AN ALKALINE D-STEREOSPECIFIC ENDOPEPTIDASE WITH BETA-LACTAMASE ACTIVITY FROM BACILLUS-CEREUS

We purified a novel extracellular D-stereospecific endopeptidase, alka line D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cer eus strain DF4-B. The M(r) of the enzyme was 37,952, and it was compos ed of a single polypeptide chain. The optimal pH for activity was simi lar to 10.3, The enzyme was strictly D-stereospecific toward oligopept ides composed of D-phenylalanine such as (D-Phe)(3) and (D-Phe)(4). Th e enzyme also acted to a lesser extent on (D-Phe)(6), Boc-(D-Phe)(4) ( where Boc is tert-butoxycarbonyl), Boc-(D-Phe)(4) methyl ester, Boc-(D -Phe)(3) methyl ester, Boc (D-Phe)(2), (D-Phe)(2), and others, but not upon their corresponding peptides composed of L-Phe, (D-Ala)(n) (n = 2-5), (D-Val)(3), and (D-Leu)(2), The mode of action of the enzyme was clarified with synthetic substrates ((D-Phe)(2)-D-Tyr and D-Tyr-(D-Ph e)(2)) and eight stereoisomers of (Phe)(3). The enzyme had beta-lactam ase activity toward ampicillin and penicillin G, although carboxypepti dase DD and D-aminopeptidase activities were undetectable. The gene co ding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, an d a 1164-base pair open reading frame consisting of 388 codons was ide ntified as the adp gene, The predicted polypeptide was similar to carb oxypeptidase DD from Streptomyces R61, penicillin-binding proteins fro m Streptomyces lactamdurans and Bacillus subtilis, and class C beta-la ctamases, Thus, the enzyme was categorized as a new ''penicillin-recog nizing enzyme.''