Isolation and some properties of an arylamidase from Flavobacterium IIb.

A constitutive L-leucylarylamidase (EC 3.4.11) hydrolase able to cleave L-aminoacyl-beta naphthylamide and L-aminoacyl-4 nitroanilide substrates, was isolated from sonicated cells of Flavobacterium IIb and partially purified with a 0.9% yield and a 159-fold recovery. Its molecular weight was estimated to be about 170,000 +/- 10%. This arylamidase exhibited optimum activity at pH 7.0 and 28 degrees C for the hydrolysis of L-leucine-4NA and is inhibited strongly by metal chelating agents, and to a weaker extent, by some sulfhydryl and reducing agents. Heavy metal ions: Cd2+, Zn2+, Cu2+, Hg2+ and Co2+, markedly inhibit it, and Zn2+ is a competitive inhibitor. This metalloenzyme, free of carboxypeptidase, proteinase and L-leucine aminopeptidase (L-leucylglycine substrate) activities, hydrolyzes aminoacyl-beta NA, aminoacyl-4NA and some dipeptides with unsubstituted amino groups of the L-configuration. The lowest Km values are associated with substrates having neutral or basic residues, with large side chains.