Modulating Enzyme Reaction by Amino Acid Substitutions

5 Modulating Enzyme Reaction by Amino Acid Substitutions 

Traditionally, the enzyme sources are limited to extraction of natural products. The low production rate of enzymes leads to relatively high cost for a biotransformation process. With the development of reeombinant DNA and DNA sequencing techniques, it is feasible to produee reeombinant enzymes in various host expression systems, such as E. coli, Bacillus subtilis, yeasts or filamentous fungi. 

Although these host systems are easy to cultivate and ean produee satisfying amounts of functional enzymes, the intrinsie defeet of enzymes still impedes their industrial application. An initially isolated enzyme, either from environmental samples or from genomie databases, has evolved naturally and been selected under the stress eonditions among diverse populations of gene variants whose sequenee variations rendered possible different enzyme properties. However, the fitness of a wild-type enzyme is usually not satisfactory, considering the aetivity, thermostability or seleetivity of an enzyme for a specific chemical reaction. 

All enzymes are proteins, which are linear sequences of amino acids linked by peptide bonds. The folding of these sequences determined the secondary structure (such as a-helix, p-sheet or p-turn) and tertiary structure. Therefore, the properties of an enzyme are actually presumed from its sequence of amino acids. Some amino acids, dubbed hot spots , especially the ones in the active site where substrate binds, are sensitive to catalytic properties of an enzyme. Substitution of these important amino acids can significantly improve the activity or enantioselectivity toward a certain reaction. Protein stability is also maintained by the intramolecular and intermolecular interactions between residues of amino acids, including van der Waals forces, hydrophobic forces, electrostatic forces, hydrogen bonds and disulfide bonds. Detailed analysis of these amino acids, usually located in the protein surface, sheds light on the protein design for better thermostability. 

DNA shuffling offers an alternative technique for directed evolution of biocatalysts. Compared with point mutagenesis using epPCR, DNA shuffling 

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