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Industrial Use of Enzymes

Industrial Use of Enzymes

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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 background 2. Enzyme classification 3. Enzyme production 3.1. Microbial production strains 3.2. Enzyme production by microbial fermentation 4. Protein engineering 5. Enzyme technology 6. Large scale enzyme applications 6.1. Detergents 6.2. Starch 6.3. Drinks 6.4. Textiles 6.5. Animal feed 6.6. Baking 6.7. Pulp and paper 6.8. Leather 7. Speciality enzymes 7.1. Enzymes in analytics 7.2. Enzymes in personal care products 7.3. Enzymes in DNA-technology 8. Enzymes in fine chemical production 8.1. Chirally pure amino acids and aspartame 8.2. Rare sugars 8.3. Semi synthetic penicillins 8.4. Lipase based reactions 8.5. Asymmetric synthesis 8.6. Enzymatic oligosaccharide synthesis 9. Future trends in industrial enzymology

Glossary Alkaline phosphatase: Amino acid amidase: Amylase: Aspartame: Beta-glucanase: Biocatalyst: Bromelain: Catalase: Cellulases: CLEC: Chirally pure: Dextran sucrase: Dextran: DNA-polymerases: Fermentor: Ficin: Formate dehydrogenase: Glucoamylase: Glucose oxidase: Glycosyltransferases: GRAS-status: Hydrolases: Hydroxynitrile lyase: Immunoassay: Isomerases: Laccase: Lactase: Ligases: Lipoxygenase: Lyases: An enzyme that degrades ester bonds in alkaline conditions. An enzyme that is used in manufacturing optically pure amino acids. It hydrolyses an amide bond in natural amino acid amides. A group of enzymes that hydrolyse chemical bonds between glucose molecules present in starch. This group includes alpha-, beta- and glucoamylase. A low calorie high intensive sweetener. An enzyme that degrades beta-glucan commonly found e.g. in barley. Isolated enzyme or a whole cell (living or dead) A protein-degrading enzyme from plants. An enzyme that degrades hydrogen peroxide to oxygen and water. A group of enzymes that synergistically degrade cellulose fibers to glucose. Enzyme crystal that has been made insoluble by chemical crosslinking; a method to immobilise and stabilise enzymes. Many organic molecules can have two chemically identical but structurally mirror image forms. Chirally pure means that only one of the forms is present. An enzyme, present in some lactic acid bacteria, that forms a glucose. polymer and fructose from the disaccharide sucrose. Glucose containing branched polymer used e.g. in blood replacements. An enzyme that synthesizes DNA polymers. A biological reactor for cultivation of microorganisms. A protein-degrading enzyme from plants. An enzyme that oxidises formate to carbon dioxide and NAD. An enzyme that splits glucose molecules from starch. An enzyme that uses oxygen to oxidise glucose to gluconic acid and hydrogen peroxide. Catalyse the transfer of monosaccharides from a donor to saccharide acceptors. is given to an organism that is Generally Regarded as Safe. Enzymes that break chemical bonds by adding water. They can be used to form chemical bonds in the absence of water. An enzyme that catalyses the addition of HCN to aldehydes and ketones. This is an analytical method in which antibodies are used to detect specific molecules. Enzymes that catalyse intramolecular reactions. 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. his enzymes degrade milk-sugar lactose to glucose and galactose. Lactose intolerant people can consume such milk. Enzymes that synthesize chemical bonds. A lipid oxidising enzyme extracted usually from soybeans. Enzymes that remove chemical groups from their substrates without addition of water 2

An aspartic protease which coagulates milk protein. An oxidative heme-containing enzyme that uses hydrogen peroxide to oxidise aromatic compounds. Usually the production organism and often also the individual enzyme have been genetically engineered for maximal productivity and optimised enzyme properties. A group of enzymes that degrade plant fibers made of xylose-sugars to xylose monomers. It is responsible for lignin biosynthesis in plants and initiates lignin biodegradation by certain rot-fungi. A sugar that is rare in nature. An enzyme that degrades proteins and is isolated from animals. Improvement of enzyme protein by genetic methods. Is widely used in animal feeds. Industrial enzyme business is steadily growing due to improved production technologies. They are important tools in gene technology. Isolated enzymes were first used in detergents in the year 1914. engineered enzyme properties and new application fields. They are also used in production of fructose and penicillin derivatives as well as several other chemicals. Enzymes that recognise specific 4-8 nucleotides long sequencies from DNA. It is used in cheese manufacturing and isolated from calf stomach or produced by recombinant fungi. A phosphatase enzyme that hydrolyses phosphoester bonds in phytic acid. Enzymes are responsible for the biocatalytic fermentation of sugar to ethanol by yeasts. An enzyme that degrades proteins and is isolated from animals. Summary Enzymes have been used since the dawn of mankind in cheese manufacturing and indirectly via yeasts and bacteria in food manufacturing. Enzymes should be considered as a part of a rapidly growing biocatalyst industry also involving genetically optimised living cells as chemical production factories. Enzymes used in special applications like diagnostics or DNA-technology need to be highly purified. Historical background Most of the reactions in living organisms are catalysed by protein molecules called enzymes. Man has indirectly used enzymes almost since the beginning of human history. a reaction that forms the bases of beer and wine 3 . Isolated enzymes have found several applications in fine chemical industry. Large volume industrial enzymes are usually not purified but sold as concentrated liquids or granulated dry products. their protein nature proven in 1926 and their large-scale microbial production started in 1960s. An antibiotic substance extracted from molds. 1.Nitrile hydratases: Oxidoreductases: Papain: Penicillin: Pepsin: Peroxidase: Phytase: Protein engineering: Rare sugar: Rennin: Restriction enzymes: Transferases: Trypsin: Xylanase: Xylitol: Enzymes that catalyse addition of water to nitrales resulting in amide formation Enzymes that oxidise or reduce chemical compounds. The major part of enzymes is produced by with GRAS-status microorganisms in large biological reactors called fermentors. Enzymes are used in production of chirally pure amino acids and rare sugars. A protein degrading enzyme from animal gut. A tooth-friendly sugar alcohol used in chewing gums. Enzymes can rightly be called the catalytic machinery of living systems.

