6.54.2.10 INDUSTRIAL USE OF ENZYMES

Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen

Laboratory of Bioprocess Engineering, Helsinki University of Technology, Finland, and

Hans Schoemaker, DSM Research, MD Geleen, The Netherlands

Keywords: Industrial enzymes, speciality enzymes, protein engineering, enzyme technology, enzyme production, biocatalysis, fine chemicals

Contents

1. Historical background2. Enzyme classification 3. Enzyme production

3.1. Microbial production strains3.2. Enzyme production by microbial fermentation

4. Protein engineering5. Enzyme technology6. Large scale enzyme applications

6.1. Detergents6.2. Starch6.3. Drinks6.4. Textiles6.5. Animal feed6.6. Baking6.7. Pulp and paper6.8. Leather

7. Speciality enzymes7.1. Enzymes in analytics 7.2. Enzymes in personal care products7.3. Enzymes in DNA-technology

8. Enzymes in fine chemical production8.1. Chirally pure amino acids and aspartame8.2. Rare sugars8.3. Semi synthetic penicillins8.4. Lipase based reactions8.5. Asymmetric synthesis8.6. Enzymatic oligosaccharide synthesis

9. Future trends in industrial enzymology

Glossary

Alkaline phosphatase: An enzyme that degrades ester bonds in alkaline conditions.Amino acid amidase: An enzyme that is used in manufacturing optically pure amino acids.

It hydrolyses an amide bond in natural amino acid amides.Amylase: A group of enzymes that hydrolyse chemical bonds between glucose

molecules present in starch. This group includes alpha-, beta- and glucoamylase.

Aspartame: A low calorie high intensive sweetener.Beta-glucanase: An enzyme that degrades beta-glucan commonly found e.g. in barley.Biocatalyst: Isolated enzyme or a whole cell (living or dead)Bromelain: A protein-degrading enzyme from plants.Catalase: An enzyme that degrades hydrogen peroxide to oxygen and water.Cellulases: A group of enzymes that synergistically degrade cellulose fibers to

glucose.CLEC: Enzyme crystal that has been made insoluble by chemical cross-

linking; a method to immobilise and stabilise enzymes.Chirally pure: Many organic molecules can have two chemically identical but

structurally mirror image forms. Chirally pure means that only one of the forms is present.

Dextran sucrase: An enzyme, present in some lactic acid bacteria, that forms a glucose. polymer and fructose from the disaccharide sucrose.

Dextran: Glucose containing branched polymer used e.g. in blood replacements.

DNA-polymerases: An enzyme that synthesizes DNA polymers. Fermentor: A biological reactor for cultivation of microorganisms.Ficin: A protein-degrading enzyme from plants.Formate dehydrogenase: An enzyme that oxidises formate to carbon dioxide and NAD.Glucoamylase: An enzyme that splits glucose molecules from starch.Glucose oxidase: An enzyme that uses oxygen to oxidise glucose to gluconic acid and

hydrogen peroxide.Glycosyltransferases: Catalyse the transfer of monosaccharides from a donor to saccharide

acceptors.GRAS-status: is given to an organism that is Generally Regarded as Safe.Hydrolases: Enzymes that break chemical bonds by adding water. They can be

used to form chemical bonds in the absence of water.Hydroxynitrile lyase: An enzyme that catalyses the addition of HCN to aldehydes and

ketones.Immunoassay: This is an analytical method in which antibodies are used to detect

specific molecules.Isomerases: Enzymes that catalyse intramolecular reactions.Laccase: A polyphenol oxidase from fungi. This enzyme can use oxygen to

oxidise different types of aromatic molecules and to form lignin type of aromatic polymers from phenolic compounds.

Lactase: his enzymes degrade milk-sugar lactose to glucose and galactose. Lactose intolerant people can consume such milk.

Ligases: Enzymes that synthesize chemical bonds.Lipoxygenase: A lipid oxidising enzyme extracted usually from soybeans.Lyases: Enzymes that remove chemical groups from their substrates without

addition of waterNitrile hydratases: Enzymes that catalyse addition of water to nitrales resulting in amide

formation

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Oxidoreductases: Enzymes that oxidise or reduce chemical compounds.Papain: A protein degrading enzyme from animal gut.Penicillin: An antibiotic substance extracted from molds.Pepsin: An enzyme that degrades proteins and is isolated from animals.Peroxidase: An oxidative heme-containing enzyme that uses hydrogen peroxide to

oxidise aromatic compounds. It is responsible for lignin biosynthesis in plants and initiates lignin biodegradation by certain rot-fungi.

Phytase: A phosphatase enzyme that hydrolyses phosphoester bonds in phytic acid. Is widely used in animal feeds.

Protein engineering: Improvement of enzyme protein by genetic methods.Rare sugar: A sugar that is rare in nature.Rennin: An aspartic protease which coagulates milk protein. It is used in

cheese manufacturing and isolated from calf stomach or produced by recombinant fungi.

Restriction enzymes: Enzymes that recognise specific 4-8 nucleotides long sequencies from DNA. They are important tools in gene technology.

Transferases: Trypsin: An enzyme that degrades proteins and is isolated from animals. Xylanase: A group of enzymes that degrade plant fibers made of xylose-sugars

to xylose monomers.Xylitol: A tooth-friendly sugar alcohol used in chewing gums.

Summary

Enzymes have been used since the dawn of mankind in cheese manufacturing and indirectly via yeasts and bacteria in food manufacturing. Isolated enzymes were first used in detergents in the year 1914, their protein nature proven in 1926 and their large-scale microbial production started in 1960s. Industrial enzyme business is steadily growing due to improved production technologies, engineered enzyme properties and new application fields. The major part of enzymes is produced by with GRAS-status microorganisms in large biological reactors called fermentors. Usually the production organism and often also the individual enzyme have been genetically engineered for maximal productivity and optimised enzyme properties. Large volume industrial enzymes are usually not purified but sold as concentrated liquids or granulated dry products. Enzymes used in special applications like diagnostics or DNA-technology need to be highly purified. Isolated enzymes have found several applications in fine chemical industry. Enzymes are used in production of chirally pure amino acids and rare sugars. They are also used in production of fructose and penicillin derivatives as well as several other chemicals. Enzymes should be considered as a part of a rapidly growing biocatalyst industry also involving genetically optimised living cells as chemical production factories.

