Molecular cloning, expression and docking studies of serine protease (SerPr) and metalloprotease (MetPr) of novel Streptomyces werraensis KN23 for keratinase activity improvement

Abstract

Because keratinases can break down keratin, keratinases have potential uses in biotechnology. Streptomyces appears to be one of the primary providers of these enzymes, yet there are still few complete genome sequences of these microorganisms. This research focuses on isolation the gene encoding serine protease (SerPr) and metalloprotease (MetPr), consisting of 3318 and 1119 bp  nucleotides (encoding 1105 and 372 amino acids, respectively), from novel Streptomyces werraensis KN23 and mutant Streptomyces werraensis SA27, the specimen was subjected to isolation, sequencing, and then submitted to the NCBI GenBank database, where it was assigned accession numbers. The structural integrity of the protein model was confirmed by validating its 3D conformation using Ramachandran's plot, which indicated that residues were in the most favourable region for S. werraensis KN23 and mutant Streptomyces werraensis SA27, respectively. Three amino acids substitution was observed in the SerPr protein, and two amino acids substitution variations in MetPr protein of the mutant strain SA27. Docking studies revealed optimal binding affinities of the serine protease (SerPr) and metalloprotease (MetPr) with beta-keratin for S. werraensis KN23 and its mutant Streptomyces werraensis SA27, respectively. Molecular cloning and expression of the mutant Streptomyces werraensis SA27 serine protease (SerPr) and metalloprotease (MetPr) genes in E. coli DH5α resulted in a recombinant E. coli DH5α pGEM-T-SerPr and E. coli DH5α pGEM-T-MetPr
significantly higher keratinase activity 193.25 and 211.32 U/mL, compared to the mutant S. werraensis SA27, which exhibited an activity of 106.92 U/ml. These results suggest that the use of Streptomyces werraensis KN23 for keratinase production seems to be very promising, since it has a diverse array of commercial uses, including animal nutrition, the leather sector, and cosmetics.