which converted starch into sugar. The traditional acid hydrolysis of starch was completely replaced by alpha-amylases and glucoamylases. The estimated value of world enzyme market is presently about US $ 1. Detergents (37%). This review concentrates on the use of isolated enzyme preparations in large scale and speciality applications and chemical manufacturing. Röhm in Germany prepared the first commercial enzyme preparation in 1914. They called the substance diastase. This trypsin enzyme isolated from animals degraded proteins and was used as a detergent. contains 470 genes of which 145 are related to gene replication and transcription. which could convert starch with over 95%. 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. which use about 75% of industrially. Scientists who found out that an alcohol precipitate of malt extract contained a thermo labile substance.enzymes were used already in 1930 in fruit juice manufacturing. The term “enzyme” comes from Latin words. Thousands of different variants of the natural enzymes are known. The use of microorganisms as biocatalysts in chemical production is. The 4 . The smallest known organism. Similar microbial enzyme reactions of acid forming bacteria and yeasts are responsible for aroma forming activities in bread making and in preserving activities in sauerkraut preparation. which contain numerous different enzyme activities. Concept and Scope of Enzyme Action]. made the first clear recognition of enzymes in 1833. Enzyme classification Presently more than 2000 different enzyme activities have been isolated and characterized [see Enzymology. The sequence information of a growing number of organisms opens the possibility to characterise all the enzymes of an organism on a genomic level. Starch industry became the second largest user of enzymes after detergent industry. Baker’s yeast has 7000 genes coding for about 3000 enzymes. The major usage of microbial enzymes in food industry started in 1960s in starch industry. The fermentative activity of microorganisms was discovered only in 18th century and finally proved by the French scientist Louis Pasteur. This name was given since enzymes where closely associated with yeast activity. an interesting and growing field.manufacturing. produced enzymes. Enzymes oxidise ethanol to acetic acid. baking (8%) and animal feed (6%) are the main industries. 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. which literally mean “in yeast”.3 billion and it has been forecasted to grow to almost US $ 2 billion by 2005. textiles (12%). They are called pectinases. 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. however. The techniques of genetic. We know now that it was an enzyme nowadays called amylase. Mycoplasma genitalium. Presently the industrial enzyme companies sell enzymes for a wide variety of applications. 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. This reaction has been used in vinegar production for thousands of years. 2. Enzymes are also indirectly used in biocatalytic processes involving living or dead and permeabilised microorganisms. These enzymes clarify the juice. yield to glucose. protein and pathway engineering are making chemical production by living cells an interesting green alternative to replace traditional chemical processes.in addition to cheese manufacturing . In food industry . The real breakthrough of enzymes occurred with the introduction of microbial proteases into washing powders. The study of enzymes is a fairly recent activity. starch (11%).

6. Enzymes used in paper industry should not contain cellulose-degrading activity as a side activity because this activity would damage the cellulose fibres. reaction rate.1. Most of the enzymes are. 3. 3. Enzymes used in animal feed industry must be thermo tolerant to survive in the hot extrusion process used in animal feed manufacturing. bromelain and ficin and some other speciality enzymes like lipoxygenase from soybeans. 3. Ideally the enzyme is secreted from the cell. The same enzymes must have maximal activity at the body temperature of the animal. produced by microorganisms in submerged cultures in large reactors called fermentors. More than 75% of industrial enzymes are hydrolases. This is especially important when the enzyme produced by the organism is used in food processes. Plant derived commercial enzymes include proteolytic enzymes papain. 6. however. 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. 2. Thirdly. 4. Selection of an enzyme Selection of a production strain Construction of an overproducing strain by genetic engineering Optimisation of culture medium and production conditions Optimisation of recovery process (and purification if needed) Formulation of a stable enzyme product Criteria used in the selection of an industrial enzyme include specificity. 5. 4. Animal derived enzymes include proteinases like pepsin and rennin. the organism should be able to produce high amount of the desired enzyme in a reasonable time frame.number of reported 3-dimensional enzyme structures is rapidly increasing. 3. Proteindegrading enzymes constitute about 40% of all enzyme sales. The industrial strains 5 . More than fifty commercial industrial enzymes are available and their number increases steadily. This makes the recovery and purification process much simpler compared to production of intracellular enzymes. Enzyme production Some enzymes are still extracted from animal or plant tissues. Microbial production strains In choosing the production strain several aspects have to be considered. Secondly. 5. 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 substrates Transferases catalyse group transfer Hydrolases catalyse bond breakage with the addition of water Lyases remove groups from their substrates Isomerases catalyse intramolecular rearrangements 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. pH and temperature optima and stability. Enzymes used in industrial applications must usually be tolerant against various heavy metals and have no need for cofactors. effect of inhibitors and affinity to substrates. The enzyme production process can be divided into following phases: 1. In the year 2000 the structure of about 1300 different proteins were known. Proteinases have found new applications but their use in detergents is the major market. which must be purified from thousands of different cell proteins and other components. 2. which means that it is Generally Regarded As Safe. the production host should have a GRAS-status.

