Enzymes and Protein Engineering

*This concerns a subgroup of genetic engineering methods which deal with targeted preparation of proteins, above all enzymes with the aim of determining and improving their properties. Enzymes are natural biocatalysts that are accelerating chemical reactions.* In practice enzymes are widely used, from the food industry where they have a range of uses, to washing powders, the pharmaceuticals industry up to use in the military sphere. One group of enzymes generally applicable in biotechnologies are dehalogenases. The use of these enzymes falls into three main categories: * decontamination of toxic materials * biosensors * synthesis of material for further processing **Decontamination** Enzymes have a range of advantages over classical chemical methods for decontamination and neutralisation of toxic substances, in particular their high effectiveness, low impact on the environment and that they are not caustic or corrosive. Like other proteins they are easily biodegradable. Their disadvantage, on the other hand, is their instability and possible allergic reactions in humans. #img_200#.<>

At present enzymes are known for more than half chemical weapons of mass destruction. For example enzymes are already known for decontamination from the better known nerve gases such as Sarin, Tabun or VX. Similarly enzymes have been developed for decontamination by, for example, oil or some waste products from chemical reactions or production. **Biosensors** Enzymes are also used as biosensors. They are widely used to monitor a wide spectrum of reactions. At present they are used for monitoring the purity of underground water, for example around places where chemical or dangerous production is carried out. The principle of such sensors is very simple. The active component in these sensors is an enzyme which reacts with the monitored substance. If this substance occurs the enzyme triggers a catalytic reaction. The products of this reaction are monitored by the detection system which analyses their amount. **Synthesis** Enzymes are used at a significant level in the production of precursors for further synthesis, for example in producing medicines. Their most interesting property is their ability to distinguish between compounds, the so called enantiomers which have the same chemical structure but in a different spatial arrangement. Such differences can result in different properties. For example, with aspartate one version is sweet while its mirror image is bitter. In normal chemical synthesis these substances occur in a ratio of 1:1, which for further use is disadvantageous. Enzymes are capable of selectively removing unwanted or even harmful component by reacting selectively with one enantiomer of the racemic mixture. Modification of enzymes by the methods of protein engineering follows two main paths to achieve enzymes with the required properties: * Rational design * Directed evolution Rational design places emphasis on structural and biochemical analysis. The structures of proteins are initially determined with the aid of X-ray crystallography or Nuclear Magnetic Resonance and complemented by the biochemical data. By combining this information, changes in the structure are identified which will lead to the required outcome. Mathematical modelling can be used to analyze the properties of an enzyme prior to its manipulation and modification. This approach is very demanding on know-how, experience and laboratory equipment and requires interdepartmental cooperation to work successfully. Directed evolution, however, is a method which tries to compress an evolutionary process which would take tens of thousands of years into just two or three. This method is derived from natural processes taking place in nature. Scientists monitor the duplication and multiplication of genes under laboratory conditions. Changes in genes are carried out using enzymes which, during genetic replication introduces mutations to the genes, and on this basis a library is built up of genes with various properties. With the aid of screening methods genes with the required properties are selected for a further round of controlled evolution. As opposed to natural selection the selection pressure is influenced by Man and the criteria are the required characteristics of the resultant enzyme. Whole approach concerns genetic breeding for selected molecules. From the technical point of view it could also be said that this method is not so demanding on know-how as rational design and is a relatively routine matter. Both methods can lead to the same outcome. With regard to the fact that not for all enzymes their structures can be determined, directed evolution has a wider applicability. The best solution, naturally, is to use both methods combined.

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