, only attractive ifthey combine with low costs. Costs and higher added value are the main driving forcesin companies to use biocatalysts. For industry, greening is no driving force for usingbiocatalysts; greening is an output.


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Europe’s scientific-technological potential in biocatalysis

From a scientific and technological perspective, biocatalysis is an interdisciplinary fieldof research including basic and applied research. A considerable percentage ofbiocatalysis research is being performed in industry and difficult to trace. This makes itvery hard to make a clear cut assessment of the quantity and quality of its potential. Thegeneral perception, based on interviews with companies in Europe, is that the quality ofpublic sector research in biotechnology, including biocatalysis research, in Europe is asgood as in the USA. An indication of the strength of biocatalysis research in Europecompared with other regions on the basis of the productivity of papers reveals thatEurope 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 isweaker than in the USA.

Patent database analysis shows that in general European patents in all sectors is laggingbehind if compared with the US and Japan. However the analysis is no basis forconclusions on Europe’s scientific potential because patenting strategies and patentingcosts differ between these regions and what is also important to note: the keywordbiocatalysis is too general.

Economic potential of Europe’s biocatalysis industry

The economic potential of Europe’s enzyme producing industry is rather strong. NovoNordisk, a Danish company, is clearly world-wide the biggest, with about 50 % of thetotal market and is for all applications the number 1 supplier. Novo is followed byGenencor - half owned by the Cultor Company in Finland and half by Eastman Kodakin the USA. Genencor has less then 20% of the total market and ‘only’ producestechnical (non food grade) enzymes. Gist Brocades, based in the Netherlands is the thirdfollowed 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 verysmall and only produce one or two enzyme products.Despite the size of the enzyme market (more than ECU 500 million world-wide), theindustry itself is not very profitable. This is primarily because enzymes make up such asmall component of a product (e.g. enzymes in washing powder) and enzymes are oftenproduced by more than one manufacturer - patenting of enzymes has historically notbeen a major issue - , hence the competition is intense.

It is likely that in the future there will be considerable consolidation of the enzymecompanies in an attempt to achieve greater profitability. Food manufacturers, feedsuppliers and the generic pharmaceutical industry will attempt to vertically integratetheir production processes. This will result in the acquisition of a number ofindependent producers. The lack of profitability and relatively small sales ofindependent enzyme producers is largely due to the nature of catalytic reactionsthemselves, for example, the amount of enzyme added in a typical biocatalyticconversion is of the order of 0.5 kg/10 tonnes of material to be converted. As each


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enzyme molecule is capable of cycling with a turnover approaching 10,000 moleculesper second, then it is easy to see why such small amounts are required. In the case ofimmobilised enzymes (which is the way the industry is moving) enzyme input is not asignificant cost in the production process. It is expected that an annual growth rate of10% within the enzyme business is realistic within the next 3-5 years.

Fine chemical/ pharmaceutical industryIn the fine chemical/ pharmaceutical industry the impact of biocatalysis is explicitlyused in order to replace traditional, stoichiometric processes in order to improve theproduct/waste ratio. Another argument is the low entry barrier, i.e. low investments, fornew technologies in this small scale industry. About 10% of all drugs arebiotechnology-based and a small proportion of the remaining 90% uses enzymes for theseparation of chiral forms or the synthesis of drug precursors. Similarly, when usingenzymes for the synthesis of drug precursors, only about 6-7% of all drugs are involvedin this set of processes. This small group of drugs use a range of enzymes such aslipases to synthesise precursors to the final drug product. The introduction of enzymeshas become established in this process because they offer a more economic productionof precursors.

Food and drinks and animal feed industriesIn the food and drink industry the use of enzymes is fully integrated. However in theexploitation of the results of new biotechnique fruits which are mostly geneticallyengineered enzymes in this sector depend largely on consumers attitude rather than ontechnological competences. The animal feed market is the fastest growing market forenzymes, and the expectations are high for the future.

Textiles and pulp-and-paper industriesIn 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, enzymaticfibre modification and biofinishing of cellulosics are considered the most promisingnew applications.