1. Historical background

Most of the reactions in living organisms are catalysed by protein molecules called enzymes. Enzymes can rightly be called the catalytic machinery of living systems. Man has indirectly used enzymes almost since the beginning of human history. Enzymes are responsible for the biocatalytic fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing. Enzymes oxidise ethanol to acetic acid. This reaction has been used in vinegar production for thousands of years. Similar microbial enzyme reactions of acid forming bacteria and

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yeasts are responsible for aroma forming activities in bread making and in preserving activities in sauerkraut preparation.

The fermentative activity of microorganisms was discovered only in 18 th century and finally proved by the French scientist Louis Pasteur. The term “enzyme” comes from Latin words, which literally mean “in yeast”. This name was given since enzymes where closely associated with yeast activity. The study of enzymes is a fairly recent activity. Scientists who found out that an alcohol precipitate of malt extract contained a thermo labile substance, which converted starch into sugar, made the first clear recognition of enzymes in 1833. They called the substance diastase. We know now that it was an enzyme nowadays called amylase. Sumner finally proved the protein nature of enzymes in 1926 when he was able to crystallize urease enzyme from jack bean.

Probably the first application of cell free enzymes was the use of rennin isolated from calf or lamb stomach in cheese making. Rennin is an aspartic protease (see Mechanisms of Enzyme Action) which coagulates milk protein and has been used for hundreds of years by cheese makers. Röhm in Germany prepared the first commercial enzyme preparation in 1914. This trypsin enzyme isolated from animals degraded proteins and was used as a detergent. It proved to be so powerful compared to traditional washing powders that the original small package size made the German housewives suspicious so that the product had to be reformulated and sold in larger packages. The real breakthrough of enzymes occurred with the introduction of microbial proteases into washing powders. The first commercial bacterial Bacillus protease was marketed in 1959 and became big business when Novozymes in Denmark started to manufacture it and major detergent manufactures started to use it around 1965.

In food industry - in addition to cheese manufacturing - enzymes were used already in 1930 in fruit juice manufacturing. These enzymes clarify the juice. They are called pectinases, which contain numerous different enzyme activities. The major usage of microbial enzymes in food industry started in 1960s in starch industry. The traditional acid hydrolysis of starch was completely replaced by alpha-amylases and glucoamylases, which could convert starch with over 95%, yield to glucose. Starch industry became the second largest user of enzymes after detergent industry.

Presently the industrial enzyme companies sell enzymes for a wide variety of applications. The estimated value of world enzyme market is presently about US $ 1.3 billion and it has been forecasted to grow to almost US $ 2 billion by 2005. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially, produced enzymes. Enzymes are also indirectly used in biocatalytic processes involving living or dead and permeabilised microorganisms. This review concentrates on the use of isolated enzyme preparations in large scale and speciality applications and chemical manufacturing. The use of microorganisms as biocatalysts in chemical production is, however, an interesting and growing field. The techniques of genetic, protein and pathway engineering are making chemical production by living cells an interesting green alternative to replace traditional chemical processes.

2. Enzyme classification

Presently more than 2000 different enzyme activities have been isolated and characterized [see Enzymology; Concept and Scope of Enzyme Action]. The sequence information of a growing number of organisms opens the possibility to characterise all the enzymes of an organism on a genomic level. The smallest known organism, Mycoplasma genitalium, contains 470 genes of which 145 are related to gene replication and transcription. Baker’s yeast has 7000 genes coding for about 3000 enzymes. Thousands of different variants of the natural enzymes are known. The number of reported 3-dimensional enzyme structures is rapidly increasing. In the year 2000 the

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structure of about 1300 different proteins were known. The enzymes are classified into six major categories based on the nature of the chemical reaction they catalyse:

1. Oxidoreductases catalyse oxidation or reduction of their substrates2. Transferases catalyse group transfer3. Hydrolases catalyse bond breakage with the addition of water4. Lyases remove groups from their substrates5. Isomerases catalyse intramolecular rearrangements6. Ligases catalyse the joining of two molecules at the expense of chemical energy

Only a limited number of all the known enzymes are commercially available and even smaller amount is used in large quantities. More than 75% of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40% of all enzyme sales. Proteinases have found new applications but their use in detergents is the major market. More than fifty commercial industrial enzymes are available and their number increases steadily.

3. Enzyme production

Some enzymes are still extracted from animal or plant tissues. Plant derived commercial enzymes include proteolytic enzymes papain, bromelain and ficin and some other speciality enzymes like lipoxygenase from soybeans. Animal derived enzymes include proteinases like pepsin and rennin. Most of the enzymes are, however, produced by microorganisms in submerged cultures in large reactors called fermentors. The enzyme production process can be divided into following phases:

1. Selection of an enzyme2. Selection of a production strain3. Construction of an overproducing strain by genetic engineering4. Optimisation of culture medium and production conditions5. Optimisation of recovery process (and purification if needed)6. Formulation of a stable enzyme product

Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH and temperature optima and stability, effect of inhibitors and affinity to substrates. Enzymes used in paper industry should not contain cellulose-degrading activity as a side activity because this activity would damage the cellulose fibres. Enzymes used in animal feed industry must be thermo tolerant to survive in the hot extrusion process used in animal feed manufacturing. The same enzymes must have maximal activity at the body temperature of the animal. Enzymes used in industrial applications must usually be tolerant against various heavy metals and have no need for cofactors. They should be maximally active already in the presence of low substrate concentration so that the desired reaction proceeds to completion in a realistic time frame.

