Cloning and expression of keratinase gene inBacillus megateriumand optimization of fermentation conditions for the production of keratinase by recombinant strain

Abstract

Aims: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. Methods and Results: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74·3 U ml−1. The ker gene was further subcloned into E. coli‐Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml−1. Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166·2 U ml−1 (specific activity, 33·25 U mg−1) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0·4 OD600 nm and xylose concentration of 0·75% w/v. Conclusions: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three‐fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. Significance and Impact of the Study: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.

Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

“…The recombinant enzyme was active at pH values from 7–9 and had optimum pH of 7.5 (Figure 8A), which is the same as the keratinase of B. licheniformis or other recombinant keratinases [8]–[9], [12]. In addition, the recombinant keratinase was stable at moderate temperature and had optimum temperature at 50°C (Figure 8B), which was also identical to the previous reports [4], [8].…”

“…The keratinase ( kerA ) gene of B. licheniformis has been sequenced and cloned [11]–[12]. The molecular mass of mature keratinase is about 30–33 kDa [8]–[9].…”

Identification and characterization of alkaline serine protease from goat skin surface metagenome

“…In standard conditions, the reaction mixture contained 480 μl of 1% (w⁄ v) azocasein, 2 mM CaCl 2 and appropriate dilution of enzyme in 50 mM Tris buffer, pH 7.5 (Radha and Gunasekaran 2007). The reaction mixture was incubated at 37°C for 30 min.…”

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

“…have been studied to improve the production and secretion of recombinant proteins in B. megaterium [17,27]. RSM has also been used as a stratagem by Radha and Ganasekaran [17] for improved production of keratinase by recombinant B. megaterium, obtaining threefold increases in keratinase production over the wild-type strain. Media components, signal peptide, and effect of affinity tags for levansucrase production by recombinant B. megaterium were optimized, which resulted in maximum levansucrase activity of 417 U/l [21].…”