Purification and characterization of a cold-active protease from psychrotrophic Serratia marcescens AP3801

Protease activity was detected in the culture medium of Serratia marcescens AP3801 grown at 10°C, which was isolated from soil collected from the top of a mountain. The enzyme, designated as CP-58 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 58 kDa, and its isoelectric point was close to 6.0. Maximal activity toward azocasein was observed at 40°C and from pH 6.5 to 8.0. The activity was strongly inhibited by 1,10-phenanthroline, suggesting that the enzyme is a metalloprotease. The N-terminal amino acid sequence was Ser-Leu-Asn-Gly-Lys-Thr-Asn-Gly-Trp-Asp-Ser-Val-Asn-Asp-Leu-Leu-Asn-Tyr-His-Asn-Arg-Gly-Asn (or Asp)-Gly-Thr-Ile-Asn-Asn-Lys-Pro-Ser-Phe-Asp-Ile-Ala. A search through databases for sequence homology aligned CP-58 protease with metalloprotease. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-58 protease has a broader specificity than other proteases against the peptide substrate.

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