A novel subtilisin-like serine protease from Thermoanaerobacter yonseiensis KB-1: its cloning, expression, and biochemical properties

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Abstract
A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases. The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252). Thermicin was overproduced in E. coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography. Thermicin from E. coli showed maximum proteolytic activity at 92.5°C and pH 9.0, and its half-life was 30 h at 80°C. In order to determine cleavage specificity, thermicin was incubated with insulin β chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved. Thermicin is well known to hydrolyze Gly- and Pro-rich collagens. The K m and k cat /K m values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 µM and 142.05 (105 s–1 M–1), respectively, at 92.5°C and pH 9.0. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins. This result indicates that thermicin is a novel enzyme different from other thermostable proteases.

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