Two different approaches are presently available: a random method called directed evolution and a protein engineering method called rational design. sampling). 3. how is the organism cultivated (batch. how to maximise cell concentration in the reactor. what are the optimal growth conditions. what needs to be measured and how is the process controlled. foam control. This is a demanding task and often involves as much effort as the intracellular engineering of the cell. purify and preserve the product. Protein engineering Often enzymes do not have the desired properties for an industrial application. cooling. Enzymes are proteins. cellulases. what is the specific growth and product formation rate. The extracellular enzymes are often recovered after cell removal (by vacuum drum filtration. Sometimes a desired property. Xylanase is a good example of an industrial enzyme. has been found but other problems have surfaced. does some of the raw materials or products inhibit the organism and finally. 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. fed-batch or continuous cultivation). which like any protein can cause and have caused in the past allergic reactions. The enzyme may not be functional in the desired temperature. The final product is either a concentrated liquid with necessary preservatives like salts or polyols or alternatively granulated to a non-dusty dry product. One of the industrial production organisms of xylanases is Trichoderma fungus. what kind of a reactor is needed (mass transfer. Enzyme production by microbial fermentation Once the biological production organism has been genetically engineered to overproduce the desired products. Another option is to engineer a commercially available enzyme to be a better industrial catalyst. If needed the purification is carried out by ion exchange or gel filtration. 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. what kind of nutrients the organism needs and what is their optimal/ economical concentration. is the product secreted out from the cells. cultivation type and process conditions. how to recover. how the nutrients should be sterilised. It may also prove very difficult to overproduce the enzyme in a suitable host. like extreme thermo stability. The optimisation of a fermentation process includes media composition. 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. a production process has to be developed. separators or microfiltration) by ultrafiltration. One can in such a case try to find a better enzyme from nature. Streptomyces fungi imperfecti and Bacillus bacteria. what is the yield and volumetric productivity.typically produce over 50-g/l extracellular enzyme proteins. By designed 6 . aeration. These include proteinases. lipases. Therefore protective measures are necessary in their production and application.2. 4. α -amylases and glucoamylases. Most of the industrial enzymes are produced by a relatively few microbial hosts like Aspergillus and Trichoderma fungi. Yeasts are not good produces of extracellular enzymes and are rarely used for this purpose. The large volume industrial enzymes are produced in 50 – 500 m3 fermentors. how to degrade the cell if the product is intracellular. Most of the industrially used microorganisms have been genetically modified to overproduce the desired activity and not to produce undesired side activities. A typical enzyme production scheme is shown in Figure 1. Its xylanase has been purified and crystallized. Several enzymes have already been engineered to function better in industrial processes. The bioprocess engineer asks questions like: is the organism in question safe or are extra precautions needed.

The concept. bleaching of cellulose pulp with xylanases or use of enzymes in animal feed. The most successful strategies to improve the stability of the Trichoderma xylanase include the stabilization of the alpha-helix region and the N-terminus. The dimensions of an enzyme reactor. are considerably smaller compared to traditional immobilized systems because the carrier matrix can be completely omitted. Many different laboratory methods for enzyme immobilization based on chemical reaction. hydrophobic areas and ionic properties. Another way to immobilise enzymes is to use ultrafiltration membranes in the reactor system. entrapment. packed with this kind of a material.mutagenesis its thermal stability has been increased about 2000 times at 70 oC and its pH-optimum shifted towards alkaline region by one pH-unit. 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. 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 is used to measure glucose concentrations in biological samples. The known structure of an enzyme is used to design and simulate mutations. In these applications the price of the enzymes must be low to make their use economical. An alternative way to use enzymes is to immobilise them so that they can be reused. Enzyme technology How are the enzymes used and applied in practical processes? This is the field of enzyme technology. In the early applications the glucose isomerase enzyme containing cells were permeabilised and immobilized on a solid support. The three dimensional structure of a xylanase enzyme is shown in Figure 2. This method has been used in production of chirally pure amino acids from racemic mixtures of amino acid derivatives. specific binding or absorption have been developed. originally developed in Finland. The largest application of an immobilised enzyme is the conversion of glucose syrup to high fructose syrup for food applications. The large enzyme molecules cannot pass the membrane but the small molecular reaction products can. All this is possible because an enzyme crystal contains water. It was based on the use of cross-linked crystalline glucose isomerase (Figure 3). The best-known example is glucose oxidase. which can be regenerated with fresh enzyme after the previous one is inactivated. pores. The enzyme containing support material was packed into a column through which the glucose solution was passed. specific separations and recently even cofactor entrapment into the crystal structure. A novel approach to use enzymes was introduced by Finnish Sugar Company in the late 80s. which is the trademark for CrossLinked Enzyme Crystals. 5. controlled release of chemicals. Extracellularly produced bulk enzyme concentrates cost only US $ 10-20/ kg protein. Therefore enzymes are retained in a reaction system and the products leave the system continuously. was later applied to other enzymes by Altus Ltd in USA. active centre. These applications include chiral separations. Enzymes have also been immobilized on membranes for analytical purposes. Enzyme crystals contain usually 30-80% free water and the enzyme is active even in the cross-linked insoluble form. Large scale enzyme applications 7 . This happens in many bulk enzyme applications like liquefaction of starch with amylases. 6. which has developed novel applications for the CLECs.

