Purification and Characterization of a DegP-Type Protease from the Marine Bacterium Cobetia amphilecti KMM 296

Abstract

A new member of the DegP-type periplasmic serine endoproteases of the S1C family from the marine bacterium Cobetia amphilecti KMM 296 (CamSP) was expressed in Escherichia coli cells. The calculated molecular weight, number of amino acids, and isoelectric point (pI) of the mature protein CamSP are 69.957 kDa, 666, and 4.84, respectively. The proteolytic activity of the purified recombinant protease CamSP was 2369.4 and 1550.9 U/mg with the use of 1% bovine serum albumin (BSA) and casein as the substrates, respectively. The enzyme CamSP exhibited maximum activity at pH 6.0–6.2, while it was stable over a wide pH range from 5.8 to 8.5. The optimal temperature for the CamSP protease activity was 50 °C. The enzyme required NaCl or KCl at concentrations of 0.3 and 0.5 M, respectively, for its maximum activity. The Michaelis constant (Km) and Vmax for BSA were determined to be 41.7 µg/mL and 0.036 µg/mL min−1, respectively. The metal ions Zn2+, Cu2+, Mn2+, Li2+, Mg2+, and Ca2+ slightly activated CamSP, while the addition of CoCl2 to the incubation mixture resulted in a twofold increase in its protease activity. Ethanol, isopropanol, glycerol, and Triton-X-100 increased the activity of CamSP from two- to four-times. The protease CamSP effectively degraded the wheat flour proteins but had no proteolytic activity towards soybean, corn, and the synthetic substrates, α-benzoyl-Arg-p-nitroanilide (BAPNA) and N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide (SAPNA).