Industrial enzymes mutation

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

For an industrial biotransformation, it is often necessary to further optimize an appropriate biocatalyst. This includes the elimination of the follow-up enzymes in the wild-type strain by mutations and the improvement of other characteristics by additional mutations and the selection of improved strains. Alternatively, the genetic information for a desired enzyme might be introduced in a host that has many of the preceding characteristics, and that has no enzymes that could modify or degrade the desired product. 

Endo-xylanases can be used industrially in two ways 1) to remove xylan from paper pulp to give purer cellulose, and 2) to convert xylan into D-xylose or xylo-oligo-sac jharides that can be further converted into useful materials. In the first case, at least, it is clear that the enzyme preparation should be free of cellulases, either by (hoosing, or producing by mutation, a strain that makes xylanases but not cellulases, or by separating the lattCT from the former after they are produced. In nearly all cases t tie use of a cellulase-firee strain is preferable. 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

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