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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 ~ 40 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. [ABSTRACT FROM AUTHOR]

We report structural characterization of proteases mined in silico from solid tannery waste (STW) metagenome. The physico-chemical analysis revealed the molecular weight of selected query proteases in the range of 34–43 kDa except 52.46 kDa for Cp-6. Secondary structure analysis suggested the dominance of α-helices (26–51 %) followed by β-sheets (13–34 %). Conserved regions in the selected proteases were identified using multiple sequence alignment. Diversity analysis of the proteases was performed by aligning with their best hits and already characterized proteases of similar families. The 3D structures of 19 selected amino acid sequences were deduced using homology modeling and evaluated following Ramachandran plots (R-plots), stability of the 3D conformation, and overall quality factor (G factor). The R-plots of all structures had 96.4–98.9 % residues in the favoured region, whereas 0-0.8 % lied in the outlier region. Fifteen modeled protein structures passed the quality assessment criteria and were subsequently used for molecular docking. All models showed two domains except Cp-6, which had an extra domain. Ligand binding sites and active sites were identified using sequence and structural homologs of the respective protease molecules. Serine, lysine, and serine of different conserved motifs were specified as the active site residues in carboxypeptidases, whereas serine, aspartate, and histidine were specific to aminopeptidases Ap-1, Ap-2, and Ap-4. Further, Ap-8 and Ap-9 required Mn2+ for enzyme activity along with histidine and glutamate in their active sites. These 3D structures were used for molecular docking with their specific peptide ligands to obtain stable docked conformations. Successfully docked complexes indicated the catalytic activity of these enzymes in hydrolyzing the peptide ligand. The structural and functional insights gained from our study may help in the identification of novel industrially important proteases after validation through experimental studies. [ABSTRACT FROM AUTHOR]