3.1. Microbial production strains

In choosing the production strain several aspects have to be considered. Ideally the enzyme is secreted from the cell. This makes the recovery and purification process much simpler compared to production of intracellular enzymes, which must be purified from thousands of different cell proteins and other components. Secondly, the production host should have a GRAS-status, which means that it is Generally Regarded As Safe. This is especially important when the enzyme produced by the organism is used in food processes. Thirdly, the organism should be able to produce high amount of the desired enzyme in a reasonable time frame. The industrial strains typically produce over 50-g/l extracellular enzyme proteins. Most of the industrial enzymes are

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produced by a relatively few microbial hosts like Aspergillus and Trichoderma fungi, Streptomyces fungi imperfecti and Bacillus bacteria. Yeasts are not good produces of extracellular enzymes and are rarely used for this purpose. Most of the industrially used microorganisms have been genetically modified to overproduce the desired activity and not to produce undesired side activities.

3.2. Enzyme production by microbial fermentation

Once the biological production organism has been genetically engineered to overproduce the desired products, a production process has to be developed. The optimisation of a fermentation process includes media composition, cultivation type and process conditions. This is a demanding task and often involves as much effort as the intracellular engineering of the cell. The bioprocess engineer asks questions like: is the organism in question safe or are extra precautions needed, what kind of nutrients the organism needs and what is their optimal/ economical concentration, how the nutrients should be sterilised, what kind of a reactor is needed (mass transfer, aeration, cooling, foam control, sampling), what needs to be measured and how is the process controlled, how is the organism cultivated (batch, fed-batch or continuous cultivation), what are the optimal growth conditions, what is the specific growth and product formation rate, what is the yield and volumetric productivity, how to maximise cell concentration in the reactor, is the product secreted out from the cells, how to degrade the cell if the product is intracellular, does some of the raw materials or products inhibit the organism and finally, how to recover, purify and preserve the product. A typical enzyme production scheme is shown in Figure 1.

The large volume industrial enzymes are produced in 50 – 500 m3 fermentors. The extracellular enzymes are often recovered after cell removal (by vacuum drum filtration, separators or microfiltration) by ultrafiltration. If needed the purification is carried out by ion exchange or gel filtration. The final product is either a concentrated liquid with necessary preservatives like salts or polyols or alternatively granulated to a non-dusty dry product. Enzymes are proteins, which like any protein can cause and have caused in the past allergic reactions. Therefore protective measures are necessary in their production and application.

4. Protein engineering

Often enzymes do not have the desired properties for an industrial application. One can in such a case try to find a better enzyme from nature. Extensive search for new enzyme variants in organisms that grow in extreme conditions has been going on for more than 20 years but has resulted in relatively few successes. Sometimes a desired property, like extreme thermo stability, has been found but other problems have surfaced. The enzyme may not be functional in the desired temperature. It may also prove very difficult to overproduce the enzyme in a suitable host. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. Two different approaches are presently available: a random method called directed evolution and a protein engineering method called rational design. Table 1 summarises some of the reasons why industrial enzymes need to be modified and table 2 describes some of the required tools used in modification work.

Several enzymes have already been engineered to function better in industrial processes. These include proteinases, lipases, cellulases, -amylases and glucoamylases. Xylanase is a good example of an industrial enzyme, which needs to be stable in high temperature and active in physiological temperatures and pHs when used as feed additive and in alkaline conditions when it is used in bleaching in pulp and paper industry. One of the industrial production organisms of xylanases is Trichoderma fungus. Its xylanase has been purified and crystallized. By designed mutagenesis its thermal stability has been increased about 2000 times at 70 oC and its pH-optimum shifted towards

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alkaline region by one pH-unit. The three dimensional structure of a xylanase enzyme is shown in Figure 2. The known structure of an enzyme is used to design and simulate mutations. The most successful strategies to improve the stability of the Trichoderma xylanase include the stabilization of the alpha-helix region and the N-terminus.

5. Enzyme technology

How are the enzymes used and applied in practical processes? This is the field of enzyme technology. The simplest way to use enzymes is to add them into a process stream where they catalyse the desired reaction and are gradually inactivated during the process. This happens in many bulk enzyme applications like liquefaction of starch with amylases, bleaching of cellulose pulp with xylanases or use of enzymes in animal feed. In these applications the price of the enzymes must be low to make their use economical. Extracellularly produced bulk enzyme concentrates cost only US $ 10-20/ kg protein.

An alternative way to use enzymes is to immobilise them so that they can be reused. The largest application of an immobilised enzyme is the conversion of glucose syrup to high fructose syrup for food applications. In the early applications the glucose isomerase enzyme containing cells were permeabilised and immobilized on a solid support. The enzyme containing support material was packed into a column through which the glucose solution was passed. Finnish Sugar Company developed in early 80s an alternative method where the intracellular glucose isomerase from Streptomyces rubiginosus was purified by crystallization and the pure enzyme was bound to an anion exchange resin, which can be regenerated with fresh enzyme after the previous one is inactivated. Another way to immobilise enzymes is to use ultrafiltration membranes in the reactor system. The large enzyme molecules cannot pass the membrane but the small molecular reaction products can. Therefore enzymes are retained in a reaction system and the products leave the system continuously. This method has been used in production of chirally pure amino acids from racemic mixtures of amino acid derivatives. Enzymes have also been immobilized on membranes for analytical purposes. The best-known example is glucose oxidase, which is used to measure glucose concentrations in biological samples. Many different laboratory methods for enzyme immobilization based on chemical reaction, entrapment, specific binding or absorption have been developed.

