Molecular Properties and Extracellular Processing of the Lipase of Staphylococcus warneri M

Abstract

Staphylococcus warneri M exhibited extracellular lipase activity. By zymogram analysis of extracellular proteins, multiple bands were detected and the profiles changed depending on the bacterial growth phase. N-terminal amino acid sequences of three bands (N1–N3) were determined. From the genome library of S. warneri M whole DNA, the gene-directing lipase activity (named gehC_WM) was cloned and characterized. The gehC_WMgene encoded a protein (GehC_WM), whose calculated molecular mass was 83.4 kDa, and the sequence was similar to the other staphylococcal lipases. Though two lipases have been known from S. warneri 863, GehC_WM differs from both of them, indicating that this enzyme is the third extracellular lipase of the S. warneri strain. The N-terminal sequences of the N1–N3 polypeptides completely coincided with the deduced amino acid sequences in GehC_WM. GehC_WM was predicted to be a prepro-protein. In vitro processing and protein sequencing suggested that pro-GehC_WM is possibly processed by extracellular glutamyl endopeptidase, PROM. Inductively coupled plasma-atomic emission spectrometer analysis showed that purified his-tagged mature GehC_WM possessed zinc ion. A gehC_WM knockout mutant was constructed by insertion of an erythromycin resistance gene into the gehC_WM. Zymogram and immunoblot analyses of the gehC_WMmutant indicated that GehC_WM was a major extracellular lipase of S. warneri M.