Cloning and expression of thermostable protease gene from Bacillus Subtilis 50a in Escherichpa Coli BL21 (DE3) pLysS

A thermostable protease producer was isolated from a hot spring in Lojing Highlands, Kelantan. It was identified as Bacillus subtilis 50a based on morphology and 16S rRNA sequencing analysis. Its native protease displayed the maximal activity at pH 9 and showed high optimum temperature of 70˚C(18 U/ml). The thermostability profile exhibited the protease was very stable at 50˚C and up to 60˚C without significant loss of enzyme activity. To exploit the potential of high temperature activity and stability, gene encoding thermostable 50a protease was cloned into pTZ57R/T vector by PCR product cloning. The open reading frame (ORF) of thermostable 50a protease was 1086 bp in length coding for 361 amino acids and the calculated protien molecular mass of 37.261 kDa. Homology searches revealed that the amino acid residues from B. subtilis 50a protease share a high homology (95%) with the subtilisin from B.subtilis 168 and subtilisin J from Geobacillus stearothermophilus. The thermostable 50a protease gene was subcloned into the pET22b(+) expression plasmid. The recombinant plasmid was then transformed into E.coli BL21 (DE3) pLysS. The expression of recombinant E.coli BL21 (DE3) pLysS was optimized by inducing it with 0.5 mM of IPTG at 12 h of induction time (53 U/ml). 50a crude protease obtained from intracellular expression was purified by a single step heat-treatment. A single band was detected by SDS-PAGE analysis with a molecular weight around 37 kDa. The purified recombinant 50a protease had a specific activity of 2428.57 U/mg and the recovery of the 50a protease increased after heat treatment because the enzyme was activated by heat. The recovery achieved was around 242.86% with a purification fold of 7.21. Characterization of the recombinant thermostable 50a protease was carried out with the purified enzyme. Recombinant thermostable 50a protease was highly active at pH 9-10 and 80 ˚C(190 U/ml). The protease was fairly stable at 70˚C even up to 8 h of treatment and had half-lives of 7 h 30 min at 70˚C and 3 h 40 min at 80˚C. The enzyme activity was more than 90% inhibited by phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor. The results confirmed that the recombinant 50a protease was a serine protease and the characteristics of the recombinant 50a protease met with that of the native 50a protease.