A novel approach to use enzymes was introduced by Finnish Sugar Company in the late 80s. It was based on the use of cross-linked crystalline glucose isomerase (Figure 3). Enzyme crystals contain usually 30-80% free water and the enzyme is active even in the cross-linked insoluble form. The dimensions of an enzyme reactor, packed with this kind of a material, are considerably smaller compared to traditional immobilized systems because the carrier matrix can be completely omitted. The concept, originally developed in Finland, was later applied to other enzymes by Altus Ltd in USA, which has developed novel applications for the CLECs, which is the trademark for Cross-Linked Enzyme Crystals. These applications include chiral separations, controlled release of chemicals, specific separations and recently even cofactor entrapment into the crystal structure. All this is possible because an enzyme crystal contains water, pores, active centre, hydrophobic areas and ionic properties.

6. Large scale enzyme applications

Table 3 summarises major large-scale enzyme applications. Each of them is discussed in the text in some detail. Industrial Enzymology is recommended as a good resource text for those who need a more comprehensive treatment of an individual subject.

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6.1. Detergents

Detergents were the first large scale application for microbial enzymes. Bacterial proteinases are still the most important detergent enzymes. Some products have been genetically engineered to be more stable in the hostile environment of washing machines with several different chemicals present. These hostile agents include anionic detergents, oxidising agents and high pH.

Late 80s lipid degrading enzymes were introduced in powder and liquid detergents. Lipases decompose fats into more water-soluble compounds by hydrolysing the ester bonds between the glycerol backbone and fatty acid. The most important lipase in the market was originally obtained from Humicola lanuginose. It is produced in large scale by Aspergillus oryzae host after cloning the Humicola gene into this organism.

Amylases are used in detergents to remove starch based stains. Amylases hydrolyse gelatinised starch, which tends to stick on textile fibres and bind other stain components. Cellulases have been part of detergents since early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose. In textile washing cellulases remove cellulose microfibrils, which are formed during washing and the use of cotton based cloths. This can be seen as colour brightening and softening of the material. Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.

6.2. Starch hydrolysis and fructose production

The use of starch degrading enzymes was the first large-scale application of microbial enzymes in food industry. Mainly two enzymes carry out conversion of starch to glucose: alpha-amylase cuts rapidly the large alpha-1,4-linked glucose polymers into shorter oligomers in high temperature. This phase is called liquefaction and is carried out by bacterial enzymes. In the next phase called saccharification, glucoamylase hydrolyses the oligomers into glucose. This is done by fungal enzymes, which operate in lower pH and temperature than alpha-amylase. Sometimes additional debranching enzymes like pullulanase are added to improve the glucose yield. Beta-amylase is commercially produced from barley grains and used for the production of the disaccharide maltose.

In the United States large volumes of glucose syrups are converted by glucose isomerase after Ca2+

(alpha-amylase needs Ca2+ for activity but it inhibits glucose isomerase) removal to fructose containing syrup. This is done by bacterial enzymes, which need Mg2+ ions for activity. Fructose is separated from glucose by large-scale chromatographic separation and crystallized. Alternatively, fructose is concentrated to 55% and used as a high fructose corn syrup in soft drink industry.

An alternative method to produce fructose is shown in Figure 4. This method is used in Europe and uses sucrose as a starting material. Sucrose is split by invertase into glucose and fructose, fructose separated and crystallized and then the glucose circulated back to the process.

6.3. Drinks

Enzymes have many applications in drink industry. The use of chymosin in cheese making to coagulate milk protein was already discussed. Another enzyme used in milk industry is beta-galactosidase or lactase, which splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers.

Enzymes are used also in fruit juice manufacturing. Fruit cell wall needs to be broken down to improve juice liberation. Pectins are polymeric substances in fruit lamella and cell walls. They are

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closely related to polysaccharides. The cell wall contains also hemicelluloses and cellulose. Addition of pectinase, xylanase and cellulase improve the liberation of the juice from the pulp. Pectinases and amylases are used in juice clarification.

Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes are liberated and they hydrolyse the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. Additional enzymes can be used to help the starch hydrolysis (typically alpha-amylases), solve filtration problems caused by beta-glucans present in malt (beta-glucanases), hydrolyse proteins (neutral proteinase), and control haze during maturation, filtration and storage (papain, alpha-amylase and beta-glucanase).

Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the yield. Enzymes hydrolyse the high molecular weight substances like pectin.

6.4. Textiles

The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology. Starch has for a long time been used as a protective glue of fibres in weaving of fabrics. This is called sizing. Enzymes are used to remove the starch in a process called desizing. Amylases are used in this process since they do not harm the textile fibres.

Enzymes have replaced the use of volcanic lava stones in the preparation of Denim (special soft cotton based fibre where the dye has been partially faded away) from an indigo-dyed cotton fibre to achieve a high degree of dye fading. The stones caused considerable damage to fibres and machines. The same effect can be obtained with cellulase enzymes. The effect is a result of alternating cycles of desizing and bleaching enzymes and chemicals in washing machines.

Recently, hydrogen peroxides have been tested as bleaching agents to replace chlorine-based chemicals. Catalase enzyme, which destroys hydrogen peroxide, may then be used to degrade excess peroxide. Another recent approach is to use oxidative enzymes directly to bleach textiles. Laccase – a polyphenol oxidase from fungi - is a new candidate in this field.Laccases are produced by white-rot fungi, which use them to degrade lignin - the aromatic polymer found in all plant materials. Laccase is a copper-containing enzyme, which is oxidised by oxygen, and which in an oxidised state can oxidatively degrade many different types of molecules like dye pigments.

Other enzymes, which interact with textiles, are often added to washing powders. These examples were discussed under detergent enzymes.

6.5. Animal feed

Intensive study to use enzymes in animal feed started in early 80s. The first commercial success was addition of beta-glucanase into barley based feed diets. Barley contains beta-glucan, which causes high viscosity in the chicken gut. The net effect of enzyme usage in feed has been increased animal weight gain with the same amount of barley resulting in increased feed conversion ratio. Finnfeeds International was the pioneer in animal feed enzymes.

