In this study, a novel metallo-β-lactamases fold hydrolase PH-1 was identified from Pelagibacterium halotolerans B2(T). This novel member of the family Hyphomicrobiaceae was isolated from the East China Sea. In silico analysis demonstrated that PH-1 and its relative homologues cluster in a unique branch and constitute a new subgroup among MBLs. PH-1 was cloned and overexpressed in Escherichia coli BL21 in a soluble form. SDS-PAGE, MALDI-TOF/TOF-MS, and size-exclusion chromatography analysis demonstrated that the PH-1 was a monomer with molecular weight of about 29 kDa. Substrate specificity study showed PH-1 preferred penicillin type β-lactams and exhibited maximum activity toward penicillin-G. Additionally, our experiments also revealed that PH-1 was a halotolerant enzyme since it is active under 4 M NaCl. The enzyme activity of PH-1 was negatively affected by 1 mM Mn(2+) and EDTA. These observations lay a foundation for further study of MBLs from marine bacterium.
Keywords: Halotolerant; Metallo-β-lactamase; Pelagibacterium halotolerans; Purification.