Stabilization of Thermolysin by Change of Reaction Conditions

Studies on stabilization of thermolysin by controlling the reaction conditions were also continued separately by a collaboration of TOSOH and DSM. Harada et al. reported such stabilization by an N-protected amino acid, such as Z-Asp, Z-Glu or Z-Gly, during storage of thermolysin solution (33). Z-amino acids seem to act as inhibitors against the autolysis of thermolysin herein. The inhibitory effect of Z-Ala was reported earlier by Morihara and Tsuzuki (10) after they had found that Z-Ala inhibited hydrolysis of Z-Ala-Leu-Ala catalyzed by thermolysin at 30 mM of Ki value. Although these results for Z-Ala are not conclusive for determining whether inhibition by protected amino acids is competitive, it now appears that Z-amino acids are likely to bind to the active site of thermolysin instead of substrates.

Moreover, Harada et al. demonstrated that thermolysin in the reaction mixture was kept stable when Z-Asp concentration is kept at a high level during the condensation reaction. They studied effects of the ratio of Z-Asp and d,l-PheOMe at the start of the condensation reaction. An example of the results is summarized in Table 2. When the reaction was started at a 1:2 ratio of Z-Asp to PheOMe, the enzyme recovery was 92%, whereas it was 44% at a ratio of 1:2.5. Remaining Z-Asp after the reactions were 55.9 and 10.5 mmol/kg, respectively. At the ratio of 1:2, much more enzyme and Z-Asp were remaining than at the ratio of 1:2.5. The remaining Z-Asp apparently protects the enzyme from inactivation. Productivity of Z-APM at the ratio of 1:2 was 0.225 mol/kg; at 1:2.5 it was 0.238. Thus, significant decrease of productivity was not observed by higher concentration of Z-Asp. This indicates that the synthesis is not inhibited by Z-Asp, whereas Z-Asp is an inhibitor against autolysis or peptide hydrolysis. Nagayasu

Table 2. Condensation Reaction at Different Z-Aspartic Acid-to-PheOMe Ratio

Z-Asp/

Initial

Conversion

Produced

Remained

Enzyme

PheOMe

[Z-Asp]

rate

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