One bacterial strain D2 (GenBank accession number CGMCC No. 1868) with a taxonomic affiliation to Stenotrophomonas maltophilia was isolated from the QINGDAO coast in China and can produce extracellular alkaline protease. In this study, the alkaline protease was purified 4.9-fold by a four-step purification protocol with 33% activity recovery and was designed S. maltophilia Protease-D2 (SmP-D2). The molecular weight of the protease was approximately 42 kDa viewed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified D2 protease exhibitedstability over pH 6–11 and in the temperature range of 0-60°C with optimal activity at pH 9and 60°C, respectively. The activity of the enzyme was enhanced with Cu2+, Ca2+, and K+respectively, while Ag+ and Mg2+ had little effect on the enzyme activity. The activity of the enzyme inhibited by ethylene diamine tetra acetate (EDTA) and phenylmethanesulfonyl fluoride (PMSF), indicating its serine type. However, the purified enzyme displayed significant stability to dithiothreitol (DTT), urea and ethanol. The enzyme activity was moderate in the presence of anionic (SDS). The N-terminal amino acid sequence of two selected tryptic peptides was determined as NH2-AHGFLPLTK and NH2-APSATGGSALYPLEFVVGK, respectively, by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The extremely high optimal pH and moderate thermaltolerance tolerance of the alkaline serine protease SmP-D2 suggested it may be potential for use as a biocatalyst in industry.
Key words: Stenotrophomonas maltophilia, alkaline serine protease, purification.
PMSF, Phenylmethanesulfonyl fluoride; EDTA, ethylene diamine tetra acetate; DTT, dithiothreitol; SDS, sodium dodecyl sulphate.
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