Cloning and characterization of a novel intracellular serine protease (IspK) from Bacillus megaterium with a potential additive for detergents.

A new intracellular serine protease gene of Bacillus megaterium, ispK, encoding a protein composed of 332 amino acid residues with a predicted pI of 4.7 was cloned into Escherichia coli. The deduced amino acid sequence of IspK showed 49-56% similarity with the other microbial intracellular serine proteases described in the literature. The enzyme was effectively purified by one-step chromatography after heat-treatment, and showed a homogeneous band corresponding to 35kDa by SDS-PAGE analysis. Amino acid analysis showed that 16 amino acids of the N-terminus of IspK were removed by post-translational protease activity. The optimum pH and temperature of IspK were 6.0-7.0 and 50°C, respectively. In the presence of 2mM of Ca2+ ion, the optimum temperature was increased to 65°C and thermostability (t1/2) increased 32.9-fold from 3.3min to 108.5min at 60°C. The enzyme was activated by Ca2+ and Mg2+, almost completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and EDTA, but tolerant to nonionic surfactants, such as, Triton X-100 or Tween 80. IspK efficiently hydrolyzed natural proteins, such as, casein and hemoglobin, and improved blood stain removal. These results suggest IspK can be used as a useful additive for detergent formulations and for deproteinizations.