Enzymes were tested later also in wheat-based diets. Xylanase enzymes were found to be the most effective ones in this case. Addition of xylanase to wheat-based broiler feed has increased the available metabolizable energy 7-10% in various studies. Xylanases are nowadays routinely used in feed formulations. Figure 2 shows the three-dimensional structure of a Trichoderma xylanase.

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Usually a feed-enzyme preparation is a multienzyme cocktail containing glucanases, xylanases, proteinases and amylases. Enzyme addition reduces viscosity, which increases absorbtion of nutrients, liberatates nutrients either by hydrolysis of non-degradable fibres or by liberating nutrients blocked by these fibres, and reduces the amount of faeces.

Another type of important feed enzyme is phytase marketed e.g. by DSM in the Netherlands. Phytase is a phosphoesterase which liberates phosphate from phytic acid which is a common compound in plant based feed materials. The net effect is reduced phosphorous in faeces resulting in reduced environmental pollution. The use of phytase reduces the need to add phosphorus to the feed diet.

Enzymes have become an important aspect of animal feed industry. In addition to poultry, enzymes are used in pig feeds and turkey feeds. They are added as enzyme premixes (enzyme-flour mixture) during the feed manufacturing process, which involves extrusion of wet feed mass in high temperature (80-90 OC). Therefore the feed enzymes need to be thermo tolerant during the feed manufacturing and operative in the animal body temperature.

6.6. Baking

Similar fibre materials are used in baking than in animal feed. It is therefore conceivable that enzymes also affect the baking process. Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. Both fungal and bacterial amylases are used. Over dosage may lead to sticky dough so the added amount needs to be carefully controlled.

One of the motivations to study the effect of enzymes on dough and bread qualities comes from the pressure to reduce other additives. In addition to starch, flour typically contains minor amounts of cellulose, glucans and hemicelluloses like arabinoxylan and arabinogalactan. There is evidence that the use of xylanases decreases the water absorption and thus reduces the amount of added water needed in baking. This leads to more stable dough. Especially xylanases are used in whole meal rye baking and dry crisps common in Scandinavia.

Proteinases can be added to improve dough-handling properties; glucose oxidase has been used to replace chemical oxidants and lipases to strengthen gluten, which leads to more stable dough and better bread quality.

6.7. Pulp and Paper

Intensive studies have been carried out during the last twenty years to apply many different enzymes in pulp and paper industry. A real excitement started with the discovery of lignin degrading peroxidases in the early 80s. In spite of extensive research no oxidative enzymes are applied in pulp and paper industry. The major application is the use of xylanases in pulp bleaching. Xylanases liberate lignin fragments by hydrolysing residual xylan. This reduces considerably the need for chlorine based bleaching chemicals. Other minor enzyme applications in pulp production include the use of enzymes to remove fine particles from pulp. This facilitates water removal.

In the use of secondary (recycled) cellulose fibre the removal of ink is important. The fibre is diluted to 1% concentration with water, flocculating surfactants and ink solvents added and the mixture is aerated. The ink particles float to the surface. There are reports that this process is facilitated by addition of cellulase enzymes.

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In paper making enzymes are used especially in modification of starch, which is used as an important additive. Starch improves the strength, stiffness and erasability of paper. The starch suspension must have a certain viscosity, which is achieved by adding amylase enzymes in a controlled process.

Pitch is a sticky substance present mainly in softwoods. It is composed of lipids. It is a special problem when mechanical pulps of red pine are used as a raw material. Pitch causes problems in paper machines and can be removed by lipases.

6.8. Leather

Leather industry uses proteolytic and lipolytic enzymes in leather processing. The use of these enzymes is associated with the structure of animal skin as a raw material. Enzymes are used to remove unwanted parts. Alkaline proteases are added in the soaking phase. This improves water uptake by the dry skins, removal and degradation of protein, dirt and fats and reduces the processing time. In some cases pancreatic trypsin is also used in this phase.

In dehairing and dewooling phases enzymes are used to assist the alkaline chemical process. This results in a more environmentally friendly process and improves the quality of the leather (cleaner and stronger surface, softer leather, less spots). The used enzymes are typically alkaline bacterial proteases. Lipases are used in this phase or in bating phase to specifically remove grease. The use of lipases is a fairly new development in leather industry.

The next phase is bating which aims at deliming and deswelling of collagen. In this phase the protein is partly degraded to make the leather soft and easier to dye. Pancreatic trypsins were originally used but they are being partly replaced by bacterial and fungal enzymes.

7. Speciality enzymes

In addition to large volume enzyme applications, there are a large number of speciality applications for enzymes. These include use of enzymes in analytical applications, flavour production, protein modification, and personal care products, DNA-technology and in fine chemical production. The latter application will be separately discussed because of its importance. Here we discuss the other aspects of speciality enzymes.

7.1. Enzymes in analytics

Enzymes are widely used in the clinical analytical methodology. Contrary to bulk industrial enzymes these enzymes need to be free from side activities. This means that elaborate purification processes are needed. Table 4 summarises some of the main analytes measured enzymatically. Normally automatic analysers carry out these measurements. The reactions normally involve either changes in NAD(P)/NAD(P)H proportions, which can be detected spectrophotometrically or production of H2O2 which can be detected in peroxidase catalysed reactions leading to coloured products, which can be easily quantified spectrophotometrically.

Immunoassays are based on detection of target molecules by specific antibodies. The detection of the antibody-antigen complex is usually based on enzymes linked to the antibodies. This enzyme is either an alkaline phosphatase, which can be detected in colour forming reaction by p-nitrophenyl phosphate or peroxidase, which is detected in the presence of H2O2 with a colour forming substrate.