This process is used for milk products that are consumed by lactose intolerant consumers. These hostile agents include anionic detergents. Starch hydrolysis and fructose production The use of starch degrading enzymes was the first large-scale application of microbial enzymes in food industry. Bacterial proteinases are still the most important detergent enzymes.1. which are formed during washing and the use of cotton based cloths. Industrial Enzymology is recommended as a good resource text for those who need a more comprehensive treatment of an individual subject. fructose is concentrated to 55% and used as a high fructose corn syrup in soft drink industry. which need Mg2+ ions for activity. Mainly two enzymes carry out conversion of starch to glucose: alpha-amylase cuts rapidly the large alpha-1. fructose separated and crystallized and then the glucose circulated back to the process. Alternatively. The use of chymosin in cheese making to coagulate milk protein was already discussed. In textile washing cellulases remove cellulose microfibrils. Another enzyme used in milk industry is betagalactosidase or lactase. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose. Lipases decompose fats into more water-soluble compounds by hydrolysing the ester bonds between the glycerol backbone and fatty acid. In the next phase called saccharification. 6. Sometimes additional debranching enzymes like pullulanase are added to improve the glucose yield. 6.2. This is done by bacterial enzymes. Cellulases have been part of detergents since early 90s. An alternative method to produce fructose is shown in Figure 4. which splits milk-sugar lactose into glucose and galactose. Fructose is separated from glucose by large-scale chromatographic separation and crystallized. which operate in lower pH and temperature than alpha-amylase. Late 80s lipid degrading enzymes were introduced in powder and liquid detergents. Sucrose is split by invertase into glucose and fructose.3. This phase is called liquefaction and is carried out by bacterial enzymes.Table 3 summarises major large-scale enzyme applications. glucoamylase hydrolyses the oligomers into glucose. Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi. 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. Beta-amylase is commercially produced from barley grains and used for the production of the disaccharide maltose. Amylases are used in detergents to remove starch based stains. Each of them is discussed in the text in some detail. which tends to stick on textile fibres and bind other stain components.4-linked glucose polymers into shorter oligomers in high temperature. This is done by fungal enzymes. 8 . Amylases hydrolyse gelatinised starch. 6. This method is used in Europe and uses sucrose as a starting material. Detergents Detergents were the first large scale application for microbial enzymes. Some products have been genetically engineered to be more stable in the hostile environment of washing machines with several different chemicals present. It is produced in large scale by Aspergillus oryzae host after cloning the Humicola gene into this organism. Drinks Enzymes have many applications in drink industry. This can be seen as colour brightening and softening of the material. oxidising agents and high pH. The most important lipase in the market was originally obtained from Humicola lanuginose.

4. Pectins are polymeric substances in fruit lamella and cell walls.Enzymes are used also in fruit juice manufacturing. Malting is a process. Catalase enzyme. filtration and storage (papain. Another recent approach is to use oxidative enzymes directly to bleach textiles. Addition of pectinase. 9 . Starch has for a long time been used as a protective glue of fibres in weaving of fabrics. Additional enzymes can be used to help the starch hydrolysis (typically alpha-amylases). alpha-amylase and beta-glucanase). Textiles The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology.5. solve filtration problems caused by betaglucans present in malt (beta-glucanases). 6. 6. Other enzymes. Laccases are produced by white-rot fungi. and which in an oxidised state can oxidatively degrade many different types of molecules like dye pigments. Enzymes hydrolyse the high molecular weight substances like pectin. which destroys hydrogen peroxide. xylanase and cellulase improve the liberation of the juice from the pulp. Laccase – a polyphenol oxidase from fungi . Recently.is a new candidate in this field. which increases the enzyme levels in the grain. are often added to washing powders. Animal feed Intensive study to use enzymes in animal feed started in early 80s. Amylases are used in this process since they do not harm the textile fibres. In the mashing process the enzymes are liberated and they hydrolyse the starch into soluble fermentable sugars like maltose. and control haze during maturation.the aromatic polymer found in all plant materials. The same effect can be obtained with cellulase enzymes. which is a glucose disaccharide. Laccase is a copper-containing enzyme. hydrolyse proteins (neutral proteinase). may then be used to degrade excess peroxide. The cell wall contains also hemicelluloses and cellulose. hydrogen peroxides have been tested as bleaching agents to replace chlorine-based chemicals. which is oxidised by oxygen. Barley contains beta-glucan. 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. which use them to degrade lignin . Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the yield. which causes high viscosity in the chicken gut. The first commercial success was addition of beta-glucanase into barley based feed diets. This is called sizing. The stones caused considerable damage to fibres and machines. Brewing is an enzymatic process. Enzymes are used to remove the starch in a process called desizing. 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. Finnfeeds International was the pioneer in animal feed enzymes. They are closely related to polysaccharides. These examples were discussed under detergent enzymes. which interact with textiles. Fruit cell wall needs to be broken down to improve juice liberation. Pectinases and amylases are used in juice clarification. The effect is a result of alternating cycles of desizing and bleaching enzymes and chemicals in washing machines.

