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Full Version: Altering Enzymes For Better Performance-Enzyme Engineering
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Enzymes are biocatalysts which are mainly biological molecules which have the ability to influence a reaction. Enzyme engineering deals with enhancement of enzyme activity of existing enzyme or inducing a new enzyme activity. This is obtained by modifying amino acids sequence of the enzyme. This is made possible by recombinant DNA technology. It modifies the genes coding for amino acids of enzymes, transfer enzyme responsible genes from low active microbes to higher active ones. Multiple genes are transferred into single organism for increased expression.
The principle behind enzyme engineering is ultimately modification of amino acids of enzymes which results in a change in the property of the enzymes. The changes brought about are mostly visible in the primary structure of proteins coded by amino acids. It is not possible to alter the protein functions by modifying any sequence of amino acids. The properties of an enzyme can be altered by introduction of changes only in the specific regions of the amino acids. Therefore, it becomes necessary that the genes coding for such portions be identified and then modified as required.
The properties which are usually targeted for improvements by enzyme engineering are- kinetic properties, allosteric regulation; specificity; effect of factors like temperature, solvents, pH; etc
Various steps involved in enzyme engineering is
(i) Study of enzymes: the particular enzyme is first isolated and the desired properties are studied. Factors affecting its properties like structure are studied extensively including the primary, secondary, tertiary structure. Various techniques like NMR are employed for these studies.
(ii) Molecular modelling: Once the structure and its coding sequences are known, it is possible to estimate the effect of amino acid modification on the structure and consecutively on the function. Gathering together all the information, molecular modelling is carried out to determine the actual level of effect and whether any other further modification is needed.
(iii) Site directed mutagenesis: this process involves controlled alteration of genes at specific sites at specific bases. The genes responsible for the amino acid is first isolated and cloned using site directed mutagenesis to obtain a gene construct. A recombinant DNA construct so produced is introduced into a host organism and expressed to produce modified amino acids. The changes in amino acids are reflected in the properties of enzymes produced by recombinant DNA from the native enzymes.
Advantages of enzyme engineering are many like, enhanced reaction rate, less influence of physical factors, greater stability. This can be used to enhance the productivity of industrially important enzymes and thus helps in high rate of production. It is also possible that different properties of same enzyme be modified by different mechanisms, to result in double the activity of same enzyme.

Successful examples of enzyme engineering;
Subtilisin: This is an enzyme isolated from Bacillus amyloliquefaciens. This serves as a proteolytic enzymes mostly having application in the production of detergents. The enzyme has been modified so as to improve its properties like stability to temperatures, pH and oxidation conditions. The modification of these properties of this enzyme helps in its enhanced activity resulting in better quality detergent.
In this enzyme, the specificity to different peptides is coded by amino acids in the P1 cleft of the enzyme. Regulation of amino acid sequence results in the increased specificity and affinity of enzyme to only related peptides. Thus the property of specificity was enhanced. Similarly, several studies revealed that the enzyme becomes inactivated in the presence of bleaches as a result of release of oxygen molecules during the reaction. This oxidation reaction resulted in affecting the methionine at position 222(Met 222) of the amino acids resulting in formation of sulphoxide and consequent inactivation of enzymes. On replacement of the met222 by serine or alanine, the enzyme was modified to become stable against oxidation by bleaches.
Lactate dehydrogenase: It is an enzyme obtained from Bacillus stearotherophilus. This enzyme can be modified to shift its specificity from lactate to maleate. The specificity was found to be linked to presence of amino acid glutamine at position 102. When this was replaced with another amino acid arginine, the enzyme underwent a specificity change from lactate to maleate.

Enzymes in its native form have many widespread applications. But often the availability becomes scarce accounting to a higher cost of production for industrially important enzymes. Enzyme engineering is looked upon to modulate or produce new enzyme with maximum efficiency and high yield.