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An important development in analytical chemistry is biosensors. They are based on H2O2 producing oxidative enzymes. Two different types of electrodes, one based on peroxide detection and the other based on oxygen consumption, can be used to quantify the analyte in question. The most widely used application is a glucose biosensor involving glucose oxidase catalysed reaction:

glucose + O2 + H2O gluconic acid + H2O2

Several commercial instruments are available which apply this principle for measurement ofmolecules like glucose, lactate, lactose, sucrose, ethanol, methanol, cholesterol and some amino acids.

7.2. Enzymes in personal care products

Personal care products are a relatively new area for enzymes and the amounts used are small but worth to mention as a future growth area. One application is contact lens cleaning. Proteinase and lipase containing enzyme solutions are used for this purpose. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by a heme containing catalase enzyme, which degrades hydrogen peroxide.

Some toothpaste contains glucoamylase and glucose oxidase. The reasoning behind this practise is that glucoamylase liberates glucose from starch-based oligomers produced by alpha-amylase and glucose oxidase converts glucose to gluconic acid and hydrogen peroxide which both function as disinfectants.

Dentures can be cleaned with protein degrading enzyme solutions. Enzymes are studied also for applications in skin and hair care products.

7.3. Enzymes in DNA-technology

DNA-technology has revolutionised both traditional biotechnology and opened totally new fields for scientific study. It is also an important tool in enzyme industry. Most traditional enzymes are produced by organisms, which have been genetically modified to overproduce the desired enzyme. Recombinant DNA-technology allows one to produce new enzymes in traditional overproducing and safe organisms. Protein engineering is used to modify and improve existing enzymes as discussed under Protein engineering. Enzymes are the tools needed in genetic engineering and are shortly discussed here. For more information the reader is referred to specific texts dealing with genetic engineering.

DNA is basically a long chain of deoxyribose sugars linked together by phosphodiester bonds. Organic bases, adenine, thymine, guanine and cytosine are linked to the sugars and form the alphabet of genes. The specific order of the organic bases in the chain constitutes the genetic language. Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process. The DNA modifying enzymes can be divided into two classes:

1. Restriction enzymes recognise specific DNA sequences and cut the chain at these recognition sites.2. DNA modifying enzymes synthesize nucleic acids, degrade them, join pieces together and remove parts of the DNA.

Restriction enzymes recognise a specific code sequence in the DNA. This is usually 4-8 nucleotides long sequence. Their role in nature is to cut foreign DNA material. These enzymes do not cut the cell’s own DNA because its recognition sites are protected. More than 150 different restriction

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enzymes have been isolated from several bacterial species and they are used in cutting the DNA in question at specific points. These enzymes are essential in gene technology.

DNA-polymerases synthesize new DNA-chains. Many of them need a model template, which they copy. Nucleases hydrolyse the phosphodiester bonds between DNA sugars. Kinases add phosphate groups and phosphatases remove them from the end of DNA chain. Ligases join adjacent nucleotides together by forming fosfodiester bonds between them.

In the cell these enzymes are involved in DNA replication, degradation of foreign DNA, repairing of mutated DNA and in recombining different DNA molecules. The enzymes used in gene technology are produced like any other enzyme but their purification needs extra attention. Many restriction enzymes from different sources are produced in Eshcerichia coli by recombinant DNA technology. They are often labile and therefore preserved at –20 OC in buffered glycerol solution.

8. Enzymes in fine chemical production

Biocatalysis has been used in fine chemical production for a long time. Usually the catalyst has been a living organism. Ethanol, acetic acid, antibiotics, vitamins, pigments, solvents are but a few examples of biotechnical products. One of the reasons to use whole cell catalysts lies in the need to combine chemical energy source (in the form of ATP) or reducing/oxidising power (in the form of NAD(P)H) to the production process. This is elegantly done in a living cell. Candida yeasts can reduce the 5-carbon sugar xylose to a tooth-friendly polyol called xylitol by a xylose reductase enzyme:

xylose + NADH xylitol + NAD

The enzyme can be isolated and the reaction proceeds easily in a test tube. However, the reducing power of NADH has to be regenerated for the reaction to proceed. This is done in a living cell by other reactions, which reduce NAD back to NADH. One can isolate another enzyme, which does the same and couples two reactions together. One suitable enzyme is formate dehydrogenase:

xylose + NADH xylitol + NAD

formate + NAD CO2 + NADH

Coupled enzymatic reactions have been extensively studied but only few commercial examples are known. Leucine dehydrogenase is used commercially to produce L-tert- leucine with a concomitant cofactor recycling using the formate reduction for cofactor regeneration. In spite of some successes, commercial production of chemicals by living cells using pathway engineering is still in many cases the best alternative to apply biocatalysis. Isolated enzymes have, however, been successfully used in fine chemical synthesis. We discuss here some of the most important examples.

8.1. Chirally pure amino acids and aspartame

Natural as well as synthetic amino acids are widely used in the food, feed, agrochemical and pharmaceutical industries. Many proteinogenic amino acids are used in infusion solutions and essential amino acids as animal feed additives. Aspartic acid and phenyl alanine methyl ester are combined to form the low calorie sweetener aspartame. In addition to natural amino acids also synthetic ones are intermediates in the production of pharmaceuticals and agrochemicals. For example several thousand tons of D-phenylglycine and D-p-hydroxyphenylglycine are produced

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annually for the synthesis of the broad-spectrum antibiotics ampicillin, amoxicillin, cefalexin and others.

Natural amino acids are usually produced by microbial fermentation. Novel enzymatic resolution methods have been developed for the production of L- as well as for D-amino acids. The concept is based on the specificity of enzymes to detect only one of the two chiral molecules of amino acid derivatives. One approach is described in scheme 1. Racemic mixture of amino acid amides is synthesized by Strecker synthesis. Permeabilised cells of Pseudomonas putida containing amino acid amidase enzyme are used to specifically hydrolyse the natural form. L-form of the amino acid is produced and separated. The D-form can then be chemically formed or recycled after racemization.

