Characterization of a Novel PepF-Like Oligopeptidase Secreted by Bacillus amyloliquefaciens 23-7A

Abstract

An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepF Ba oligopeptidase is a secreted protein and may facilitate the process of sporulation.In various species and tissues, peptidases of family M3 are involved in peptide degradation, bioactive neural-peptide synthesis, and cleavage of signal peptides (3,4,14,16). This kind of endopeptidase only hydrolyzes oligopeptides that contain no more than 20 amino acid residues. Bacterial PepFs also belong to the M3 family of peptidases. In this report, another PepFlike oligopeptidase was identified in a collagen-degrading strain, Bacillus amyloliquefaciens 23-7A. This peptidase, designated as PepF Ba , was characterized in great detail, and its potential physiological roles were also discussed.Bacterial cultivation and enzyme purification. The microbial strain 23-7A bearing collagenolytic activity was screened from soil in Taiwan. The bacterium was spore-forming, grampositive, and taxonomically identified as Bacillus amyloliquefaciens by 16S rRNA gene sequence and API strips. During cultivation of this strain in the basal medium (1% defatted soybean as the nitrogen source, 1% glucose, 0.5% yeast extract, 0.1% K 2 HPO 4 , and 0.2% MgSO 4 ) at 37°C using a Biostat B 5-liter fermentor (Sartorius BBI Systems Inc.), the cell density and number of spores were determined. Since PepF Ba is the only secreted protease that is able to cleave synthetic substrate N- (3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) in this strain (data not shown), proteolysis of FALGPA was used to monitor PepF Ba that was present in the supernatant of collected cell pellets. PepF Ba activity appeared at the beginning of exponential phase and lasted until spores began to form (Fig.

Έλεγχος Της Δομής Και Της Δράσης Ενζύμων Με Υπερυψηλή Πίεση

“…Η PepF δεν μπορεί να υδρολύσει τη Μετενκεφαλίνη, σε αντίθεση με την PepO, για την οποία αποτελεί πολύ καλό υπόστρωμα. Οι βέλτιστες συνθήκες για τη δραστικότητα της PepF είναι pH 7-9 και θερμοκρασία ~40 ο C. Η PepF απενεργοποιείται σε θερμοκρασία πάνω από 55 ο C(Asdornithee et al, 1995;Chao et al, 2006). Η ολιγοπεπτιδάση F βρίσκεται στο κυτταρόπλασμα του L. lactis, ο οποίος χρησιμοποιείται ευρέως στην τεχνολογία παρασκευής τυριού.…”