Isolation and biochemical characterization of a novel serine protease identified from solid tannery waste metagenome

Abstract

Metagenomics facilitates the isolation of novel enzymes from the unculturable microorganisms inhabiting a particular ecosystem. In the present study, solid tannery waste (STW) metagenome was explored as a potential source of microbial serine proteases. KEGG annotated functional profile of the UNK metagenome was exploited for in silico mining of candidate serine proteases. A novel serine protease was identified, namely Prt1SU, corresponding to an 1149 bp full-length gene encoding a 383 amino acid protein. The identity of the Prt1SU was evaluated by PSI-BLAST against the databases at NCBI and BLAST against the MEROPS database. Identities ranged from 30–45.7% to their respective nearest homologs of the serine protease family. Utilizing the DNA template isolated from the UNK metagenome, the candidate serine protease gene was cloned in the pET28a(+) vector and expressed in E. coli BL21 (DE3) strain. Although the Prt1SU was overexpressed at 18oC using 0.1 mM IPTG, it remained inactive because of the formation of inclusion body. After solubilization with 8 M urea, the Prt1SU enzyme was purified to homogeneity using Ni-NTA chromatography under denaturing conditions and then renatured to retain protease activity. The purified Prt1SU with a molecular weight of ~\u200940 kDa exhibited the maximum enzyme activity at pH 9.0 and temperature 37oC. The enzyme activity of the Prt1SU was enhanced by the addition of organic solvents, and the enzyme was found stable in the presence of anionic and non-ionic surfactants and oxidizing agents, suggesting that Prt1SU is suitable for various industrial applications as well as wastewater and STW treatment.