Cloning, heterologous expression and characterization of a novel streptomyces trypsin in Bacillus subtilis SCK6.

A novel streptomyces trypsin GM2938 was selected as the object of study. The active GM2938 contains 223 amino acid residues. Constructing recombinant plasmid and transforming Bacillus subtilis SCK6, the heterogenous expression of GM2938 was achieved. Through optimization of fermentation conditions, the expression level of GM2938 reached 1622.2 U/mL (esterase activity) and 33.8 U/mL (amidase activity). The recombinant trypsin was purified and measured: the specific activity of esterase was 5.6 × 103 U/mg, and the specific activity of amidase was 1.1 × 103 U/mg. Furthermore, the enzymatic properties of GM2938 were explore: the optimal reaction temperature and pH were 50 °C and 9.0, respectively; the recombinant enzyme show high stability at 25 °C and range of pH 5.0-9.0; Ca2+, K+, Mg2+, EDTA, DTT, DMSO, methanol, glycerin and ethanediol could promote the esterase and amidase activities at the investigated concentrations, while Fe2+, SDS, tritonx-100, acetone, chloroform and n-hexane inhibited the trypsin activities. Kinetic parameters of GM2938 were calculated: the Km of BAEE was 3.15 × 10-5 mol·L-1, Vmax value was 2.87 × 10-4 mol·L-1·min-1; the Km of BAPAN was 2.20 × 10-4 mol·L-1, the Vmax was 2.40 × 10-4 mol·L-1·min-1. These properties give trypsin GM2938 a potential application prospect.