Enzyme Improvement by Site Directed Mutagenesis

Improvement of enzymes by site-directed amino acid substitutions at selected sites has recently become possible by the progress in genetic engineering and of computer simulation of the three-dimensional structure of the protein molecules. These so-called protein engineering techniques are becoming an important tool for the improvement of enzyme character. A first example of improved thermolysin-like metalloproteases was shown by Imanaka et al. (24) using a metalloprotease from Bacillus stearothermophilus CU21. This protease exhibited 85% homology in amino acid sequence with thermolysin. They substituted the 141st glycine of this protease to alanine and expected to find enhancement of the internal hydrophobicity and stabilization of the internal a-helix. The resulting mutant protease indeed exhibited higher thermostability than did wild-type protease (25). Another example of stabilization of this metalloprotease was reported by Hardy et al. (26), who introduced proline at the 65th or 69th site. Although the stability of this protease was lower than that of ther-molysin even with introduction of these mutations, it may be expected that thermolysin can be improved by a similar procedure. Thus, studies for the improvement of thermo-lysin by protein engineering were started by a collaboration of TOSOH and Sagami Chemical Research Center.

In this research, attention was focused to enhancing the activity of thermolysin rather than its stability. Even when the stability of thermolysin is enhanced until the enzyme inactivation during the condensation reaction is at a negligible level, the enzyme has to be recycled for reduction of enzyme costs. This requires additional equipment or procedures to recover the enzyme from the process solution, such as concentration of enzyme by ultrafiltration, salting out, or immobilization of enzyme (9). This will increase the fixed costs at an APM plant. On the other hand, if the activity is enhanced adequately and if the enzyme amount in the APM production can be reduced to a negligible level by application of highly active mutant thermolysins, it would be possible to use the enzyme without recycling. A highly active mutant thermolysin would be more effective than a highly stable one. Additionally, useful knowledge would be gained about the mechanism determining the relationships among the proteins' structures, properties, and functions from the improvement of thermolysin activity.

Thus, studies for improvements of thermolysin were started, and highly active mutant thermolysins were successfully constructed. One of these mutants exhibited a five times higher activity toward Z-APM synthesis when it is evaluated by the initial reaction rates (27). Details of the improvements of thermolysin are described in the article entitled THERMOLYSIN. The stability of these mutant ther-molysins was the same as that of wild-type thermolysins when it was determined by calorimetry (28). It is therefore expected that mutant thermolysin can be used for APM production in the same way as wild-type thermolysin, except that the enzyme amount to obtain the same reaction rate is lower with mutant thermolysin.

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