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.
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.
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 of
molecules 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 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.
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.
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 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.
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.
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.
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.
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.
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.