An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus

作者：Asano Y^*; Ito H; Dairi T; Kato Y

来源：Journal of Biological Chemistry, 1996, 271(47): 30256-30262.

We purified a novel extracellular D-stereospecific endopeptidase, alkaline D-peptidase (D-stereospecific peptide hydrolase, EC 3.4.11.-), to homogeneity from the culture broth of the soil bacterium Bacillus cereus strain DF4-B. The M(r) of the enzyme was 37,952, and it was composed of a single polypeptide chain. The optimal pH for activity was similar to 10.3, The enzyme was strictly D-stereospecific toward oligopeptides composed of D-phenylalanine such as (D-Phe)(3) and (D-Phe)(4). The 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-Phe)(2)) and eight stereoisomers of (Phe)(3). The enzyme had beta-lactamase activity toward ampicillin and penicillin G, although carboxypeptidase DD and D-aminopeptidase activities were undetectable. The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1164-base pair open reading frame consisting of 388 codons was identified as the adp gene, The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from Streptomyces lactamdurans and Bacillus subtilis, and class C beta-lactamases, Thus, the enzyme was categorized as a new ''penicillin-recognizing enzyme.''

• 出版日期1996-11-22