To improve the condensation reaction in the enzymatic process, reduction of enzyme consumption may be of great importance. Although thermolysin is currently the most stable of the proteases industrially available (16), some portion of it is inactivated during the condensation reaction. This enzyme inactivation, of course, influences production costs. Stabilization of the enzyme during the condensation reaction thus was one of the most important aspects of cost reduction.
It is well known that calcium ion stabilizes many mi-crobial proteases, and thermolysin is also stabilized by it (14). If the concentration of calcium ions in the reaction mixture is increased, it will diminish the inactivation of thermolysin. However, this would not be suitable to improve this process, because high concentration of calcium will frequently cause troubles caused by scaling.
If thermolysin can be replaced by a more stable protease, the enzyme loss can be reduced further. Extremely stable proteases such as archaelysin (17) and a serine protease from Desulfurococcus strain SY (18) were recently found in cultures of hyperthermophilic archaea (formerly called archaeobacteria). The archaea, such as Desulfurococcus, Pyrococcus, or Thermococcus, showed an optimum temperature for growth around 90 °C and even can grow above 100 °C. Their enzymes exhibited extremely highsta-bility against extreme conditions; archaelysin and the protease from D. strain SY retained 50% of their activity when they were heated at 95 °C for 80 min and 450 min, respectively (17,18). These proteases are expected to have applicability in various industrial fields (19). Unfortunately, no metalloprotease has yet been found from hyperthermo-philic archaea. The proteases separated from the archaea up to now belong to serine proteases (17,18) or cysteine proteases (20). Because their specificity toward Z-APM is similar to that of subtilisin or papain, they are not appropriate for APM production. They will hydrolyze the ester bond of PheOMe or APM in the same way as subtilisn or papain. Although finding a metalloprotease from such ar-chaea would be desirable, thermolysin remains the most stable protease currently available for APM production. Recently a metalloprotease was found from a culture broth of a hyperthermophilic aerobic archaeon "Aeropyrum pernix K1" that also exhibited extremely high stability against thermal denaturation with a half-life of 70 min at 125 °C (21), although its specificity for APM relating pep-tides has not been solved.
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