The expected growth rate of biocatalysts in the pulp-and-paper is highly dependent onbreak-throughs within the field. According to inquiries made, the growth may be fast(when expecting successful R&D and commercialisation) multiplying the presentmarket figures by several decades within 10 years - or not. Especially in the past, therehave 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 costsfor developing new enzymes. However, the biocatalysts developed by the academia orenzyme companies must offer clear technical or economic improvements. The mostrelevant application areas are still bleaching, modification of fibres for e.g. improvingthe deinking process and applications of new oxidative enzymes.

BottlenecksThe potential consumer resistance to the use of enzymes that are essentially producedby biotechnology in food, drink and animal feeds may have the effect of limiting themarket growth. The public acceptance of using enzymes produced with genetically


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modified organisms, constitutes a very serious bottleneck for the diffusion of newtechnologies in a number of EU member states.The regulations which cover the use of biotechnology and specific marketing approvalsalso influence the scope of markets. The availability of experienced entrepreneurialmanagers and high quality staff are also important.

Barriers to Biocatalysis

Notwithstanding the high promises of the use of biocatalysts in industry forenvironmentally savings, the factual implementation is still rather limited. One veryimportant barrier are the sunk investments in existing processes. This is a generalproblem which is met by policymakers when they try to introduce clean technologies inmature industries. In these industries far reaching optimisation of existing processes isthe main cost-saving driving force for innovation.

Another barrier are the other competing technologies. However, does biocatalysisalready have such a fixed position in the setting of competing technologies that a faircompetition can be made? In this respect it can be observed that not only sunkinvestments, but sunk experiences and cultures can make it more difficult to integratenew ‘scientific cultures’, like the biological sciences, into the existing culture inindustry4. It must be concluded that the integration of modern biotechnologies not onlymeets difficulties by the public, but for other reasons, it also seems a not veryapproachable and attractive issue to policymakers in companies and government5.Nevertheless there are companies where barriers between chemical and biologicalcultures 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 avery 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 isgreen, Bio should, because Bio is good” is not correct. However it is quite clear thatbiocatalysis has a number of advantages for environmental savings and that biocatalysisshould be in an equal position to other technologies in the race for the most competitivetechnology. As we concluded, this position has not been reached yet and this legitimatesorganisations including government bodies, who give a high priority to environmentalissues, 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 inbars. Biotechnology has a soft image and it has no such gadgets as mobile telephones, powerfullcomputers, etc. Also in the Advisory Board of this project, it was acknowledged by one of the membersfrom industry that knowledge and perception of a technology play a major role in the uptake of newtechnologies.

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 innovativeresearch on the borderline of biocatalysis and organic-chemical synthesis (Chemisch Weekblad, 1 Maart1997). ‘Better be bio’ is the slogan for DSM, a Dutch chemical company that already practicedbiocatalysis, but now also starts to work on fermentation (Chemisch Weekblad, 11 Oktober 1997).


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Recommendations

In order to develop this stimulus, one should bear in mind that the decisions taken inindustries as to which technologies should be used are based on the knowledge ofexisting 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 accessibledatabase with the economic and environmental benefits of biocatalysts in specificapplications.

2. Stimulate innovative entrepreneurs inside and outside companies to build uppractices with these new process integrated biotechnologies and use these asillustration projects for other companies.

3. Stimulate the development and use of methods for the evaluation of theenvironmental costs/benefits of competing (bio)technologies from an economicalperspective.

4. In those cases were the cost/benefits evaluation shows that from an environmentalperspective a specific technology, i.e. biocatalysis, is more favourable than othertechnologies, regulation can be tuned into direct the economic driving forces togreen production processes.

Regulations promoting the industrial application of biocatalysts must be clearly basedon the acknowledged facts on the properties of biocatalysts and their customary use.Regulations should however, not unfavourably prohibit or hamper the use ofbiocatalysts. At present this trend may be foreseen, especially in the food and feedsectors.

One last recommendation is based on the fact that the enzyme producing companiesconsider their participation difficult in public, e.g. EU projects due to confidentialityaspects, and due to participation of their competitors in the projects. This is especiallyproblematic in applied areas as in most biocatalysis research.

However, it is seen desirable to support more applied oriented work within the sector. Itis generally considered that the applying industry should be more efficiently informedabout the projects, and consequently be more involved in the development phase. It isalso considered of utmost importance to promote further the co-operation of industryand research institutes. Within research programmes, however, the fundamental andapplied research should be more clearly separated.

Documents

Biocatalysis: State of the Art in Europe