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Figure 3. Effect of pH on the initial reaction rate in thermolysin catalyzed condensation reaction. Curves are shown for TZ-1 (open circle) and wild type (closed circle), respectively, for 0.2 M of Z-l-Asp and 0.5 M of d,l-PheOMe and containing 5.1 mM CaCl2 and 0.17 M NaCl at 40 °C.

of thermolysin activity, because the pKa value of the 231st histidine is likely to be shifted toward the acidic side by introduction of a plus charge of histidine at the 227th site. This shift in optimum pH is not observed, however, when the activity is evaluated under the model conditions where the optimum pH of both wild type and TZ-1 are pH 7.0, as is mentioned in Thermolysin. An explanation for the difference of the optimum pH between the model conditions described in there and the production conditions described herein have not yet been found, although important factors may be (1) concentration of substrates, (2) ratio of the two substrates, and (3) optical purity of PheOMe.

Typical time courses of the Z-APM synthesis by TZ-1 and wild type at pH 6.0 are shown in Figure 4. Reaction rate and production yield by 50 mg of TZ-1 are the same as those by 150 mg of wild type. When the reaction is performed with 150 mg of TZ-1, the initial reaction rate is three times higher than that by the same amount of wild type. It seems that activity of TZ-1 is three times higher than that of wild type under this condition. Remaining activities of both wild type and TZ-1 after the condensation reaction were 70%. This indicates that the stability of TZ-1 is the same as that of wild type. Moreover, there are no differences in thermostability between TZ-1 and wild type as has been demonstrated by heat capacity analysis with the calorimeter as described earlier. From these results, it follows that mutant thermolysins can be useful for cost reduction in APM production.

Thus, studies for improvement of thermolysin were continued. Another important amino acid substitution, the 150th aspartic acid to tryptophan (D150W) (32) was recently found. The activity of the single site mutant D150W was five times higher than that of wild type. This is the same as the effect of the combination of three mutations, L144S, D150H, and N227H in TZ-1. Combination of D150W with L144S and N227H resulted in a new mutant, L144S-D150W-N227H, which exhibits about 10 times higher activity than wild type under the model conditions

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