In Silico Characterization And Recombinant Protein Analysis Of Two Subtilisin-Like Serine Proteases From The Mushroom Lignosus Rhinocerus

Serine proteases are a class of proteolytic enzymes with important physiological functions in living organisms. Due to their wide medical and industrial applications, there is an increasing interest in isolation and characterization of novel serine proteases from natural sources. Two serine proteases (4347-SPL and 8711-SPL) were isolated from the medicinal mushroom Lignosus rhinocerus for in silico characterisation and proteolytic analysis. Both the cDNA encoding for zymogens consist of 399 amino acids residues with 67.2% similiarity. Clustal sequence alignments of 4347-SPL and 8711-SPL with other serine proteases reveal active catalytic triads of Asp-His-Ser residues.Based on SBT3 (PDB:3I74), the predicted 3D structures of the enzymes comprise seven beta-sheets with four/five alpha-helices. Expression of 4347-SPL and 8711-SPL in pET-28a(+)/Escherichia coli produces insoluble recombinant proteins. After refolding, only 8711-SPL provided substantial azocasein digestion from pH 6-11 at 60oC and was thermostable. The proteolytic activity was inhibited by EDTA and PMSF and mildly affected by Mg2+ and Ca2+. Both are subtilisin-like monomers. Further studies are warranted to explore potential industrial and pharmaceutical applications.