Enzyme Engineering

Four primary goals are usually pursued when engineering a protease for synthesis. First, one needs to make the enzyme a more stable catalyst especially towards organic solvents. Second, and usually more difficult to accomplish, one needs to improve the enzyme's synthetic efficiency, mainly by reducing the rate of competitive hydrolysis reactions. Unfortunately, the molecular basis of control of the rate of hydrolysis of the acyl enzyme intermediate is only poorly understood so there is, currently, no general approach enabling direct suppression of competitive hydrolysis reactions by enzyme engineering and optimization of active-site specificity for better binding of the acyl acceptor is the only practical way of minimizing un-desired hydrolysis of the acyl donor. Third, one needs to reduce the usually unwanted proteolytic activity of proteases to prevent competitive peptide cleavage during synthesis. Several approaches have been developed and a few have been found to be synthetically useful. Nevertheless, practically all the resulting enzyme variants suffer from significantly lower overall enzyme activity making it questionable whether they will reach industrial use in large-scale synthesis. Fourth, one needs to extend or alter the native specificity of the enzyme to fit the requirements of the synthesis. As for all other goals, chemical and genetic modification can both be used to create biocatalysts with the desired function different from that of the original parent enzyme. Selected examples related to proteases will be presented below.

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