Gene Cloning of Bacillus licheniformis for Increased Production of Keratinase for Industrial Applications

Abstract

Keratinases are a special group of proteases which are well recognized for their action on recalcitrant proteins such as feather, nail, hair, hoof etc. They have also great applications in eco-friendly dehairing processing of leathers, production of poultry feed, medical treatment and cosmetics products. So it is important to increase the production level of these enzyme. Therefore, present study was aimed at improving the production level of the enzyme through cloning of keratinase encoded gene of Bacillus licheniformis MZK05 and its expression in E.coli BL21. Bacillus licheniformis strain MZK05, isolated from feather-decomposed soil and was identified by 16S rRNA gene sequencing. The primers were designed from the alignment of the sequences obtained for kerA genes from different Bacillus licheniformis strains. PCR amplification with the designed primers revealed a distinctive 1156 bp fragment from B. licheniformis MZK05. PCR product was purified and cloned into two vectors viz: pGEX-6P-2 induced on IPTG and pET-30a(+) induced on lactose expressed in E.coli BL21. Escherichia coli BL21 cells were transformed with the respective recombinant vector for the expression of protein. In pGEX-6P-2, GST-KerA fusion protein was expressed and optimized in different IPTG concentrations and the maximum expression of the desired protein was exhibited on 3 hours with 0.3 mM IPTG. From SDS-PAGE analysis, the molecular weight of the GST-KerA fusion protein was determined to be 58 kDa. Its purification by Glutathione Sepharose 4B followed by PreScission protease cleavage produced the KerA protein of about 39 kDa. The yield of purified protein was approximately 119 mg/L with the corresponding keratinase activity of 312 U/ml. A 4-fold increase in keratinase activity was obtained by heterologous expression of keratinase over the wild-type strain. The pET-30a(+)-kerA protein expression was observed within 1-3 hours of induction at 2 mM lactose concentration. The time course induction produced gradual increase of expressed protein and was achieved into highest level after 3 hours of post-induction. A 40 kDa His-tag protein was expressed upon lactose induction which upon successful purification using Ni 2+ -NTA resin, gave a 39 kDa KerA protein. The purified KerA protein showed 4.5 fold higher keratinase activity than that of wild strain of B. licheniformis MZK05. The keratinase expression by the recombinants pGEX-6P-2-kerA and pET-30a(+)-kerA were influenced by temperature, pH, metal ions and inhibitors in the liquid culture. The activity of both the recombinants pGEX-6P-2-kerA and pET-30a(+)-kerA appeared highest at pH 8.0 and temperature at 40°C. While the keratinolytic activity was augmented following addition of metal ions: Ca , it were decreased with Co 2+ 2+ 2+ , Mg and Mn 2+ 2+ 2+ . Both pGEX-6P-2-kerA and pET-30a(+)-kerA were strongly inhibited by PMSF and slightly inhibited by EDTA and DDT indicating it serine like protease. , Zn and Cu The whole genome sequencing of Bacillus licheniformis MZK05 was performed using Illumina MiSeq technique. The genome of strain Bacillus licheniformis MZK05 is 4,145,737 bp long with a GC content of 43.05%, containing 85 tRNA genes and 6 rRNA operons. The number of known protein coding gene is 2796 and hypothetical gene is 1282. The position of KerA gene in whole genome sequencing of Bacillus licheniformis MZK05 is (1211369-1212344) bp. Keratinase productivity was compared between shake flask and bioreactor cultivations. These recombinants pGEX-6P-2-kerA and pET-30a(+)-kerA showed highest keratinase expression at 5 hrs which were 240 U/ml and 310 U/ml respectively in bioreactor cultivation. The result showed that the productivity of pGEX-6P-2-kerA and pET-30a(+)kerA in bioreactor were 1.5 and 1.9 times higher as compared to the shake flask. The application of keratinase enzyme in human hair rebonding test demonstrated that the hair treated with enzyme applied alone showed better performance to only rebonding cream used hair. This suggest that the keratinase produced can successfully be applied in the business of beauty parlour. In whole feathers hydrolysis test, after 48 hrs, both the enzymes used completely hydrolyzed the whole feathers comparing to wild MZK05. In dehairing of goat skins, processing demonstrated that the treatment with purified recombinant keratinase removed 95% of hair from goat skins after 26 hrs whereas the treatment with the enzyme (keratinase) and 5% lime together resulted in 85% dehairing under similar condition. Sodium sulfide along with lime removed 100% hair as done in conventional chemical methods. This test also suggest the enzyme can also be used in dehairing processing of leathers in tanneries. The overall findings of the present study thus will be an useful basis for developing a bioprocess for commercial production and application of keratinase in Bangladesh.