The major application is the use of xylanases in pulp bleaching. and reduces the amount of faeces. This facilitates water removal. The fibre is diluted to 1% concentration with water. In spite of extensive research no oxidative enzymes are applied in pulp and paper industry. Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. Usually a feed-enzyme preparation is a multienzyme cocktail containing glucanases. Therefore the feed enzymes need to be thermo tolerant during the feed manufacturing and operative in the animal body temperature. by DSM in the Netherlands. glucose oxidase has been used to replace chemical oxidants and lipases to strengthen gluten. Proteinases can be added to improve dough-handling properties. This leads to more stable dough. Both fungal and bacterial amylases are used. Xylanases are nowadays routinely used in feed formulations. flour typically contains minor amounts of cellulose. flocculating surfactants and ink solvents added and the 10 . It is therefore conceivable that enzymes also affect the baking process. Xylanase enzymes were found to be the most effective ones in this case. Other minor enzyme applications in pulp production include the use of enzymes to remove fine particles from pulp. which increases absorbtion of nutrients. Baking Similar fibre materials are used in baking than in animal feed.g. One of the motivations to study the effect of enzymes on dough and bread qualities comes from the pressure to reduce other additives. The net effect is reduced phosphorous in faeces resulting in reduced environmental pollution. There is evidence that the use of xylanases decreases the water absorption and thus reduces the amount of added water needed in baking. This reduces considerably the need for chlorine based bleaching chemicals. Overdosage may lead to sticky dough so the added amount needs to be carefully controlled. Addition of xylanase to wheat-based broiler feed has increased the available metabolizable energy 7-10% in various studies. Enzymes have become an important aspect of animal feed industry. In addition to starch. Another type of important feed enzyme is phytase marketed e. proteinases and amylases. 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). Enzyme addition reduces viscosity. 6. Pulp and Paper Intensive studies have been carried out during the last twenty years to apply many different enzymes in pulp and paper industry.Enzymes were tested later also in wheat-based diets. 6. In the use of secondary (recycled) cellulose fibre the removal of ink is important. which leads to more stable dough and better bread quality. Xylanases liberate lignin fragments by hydrolysing residual xylan. Phytase is a phosphoesterase which liberates phosphate from phytic acid which is a common compound in plant based feed materials. enzymes are used in pig feeds and turkey feeds. liberatates nutrients either by hydrolysis of non-degradable fibres or by liberating nutrients blocked by these fibres. glucans and hemicelluloses like arabinoxylan and arabinogalactan. A real excitement started with the discovery of lignin degrading peroxidases in the early 80s. The use of phytase reduces the need to add phosphorus to the feed diet. Especially xylanases are used in whole meal rye baking and dry crisps common in Scandinavia.7.6. In addition to poultry. xylanases. Figure 2 shows the three-dimensional structure of a Trichoderma xylanase.

DNA-technology and in fine chemical production. Normally automatic analysers carry out these measurements. dirt and fats and reduces the processing time. The ink particles float to the surface. In paper making enzymes are used especially in modification of starch. and personal care products. In some cases pancreatic trypsin is also used in this phase. Lipases are used in this phase or in bating phase to specifically remove grease. Speciality enzymes In addition to large volume enzyme applications.1. there are a large number of speciality applications for enzymes. In this phase the protein is partly degraded to make the leather soft and easier to dye. The use of these enzymes is associated with the structure of animal skin as a raw material. This means that elaborate purification processes are needed. Here we discuss the other aspects of speciality enzymes. stiffness and erasability of paper. which is achieved by adding amylase enzymes in a controlled process.mixture is aerated. Alkaline proteases are added in the soaking phase. Starch improves the strength. The use of lipases is a fairly new development in leather industry. 7. Enzymes in analytics Enzymes are widely used in the clinical analytical methodology. protein modification. The starch suspension must have a certain viscosity. which can be detected spectrophotometrically or production of H2O2 which can be detected in peroxidase catalysed reactions leading to coloured products. The detection of the antibody-antigen complex is usually based on enzymes linked to the antibodies. Pitch is a sticky substance present mainly in softwoods. It is composed of lipids.8. 6. This results in a more environmentally friendly process and improves the quality of the leather (cleaner and stronger surface. This enzyme is 11 . In dehairing and dewooling phases enzymes are used to assist the alkaline chemical process. These include use of enzymes in analytical applications. Leather Leather industry uses proteolytic and lipolytic enzymes in leather processing. Table 4 summarises some of the main analytes measured enzymatically. Immunoassays are based on detection of target molecules by specific antibodies. Enzymes are used to remove unwanted parts. There are reports that this process is facilitated by addition of cellulase enzymes. The used enzymes are typically alkaline bacterial proteases. 7. flavour production. Contrary to bulk industrial enzymes these enzymes need to be free from side activities. which is used as an important additive. The reactions normally involve either changes in NAD(P)/NAD(P)H proportions. It is a special problem when mechanical pulps of red pine are used as a raw material. which can be easily quantified spectrophotometrically. removal and degradation of protein. The latter application will be separately discussed because of its importance. This improves water uptake by the dry skins. less spots). The next phase is bating which aims at deliming and deswelling of collagen. Pancreatic trypsins were originally used but they are being partly replaced by bacterial and fungal enzymes. Pitch causes problems in paper machines and can be removed by lipases. softer leather.

