Evaluation of Mutant Thermolysins in APM Production

The highly active thermolysin exhibiting a five times higher activity than wild-type thermolysin was called TZ-1. TZ-1 has substituted amino acid residues at three sites, namely the 144th leucine to serine (L144S), the 150th aspartic acid to histidine (D150H) and the 227th as-paragine to histidine (N227H). Although this mutant exhibits five times higher activity toward Z-APM synthesis, it should be noticed that its activity was evaluated at conditions different from real APM production conditions as described in the article on thermolysin referred to earlier. The evaluation conditions were chosen to reduce the viscosity of the reaction solution; the concentration of substrates was lower than that under conventional production conditions, and the ratio of Z-Asp and PheOMe was set at about 1:1. These conditions enabled to obtain exact data in a milliliter scale of reaction mixture. These conditions are referred to as "model conditions." The model conditions enabled evaluating several candidates during molecular engineering of mutants. Because TZ-1 showed very high activity under the model conditions, its efficiency in small-scale APM production under the conventional production conditions was also examined.

The reactions were performed in 200 mL of a mixture containing 0.2 M of Z-L-Asp, 0.5 M of D,L-PheOMe, 5.1 mM CaCl2, 0.17 M NaCl and 50-150 mg of the thermolysin at 40 °C and in a range of pH between 5 and 7. Mutant ther-molysins used here were produced by fermentation of Bacillus subtilis MT-2, harboring recombinant plasmid p-UBTZ2 in a jar fermentor equipped with a 16-L vessel (New Brunswick Scientific Co. Ltd., U.S.). Other conditions for the fermentation were the same as described in the article on thermolysin. Enzyme amounts in the reaction mixture were determined by casein hydrolytic activity according to the method of Endo (29) as are also described in the article on thermolysin. The condensation reactions were monitored by HPLC equipped with TSK gel G2000SW (TOSOH, Japan) with 5% acetonitrile in 0.05% trifluoroacetic acid as solvent for determining the amount of the product and the remaining substrates. Before HPLC, the samples were diluted by appropriate solution, such as 10 mM calcium acetate, and the insoluble addition compound of Z-APM and PheOMe was removed by centrifu-gation. The conversion rates were calculated from remaining Z-Asp using the following formula:

Herein, [Z-Asp]0 and [Z-Asp]t refer to the concentration of Z-Asp at the initial and at an appropriate later time, respectively.

Effect of pH on the initial reaction rates by 50 mg of TZ-1 or wild type is shown in Figure 3. The optimum pH of TZ-1 was 6.0, and that of wild type was 6.5. At pH 6.0, the activity of the TZ-1 was about fivefold that of wild type. The optimum pH shift of TZ-1 seems to result from the introduction of a plus charge of histidine instead of the 150th aspartic acid and the 227th asparagine. Because the 150th residue and the 227th residue exist at vicinity of the Zn ion and the 231st histidine residue in the active center of thermolysin, respectively, the plus charge of histidine may influence the electrostatic interactions in this center. Both Zn ion and the 231st histidine are regarded as important to express thermolysin activity (30,31). The mutation N227H especially seems to influence the pH profile

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