Aspartame, the intensive non-calorie sweetener, is synthesized in non-aqueous conditions by thermolysin, a proteolytic enzyme, from N-protected aspartic acid and phenylalanine methyl ester (Scheme 2). The enzyme catalyses not only a typical condensation reaction in the absence of water but shows remarkable selectivity in forming the correct bond to form aspartame. After the condensation reaction the protective group is removed.

8.2. Rare sugars

Non-natural monosaccharides are needed as starting materials for new chemicals and pharmaceuticals. Examples are L-ribose, D-psicose, L-xylose, D-tagatose and others. Some of the sugars are presently produced by chemical isomerization or epimerisation. Recently enzymatic methods have been developed to manufacture practically all D- and L-forms of simple sugars. Figure 4 gives an example how enzymes can be used to convert sucrose into various natural sugars and a rare sugar psicose.

Glucose isomerase is one of the important industrial enzymes used in fructose manufacturing. Recently it has been shown that it can catalyse previously unknown conversions. For example L-arabinose is isomerised to L-ribulose and slowly also to L-ribose. D-xylose is isomerised to D-xylulose and slowly to D-lyxose. Also 4-carbon sugars are good substrates. Enzymatic methods are an important tool in production of rare sugars.

8.3. Semisynthetic penicillins

Penicillin is produced by genetically modified strains of Penicillium strains. Most of the penicillin is converted by immobilised acylase enzyme to 6-aminopenicillanic acid, which serves as a backbone for many semisynthetic penicillins. These can be synthesized by chemical or enzymatic methods.

8.4. Lipase based reactions

In addition to detergent applications lipases can be used in versatile chemical reactions since they are active in organic solvents. Thus water can be replaced by other nucleophiles like alcohols. The transferase activity of lipases is used to convert low value fats into more valuable ones in transesterification reactions. This occurs when low value fats are incubated in the presence of lipases and fatty acids. Lipases have also been used to form aromatic and aliphatic polymers. The enzyme can be used for enantiomeric separation of alcohols. In place of alcohols also amines can be used as the nucleophile. This makes it possible to separate rasemic amine mixtures. Chirally pure amines can be used as building blocks for bioactive molecules. Several other intensively studied synthetic reactions are possible in lipase-catalysed reactions.

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8.5. Asymmetric synthesis

Proteases and lipases are used in biocatalytic chiral hydrolytic resolutions as shown in scheme 1. Chiral compounds can alternatively be produced in biocatalytic asymmetric syntheses in which a prochiral precursor is converted to a chiral molecule by enantioselective addition reaction. Lyases catalyse the addition of a substance to a double bond or the elimination of a group resulting in an unsaturated bond. A chiral compound is formed in such a reaction. Ammonia lyases are used to produce amino acids from alpha-keto acid precursors. Example is L-aspartate ammonia lyase in production of L-aspartic acid.

A novel lyase application involves hydroxynitrile lyase, which catalyses the addition of HCN to aldehydes and ketones. The enzyme from rubber tree has been cloned and overexpressed in microorganisms. This enzyme produces valuable chemical intermediates.

A third important biocatalytic enzyme group is nitrile hydratases. They catalyse the addition of water to nitriles resulting in the formation of amides. They are used for example in the production of acrylamide from acrylonitrile and nicotine amide.

8.6. Enzymatic oligosaccharide synthesis

The chemical synthesis of oligosaccharides is a complicated multi-step effort. The saccharide building blocks must be selectively protected then coupled and finally deprotected to obtain desired stereochemistry and regiochemistry. Biocatalytic synthesis with isolated enzymes like glycosyltransferases and glycosidases or engineered whole cells are powerful alternatives to chemical methods.

Glycosyltransferases catalyse the transfer of monosaccharides from a donor to saccharide acceptors. Typically the donor is a nucleotide. The type of donor that the enzyme utilises and the position and stereochemistry of the transfer to the acceptor classify these enzymes. These enzymes can also be extracellular. Leuconostoc lactic acid bacteria produce an enzyme called dextran sucrase. It converts sucrose into fructose and a glucose polymer called dextran (Figure 4). Dextran is used in biomedical applications and as a matrix in separation processes. The enzyme can use other molecules than glucose as acceptor and thus novel oligomers with e.g. antibacterial properties can be produced. Glycosidases are hydrolytic enzymes, which can be used for synthetic reactions in a similar manner than thermolysin is used for aspartame synthesis. Oligosaccharides have found applications in cosmetics, medicines and as functional foods.

9. Future trends in industrial enzymology

Industrial enzyme market grows steadily. The reason for this lies in improved production efficiency resulting in cheaper enzymes, in new application fields and in new enzymes from screening programmes or in engineered properties of traditional enzymes. New applications are to be expected in the field of textiles, new animal diets like ruminant and fish feed. It can be expected that breakthroughs in pulp and paper will materialise. The use of cellulases to convert waste cellulose into sugars and further to ethanol by fermentative organisms has been a major study topic for years. Increasing environmental pressures and energy prices will make this application a real possibility one day.

Tailoring enzymes for specific applications will be a future trend with continuously improving tools and understanding of structure-function relationships and increased search for enzymes from exotic

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environments. This means that there will be a specifically tailored xylanase for baking, another for feed and a third one for pulp bleaching.

New technical tools to use enzymes as crystalline catalysts, ability to recycle cofactors, and engineering enzymes to function in various solvents with multiple activities are important technological developments, which will steadily create new applications.

Enzymes should, however, not be considered alone but rather as a part of a biocatalyst technology. Whole cell catalysts, increased ability to engineer metabolic pathways and a combination of specific biocatalytic reactions with organic chemistry form a basis to develop new technologies for chemical production.