Proteinase and lipase containing enzyme solutions are used for this purpose. can be used to quantify the analyte in question. Restriction enzymes recognise specific DNA sequences and cut the chain at these recognition sites. Recombinant DNA-technology allows one to produce new enzymes in traditional overproducing and safe organisms. Enzymes are the tools needed in genetic engineering and are shortly discussed here. Enzymes are studied also for applications in skin and hair care products. Most traditional enzymes are produced by organisms. Enzymes are crucial tools in this process. methanol. adenine.2. one based on peroxide detection and the other based on oxygen consumption. Enzymes in DNA-technology DNA-technology has revolutionised both traditional biotechnology and opened totally new fields for scientific study. The DNA modifying enzymes can be divided into two classes: 1. Two different types of electrodes. 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. The residual hydrogen peroxide after disinfections can be removed by a heme containing catalase enzyme.either an alkaline phosphatase. degrade them. 12 . sucrose. An important development in analytical chemistry is biosensors. Some toothpaste contains glucoamylase and glucose oxidase. 7. 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 of molecules like glucose. which degrades hydrogen peroxide. It is also an important tool in enzyme industry. The specific order of the organic bases in the chain constitutes the genetic language. Genetic engineering means reading and modifying this language. cholesterol and some amino acids. which have been genetically modified to overproduce the desired enzyme. which can be detected in colour forming reaction by p-nitrophenyl phosphate or peroxidase. thymine. Organic bases. 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. DNA is basically a long chain of deoxyribose sugars linked together by phosphodiester bonds. One application is contact lens cleaning. They are based on H 2O2 producing oxidative enzymes. Dentures can be cleaned with protein degrading enzyme solutions. Protein engineering is used to modify and improve existing enzymes as discussed under Protein engineering. 2.3. lactate. ethanol. join pieces together and remove parts of the DNA. Hydrogen peroxide is used in disinfections of contact lenses. guanine and cytosine are linked to the sugars and form the alphabet of genes. DNA modifying enzymes synthesize nucleic acids. For more information the reader is referred to specific texts dealing with genetic engineering. 7. lactose. which is detected in the presence of H2O2 with a colour forming substrate.

which does the same and couples two reactions together. been successfully used in fine chemical synthesis. 8. We discuss here some of the most important examples. The enzymes used in gene technology are produced like any other enzyme but their purification needs extra attention. pigments. Chirally pure amino acids and aspartame Natural as well as synthetic amino acids are widely used in the food. 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. Enzymes in fine chemical production Biocatalysis has been used in fine chemical production for a long time. Leucine dehydrogenase is used commercially to produce L-tert. These enzymes do not cut the cell’s own DNA because its recognition sites are protected. however. 8. They are often labile and therefore preserved at –20 OC in buffered glycerol solution. This is done in a living cell by other reactions. This is elegantly done in a living cell. agrochemical and pharmaceutical industries. In addition to natural amino acids also 13 . antibiotics. feed. which reduce NAD back to NADH. Many proteinogenic amino acids are used in infusion solutions and essential amino acids as animal feed additives. In the cell these enzymes are involved in DNA replication. commercial production of chemicals by living cells using pathway engineering is still in many cases the best alternative to apply biocatalysis. 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. vitamins. Ethanol. solvents are but a few examples of biotechnical products. Kinases add phosphate groups and phosphatases remove them from the end of DNA chain. These enzymes are essential in gene technology. DNA-polymerases synthesize new DNA-chains. which they copy. Usually the catalyst has been a living organism. Many of them need a model template. Isolated enzymes have. repairing of mutated DNA and in recombining different DNA molecules. Ligases join adjacent nucleotides together by forming fosfodiester bonds between them. However. Their role in nature is to cut foreign DNA material. Many restriction enzymes from different sources are produced in Eshcerichia coli by recombinant DNA technology. Nucleases hydrolyse the phosphodiester bonds between DNA sugars.Restriction enzymes recognise a specific code sequence in the DNA. More than 150 different restriction enzymes have been isolated from several bacterial species and they are used in cutting the DNA in question at specific points. One can isolate another enzyme. degradation of foreign DNA. In spite of some successes. This is usually 4-8 nucleotides long sequence. Aspartic acid and phenyl alanine methyl ester are combined to form the low calorie sweetener aspartame. acetic acid.leucine with a concomitant cofactor recycling using the formate reduction for cofactor regeneration.1. 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. the reducing power of NADH has to be regenerated for the reaction to proceed.

