et al., however, reported that Z-Asp inhibited the condensation reaction by immobilized thermolysin (24). The inhibition effect toward the condensation reaction can be avoided by choosing the reaction conditions, as suggested by Harada et al. According to Harada et al. (33), enzyme inactivation may be caused by adsorption of thermolysin on the surface of crystals of the addition compound of Z-APM and PheOMe. Apparently, Z-Asp is likely to prevent such adsorption.

In contrast to the protection by Z-Asp, thermolysin is not protected from inactivation by L-PheOMe, although the latter is another substrate of the condensation reaction; this may be because of much higher Km value of ther-molysin toward L-PheOMe. According to results of earlier kinetic studies of the condensation reaction by Oyama et al. (34), a double reciprocal plot toward L-PheOMe and D,L-PheOMe showed no apparent Km value, whereas that toward Z-Asp is 10.3 mM. These authors presented a reaction model where thermolysin at first forms an enzyme-Z-Asp complex, and then Z-APM is produced by the attack of L-PheOMe on the complex, as shown in Scheme 3. In this scheme, TLN indicates thermolysin. This model suggests that thermolysin cannot form an enzyme-L-PheOMe complex to prevent autolysis and adsorption on crystals of the addition compound. Therefore, thermolysin does not seem to be stabilized by L-PheOMe.

The stabilization by Z-Asp is quite effective for ther-molysin stabilization during APM production because the effect can be obtained quite simply by adjusting the concentration of the substrates. Moreover, Harada et al. mentioned (33) that Z-Asp stabilized thermolysin without presence of calcium ions. This is another advantage of this stabilization method because various problems caused by calcium scaling can be avoided by this method. As described earlier, enzyme inactivation during the condensation reaction can be prevented by the adjusting the ratio of the two substrates. Recently another method to stabilize thermolysin during condensation was revealed by Harada et al. (35): an addition of toluene into the reaction mixture at about 30% (w/w) of low materials. It prevented adsorption of thermolysin onto the crystal surface of the addition compound of Z-APM and PheOMe and resulted in an increase of thermolysin recovery after the reaction.


Scheme 3.


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