Substrate specificity of a new alkaline elastase from an alkalophilic bacillus.

The substrate specificity of alkaline elastase from alkalophilic Bacillus sp. Ya-B was studied by using a number of synthetic substrates. From the relative hydrolysis rate for p-nitrophenyl esters and t-butoxycarbonyl-L-Phe-L-Arg(NO2)-X-L-Phe-p-nitroanilide (X = L-Ala, Val, Leu, Ile, and Gly), the subsite S1 and S2 were concluded to be specific for L-alanine and glycine. The alkaline elastase rapidly hydrolyzed elastase specific substrate succinyl-L-Ala3-p-nitroanilide and succinyl-L-Ala-L-Pro-L-Ala-p-nitroanilide. These results prompted us to characterize our enzyme as a microbial elastase. Inhibition study with carbobenzoxy-L-Phe-chloromethyl ketone (ZPCK), Z-L-Ala-L-Phe-CK (ZAPCK), Z-L-Ala-Gly-L-Phe-CK (ZAGPCK), and kinetic study with succimyl-L-Ala2(3)-p-nitroanilide revealed that the enzyme has at least four subsites.