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. The enzyme can be used for enantiomeric separation of alcohols. Natural amino acids are usually produced by microbial fermentation. The concept is based on the specificity of enzymes to detect only one of the two chiral molecules of amino acid derivatives. These can be synthesized by chemical or enzymatic methods. Recently it has been shown that it can catalyse previously unknown conversions. Novel enzymatic resolution methods have been developed for the production of L. For example Larabinose is isomerised to L-ribulose and slowly also to L-ribose. is synthesized in non-aqueous conditions by thermolysin. a proteolytic enzyme. the intensive non-calorie sweetener. Examples are L-ribose. D-tagatose and others. In place of alcohols also amines can be used as the nucleophile. from N-protected aspartic acid and phenylalanine methyl ester (Scheme 2). 8.2. Aspartame. Chirally pure 14 .as well as for D-amino acids. Racemic mixture of amino acid amides is synthesized by Strecker synthesis. After the condensation reaction the protective group is removed. Rare sugars Non-natural monosaccharides are needed as starting materials for new chemicals and pharmaceuticals. Lipases have also been used to form aromatic and aliphatic polymers. Also 4-carbon sugars are good substrates. One approach is described in scheme 1. D-psicose.4. 8. Most of the penicillin is converted by immobilised acylase enzyme to 6-aminopenicillanic acid. For example several thousand tons of D-phenylglycine and D-p-hydroxyphenylglycine are produced annually for the synthesis of the broad-spectrum antibiotics ampicillin. This occurs when low value fats are incubated in the presence of lipases and fatty acids. Glucose isomerase is one of the important industrial enzymes used in fructose manufacturing. Recently enzymatic methods have been developed to manufacture practically all D. This makes it possible to separate rasemic amine mixtures. Lipase based reactions In addition to detergent applications lipases can be used in versatile chemical reactions since they are active in organic solvents. L-xylose. D-xylose is isomerised to Dxylulose and slowly to D-lyxose.synthetic ones are intermediates in the production of pharmaceuticals and agrochemicals. Permeabilised cells of Pseudomonas putida containing amino acid amidase enzyme are used to specifically hydrolyse the natural form. 8. Semisynthetic penicillins Penicillin is produced by genetically modified strains of Penicillium strains. cefalexin and others.and L-forms of simple sugars. Enzymatic methods are an important tool in production of rare sugars. L-form of the amino acid is produced and separated. which serves as a backbone for many semisynthetic penicillins. amoxicillin. Some of the sugars are presently produced by chemical isomerization or epimerisation. The transferase activity of lipases is used to convert low value fats into more valuable ones in transesterification reactions.3. Figure 4 gives an example how enzymes can be used to convert sucrose into various natural sugars and a rare sugar psicose. Thus water can be replaced by other nucleophiles like alcohols. The D-form can then be chemically formed or recycled after racemization.

Asymmetric synthesis Proteases and lipases are used in biocatalytic chiral hydrolytic resolutions as shown in scheme 1. Leuconostoc lactic acid bacteria produce an enzyme called dextran sucrase. Example is L-aspartate ammonia lyase in production of L-aspartic acid. antibacterial properties can be produced. The type of donor that the enzyme utilises and the position and stereochemistry of the transfer to the acceptor classify these enzymes.g. These enzymes can also be extracellular. 8. They are used for example in the production of acrylamide from acrylonitrile and nicotine amide. It can be expected that breakthroughs in pulp and paper will materialise. The reason for this lies in improved production efficiency resulting in cheaper enzymes. 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. Typically the donor is a nucleotide. 15 . new animal diets like ruminant and fish feed. medicines and as functional foods. 8. Biocatalytic synthesis with isolated enzymes like glycosyltransferases and glycosidases or engineered whole cells are powerful alternatives to chemical methods. 9. Future trends in industrial enzymology Industrial enzyme market grows steadily.amines can be used as building blocks for bioactive molecules. This enzyme produces valuable chemical intermediates. Dextran is used in biomedical applications and as a matrix in separation processes. New applications are to be expected in the field of textiles. 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. The enzyme from rubber tree has been cloned and overexpressed in microorganisms. They catalyse the addition of water to nitriles resulting in the formation of amides. Several other intensively studied synthetic reactions are possible in lipase-catalysed reactions. 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. A chiral compound is formed in such a reaction. which can be used for synthetic reactions in a similar manner than thermolysin is used for aspartame synthesis. in new application fields and in new enzymes from screening programmes or in engineered properties of traditional enzymes.5.6. The enzyme can use other molecules than glucose as acceptor and thus novel oligomers with e. which catalyses the addition of HCN to aldehydes and ketones. Increasing environmental pressures and energy prices will make this application a real possibility one day. A novel lyase application involves hydroxynitrile lyase. Oligosaccharides have found applications in cosmetics. It converts sucrose into fructose and a glucose polymer called dextran (Figure 4). Lyases catalyse the addition of a substance to a double bond or the elimination of a group resulting in an unsaturated bond. A third important biocatalytic enzyme group is nitrile hydratases. Glycosyltransferases catalyse the transfer of monosaccharides from a donor to saccharide acceptors. Glycosidases are hydrolytic enzymes. Ammonia lyases are used to produce amino acids from alpha-keto acid precursors.