Bibliography

Bhat M (2000) Cellulases and related enzymes in biotechnology. Biotechnology Advances 18, 355-383. [This review paper discusses present and possible future trends; xylanases were first suggested for pulp bleaching by VTT-Biotechnology in Finland, Röhm Enzyme is the leader in the field see http://www.roehmenzyme.com/]Biochimica et Biophysica Acta (2000) 1543, 203-252 [Contains several reviews of engineered enzymes of industrial importance]Chotani G., Dodge T., Hsu A., Kumar M., LaDuca R., Trimbur D., Weyler W. and Sandford K. (2000) The commercial production of chemicals using pathway engineering. Biochimica et Biophysica Acta 1543, 434-455. [This review article discusses the latest trends in using engineered organisms in chemical production]Doran P.M. (1999) Bioprocess Engineering Principles. 439 pp. Academic Press. [A textbook, which describes engineering aspects of bioprocesses]Flickinger M.C. and Drew S.W. (eds., 1999) Encyclopedia of Bioprocess Technology: fermentation, biocatalysis, and bioseparation (5 volumes), John Wiley & Sons [Good resource book on all aspects of modern bioprocess technologies]Godfrey T. and West S. (eds, 1996) Industrial Enzymology, Macmillan [This is a basic textbook on industrial enzymes, authored by industrial experts; www.novozymes.com is a webpage of world´s largest enzyme company, second largest is Genencor Int. Inc., www.genencor.com]; Palcic M.M. (1999) Biocatalytic synthesis of oligosaccharides. Current Opinion in Biotechnology 10, 616-624 [A good review with several references about the topic]Pastinen O, Visuri K, Schoemaker H and Leisola M (1999) Novel reactions of xylose isomerase from Streptomyces rubiginosus. Enzyme and Microbial Technology 25, 695-700. [Glucose isomerase is a traditional name; xylose isomerase would be a more correct name although it has recently been shown to catalyse many monosaccharide isomerizations]Schmid A., Dordick J.S., Hauer B., Kiener A., Wubbolts M. and Witholt B. (2001) Industrial biocatalysis today and tomorrow. Nature 409, 258-268 [A good review article on developments of enzymatic and whole cell biocatalytic applications and trends in chemical industry; http://www.isrs.kagawa-u.ac.jp/ is a page of a recently formed International Society of Rare Sugars]Visuri, K. (1987) Stable glucose isomerase concentrate and a process for the preparation thereof. US Patent 4,699,882 [This patent describes the first large scale crystallization process of an intracellular industrial enzyme] and Visuri, K. (1995) Preparation of cross-linked glucose isomerase crystals. US Pat. 5437993. [This patent describes the first preparation of a cross-linked enzyme crystal catalyst]

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Figure 1. A typical enzyme production scheme. Large volume industrial enzymes are usually not purified. Their recovery is often finalised by an ultrafiltration step. Speciality enzymes need more purification.

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Figure 2. Three-dimensional structure of a Trichoderma xylanase II. This enzyme is used in baking to improve bread quality, in animal feed to improve digestibility of feed, in cellulose pulp bleaching to reduce the use of chlorine chemicals and in fruit juice manufacturing to facilitate juice extraction and clarification. The two active centre glutamates and the one alpha helix are shown in a green colour.

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Figure 3. Cross-linked glucose isomerase crystals. The average crystal size of these crystals is 86 m. They can be used in chiral separations and as an immobilized enzyme in a backed-bed or fluidised bed column. Cross-linking makes the enzyme insoluble but it retains its activity as water containing porous material.

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Scheme 1. Formation of a racemic amino acid amide (1) synthetically by Strecker reaction and enzymatic resolution of the racemic amide mixture by amidase to form L-amino acid (L-2) and D-amide (D-1) which can be hydrolysed to D-amino acid (D-2).

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Scheme 2. L-aspartic acid (3) is formed from fumaric acid by aspartase which catalyses the addition of ammonia to fumaric acid. A protective group is added to form Z-aspartic acic (Z-3) which is combined using a racemic mixture of D/L-phenylalanine methyl ester (rac-4) by thermolysin to give Z-aspartame (Z-5). By removing the protective group by catalytic hydrogenation, aspartame (5) is obtained.

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Table 1. Change in enzyme characteristics by protein engineering

Enzyme Industry Need

xylanase feed temperature stabilityacid activity

pulp and paper

temperature and alkali stability higher pH-optimum

glucoamylase starch higher pH-optimum

glucose isomerase fructose substrate specificity

acid stabilitythermo stability

proteinase detergent thermostabilityalkali stabilityoxidative stability

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Table 2. Tools in protein engineering

Target Method

protein structure crystallizationx-ray crystallographyNMR

modelling and simulation

computational methods

gene plasmids expression systemstargeted mutagenesisPCRDNA shufflingrandom mutagenesis

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Table 3. Large scale enzyme applications

Industry Effect

Detergentproteinase protein degradationlipasecellulase

fat removalcolor brightening

Textilecellulase microfibril removallaccase color brightening

Animal feedxylanase fiber solubilityphytase release of phosphate

Starch amylases glucose formationglucose isomerase fructose formation

Pulp and paper xylanase biobleaching

Fruit juicepectinase cellulase, xylanase

juice clarification, juice extraction

Bakingxylanase dough conditioningalpha-amylaseglucose oxidase

loaf volume; shelf-lifedough quality

Dairyrennin protein coagulation

Brewing

lactase

glucanasepapain

lactose hydrolysis

filter aidhaze control

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Table 4. Major enzymatically measured analytes

Analyte Enzyme

alcohol alcohol dehydrogenaseammonia L-glutamate dehydrogenasecarbon dioxide phosphoenolpyruvate carboxylasecholesterol cholesterol oxidaseglucose glucose oxidaseoxalate oxalate oxidaseurea ureaseuric acid uricase

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