The history of genetic enzyme modification is closely connected with proteases and with subtilisin in particular. The first genetic modifications in this enzyme were conducted soon after the gene was cloned in the early 1980s . Two decades later mutations in well over 50% of the 275 amino acids of subtilisin have been reported. Several other proteases also became targets of genetic modification and a variety of useful techniques now exists for introducing changes into the enzyme at the genetic level. Basic principles and selected examples related to proteases will be summarized below.
Site-directed mutagenesis can be regarded as one of the first genetic modification techniques; it has proved to be useful for engineering a protease for synthesis. Although simple to perform, it does, however, require that one have some idea of which residues are important. Having a three-dimensional structure of the enzyme in question is particularly helpful in selecting those important moieties. Because of the inability to predict long range structural changes, most protease engineering involves catalytic amino acids, substrate binding regions, and direct stabilizing mutations. With regard to peptide synthesis, subtilisin species with enhanced synthetic efficiency, modified specificity, improved stability, and altered pH profile have been designed by this method [2, 19]. Efforts in this field led, for example, to a double mutant ("subtiligase"), in which the catalytic Ser221 is exchanged with Cys, and Pro225 with Ala . The enzyme variant was used to synthesize wild-type and mutant ribonuclease A in milligram quantities by stepwise ligation of six es-terified peptide fragments [20b]. Single and multiple site-specific mutations have also been extensively used to obtain subtilisin variants with increased stability toward inactivation by organic solvents . Although several aspects of the stability of the enzyme have been significantly improved, the successful rational design of
Tab. 5.1.1. Effect of active-site glycosylation at the artificial cysteine166 at the base of the primary specificity Sj pocket on the substrate tolerance of subtilisin (according to ref. [1 7b], with permission from the Royal Society of Chemistry).a
Acyl donor Acyl acceptor Product Yieldh (%)
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