Biotechnology Advances 18. and West S. and engineering enzymes to function in various solvents with multiple activities are important technological developments.. New technical tools to use enzymes as crystalline catalysts.. Current Opinion in Biotechnology 10. Schoemaker H and Leisola M (1999) Novel reactions of xylose isomerase from Streptomyces rubiginosus. K. [Glucose isomerase is a traditional name. however.. Enzymes should. (1995) Preparation of cross-linked glucose isomerase crystals. 434-455. http://www. (eds. Bibliography Bhat M (2000) Cellulases and related enzymes in biotechnology. biocatalysis. Enzyme and Microbial Technology 25... 695-700. Wubbolts M. [This review article discusses the latest trends in using engineered organisms in chemical production] Doran P. K..novozymes.882 [This patent describes the first large scale crystallization process of an intracellular industrial enzyme] and Visuri.. Visuri K. 1999) Encyclopedia of Bioprocess Technology: fermentation.C. (1999) Biocatalytic synthesis of oligosaccharides.isrs. 355383. John Wiley & Sons [Good resource book on all aspects of modern bioprocess technologies] Godfrey T. 258-268 [A good review article on developments of enzymatic and whole cell biocatalytic applications and trends in chemical industry. www. xylanases were first suggested for pulp bleaching by VTT-Biotechnology in Finland. which describes engineering aspects of bioprocesses] Flickinger M. (1987) Stable glucose isomerase concentrate and a process for the preparation thereof. www..roehmenzyme.M.. Palcic M. 616-624 [A good review with several references about the topic] Pastinen O. Whole cell catalysts. second largest is Genencor Int. 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.kagawa-u. ability to recycle cofactors. LaDuca R. and bioseparation (5 volumes). [A textbook. and Sandford K. Hauer B. 1996) Industrial Enzymology. Dordick J. Trimbur D.S. (2001) Industrial biocatalysis today and tomorrow.com is a webpage of world´s largest enzyme company. Nature 409. and Witholt B.com/] Biochimica et Biophysica Acta (2000) 1543. and Drew S. [This review paper discusses present and possible future trends. Kiener A. another for feed and a third one for pulp bleaching. which will steadily create new applications.. Academic Press. US Patent 4. 203-252 [Contains several reviews of engineered enzymes of industrial importance] Chotani G. (eds. Macmillan [This is a basic textbook on industrial enzymes. authored by industrial experts.ac. Biochimica et Biophysica Acta 1543.W.jp/ is a page of a recently formed International Society of Rare Sugars] Visuri. (1999) Bioprocess Engineering Principles.699. Hsu A. US Pat.M... Weyler W. Röhm Enzyme is the leader in the field see http://www. This means that there will be a specifically tailored xylanase for baking. not be considered alone but rather as a part of a biocatalyst technology.com]. [This patent describes the first preparation of a cross-linked enzyme crystal catalyst] 16 . xylose isomerase would be a more correct name although it has recently been shown to catalyse many monosaccharide isomerizations] Schmid A.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 environments. 5437993. Inc. Kumar M. (2000) The commercial production of chemicals using pathway engineering. Dodge T. 439 pp.genencor.

Their recovery is often finalised by an ultrafiltration step. Large volume industrial enzymes are usually not purified. Speciality enzymes need more purification.Figure 1. A typical enzyme production scheme. 17 .

in animal feed to improve digestibility of feed.Figure 2. 18 . in cellulose pulp bleaching to reduce the use of chlorine chemicals and in fruit juice manufacturing to facilitate juice extraction and clarification. Three-dimensional structure of a Trichoderma xylanase II. The two active centre glutamates and the one alpha helix are shown in a green colour. This enzyme is used in baking to improve bread quality.

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. Cross-linked glucose isomerase crystals. 19 .Figure 3. The average crystal size of these crystals is 86 µ m.

neoaurum NH2 OH O L-2 R NH2 NH2 O D-1 R NH2 OH O D-2 Scheme 1.O Strecker reacton: R HCN NH3 R NH2 N H3O+ R NH2 NH2 O 1 NH2 R O rac-1 NH2 Amidase R P. 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). 20 . antropii M. putida O.

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). O H2N N H O O HO O Z-5 5 Scheme 2. L-aspartic acid (3) is formed from fumaric acid by aspartase which catalyses the addition of ammonia to fumaric acid.O HO OH O Aspartase HO O 3 O OH NH2 PhCH2OCOCl O HO O N H HO Z-3 O O Z-3 H2N O rac-4 O Thermolysin O O H N O N H OH O O O deprot. By removing the protective group by catalytic hydrogenation. 21 . aspartame (5) is obtained.

Change in enzyme characteristics by protein engineering Enzyme xylanase Industry feed pulp and paper glucoamylase starch Need temperature stability acid activity temperature and alkali stability higher pH-optimum higher pH-optimum substrate specificity acid stability thermo stability thermostability alkali stability oxidative stability glucose isomerase fructose proteinase detergent 22 .Table 1.

Table 2. Tools in protein engineering Target protein structure Method crystallization x-ray crystallography NMR computational methods plasmids expression systems targeted mutagenesis PCR DNA shuffling random mutagenesis modelling and simulation gene 23 .

xylanase Baking xylanase alpha-amylase glucose oxidase Dairy rennin lactase Brewing glucanase papain filter aid haze control protein coagulation lactose hydrolysis dough conditioning loaf volume. juice extraction glucose formation fructose formation biobleaching microfibril removal color brightening fiber solubility release of phosphate protein degradation fat removal color brightening Effect 24 . Large scale enzyme applications Industry Detergent proteinase lipase cellulase Textile cellulase laccase Animal feed xylanase phytase Starch amylases glucose isomerase Pulp and paper xylanase Fruit juice pectinase cellulase.Table 3. shelf-life dough quality juice clarification.

Major enzymatically measured analytes Analyte alcohol ammonia carbon dioxide cholesterol glucose oxalate urea uric acid Enzyme alcohol dehydrogenase L-glutamate dehydrogenase phosphoenolpyruvate carboxylase cholesterol oxidase glucose oxidase oxalate oxidase urease uricase 25 .Table 4.

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