Keratinase gene Cloning in Escherichia coli and investigating some of its bioinformatics features

Keratinases is the proteolytic enzymes in the nature. This enzyme is produced only in the presence of keratin substrate. That targets the disulfide bonds. In this study, keratinases gene was extracted from the bacterial strain Bacillus licheniformis was previously isolated from poultry litter. Amplification reaction was performed using DNA extracted from native bacteria as the template. After purification, keratinases gene was cloned in pTZ57R/T vector. After ligation, ker-pTZ57R/T construct was transferred into susceptible E. coli bacteria strain DH5α. PCR reactions using primers FB1 and RB1 on ker-pTZ57R/T construct, leading to gene amplification keratinases 1164 bp length with cut site XhoI and NcoI restriction enzymes for the transfer into appropriate expression pET-26b() vector. The recombinant plasmid, ker-pET-26b(), was extracted from positive colonies on LB agar with kanamycin antibiotic and was transferred into E. coli strain BL21. Keratinases cloned gene was sequenced. In the analysis of bioinformatics, molecular weight protein sequence of the nucleotide sequence was estimated at ~ 40 kDa. Protein net charge equal to -0.009 with aliphatic ratio is 84/73 and has the signal peptide. This enzyme is a serine protease with active sites aspartic acid 137, amino acid histidine 168 and serine 325. In general, the results of this bioinformatics studies indicated that the keratinase enzyme is considered to be a category of endogenous enzymes that can be used in various industries.