Cloning, expression and structure simulation of keratinase from Bacillus licheniformis strain MZK05

Abstract

Aims: Bacillus licheniformis MZK-05 is a keratinolytic bacterium having potential in dehairing of leather and feather hydrolysis. The present study aimed at to improving the production level of keratinase through gene cloning and expression of recombinant keratinase. Methodology and results: Bacillus licheniformis MZK-05 produced an amplicon of 1,156 bp in a polymerase chain reaction while targeting the gene, kerA, responsible for the enzyme keratinase. The amplicon was subsequently cloned into the plasmid vector pGEX-6p-2 for expression in Escherichia coli BL21. A 58 kD GST-KerA fusion protein was expressed upon IPTG induction which was eventually cleaved by PreScission protease that produced a 39 kD protein.A corresponding increase in proteolytic (312 U/mL) and keratinolytic (196 U/mL) activity were observed with the expressed keratinase. Specific enzyme activities for protease and keratinase, an indication of efficiency of the enzyme, were 2621.84 U/mg and 1647 U/mg, respectively and the specific keratinase activity was the highest activity ever reported by any recombinant bacterial strain. Conclusion, significance and impact study: Since the production of keratinase by wild type strain is limited to a certain level, the industrial need could be met by improving the production level through gene cloning and expression of recombinant keratinase. In this connection, the cloning of kerA gene from B. licheniformis MZK-05 into pGEX-6p-2 vector, its expression in Escherichia coli BL21 host and prediction of 3-D model of the expressed protein were performed which will be the basis for industrial production of keratinase in Bangladesh.

Microbial enzymes catalyzing keratin degradation: Classification, structure, function

“…Azo-keratin is not commercially available, but it is possible to prepare this keratinase assay substrate by coupling keratin (derived from various keratin sources) with a diazotized aryl amine to produce a chromophoric derivative, sulfanilic acid-azokeratin ( Riffel et al, 2003 ). Another synthetic colored keratin substrate is keratin azure ( Habbeche et al, 2014 ; Nahar et al, 2016 ). This substrate is available commercially from Sigma–Aldrich (St. Louis, MO, USA) and is presumably prepared from sheep wool ( Scott and Untereiner, 2004 ).…”

Combining Pro-peptide Engineering and Multisite Saturation Mutagenesis To Improve the Catalytic Potential of Keratinase

“…Hu et al 19 expressed the keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli with two vectors, pET30a and pET32a, which achieved specific keratinase activities of 9.8 units/mg (total activity of 26.8 units/mL) and 7.2 units/mg (19.4 units/mL), respectively. Nahar et al 20 cloned the keratinase gene from B. licheniformis strain MZK05 and subsequently expressed it in E. coli BL21, resulting in an increased keratinolytic activity of 196 units/mL. Dong et al 21 overexpressed a novel keratinase from Bacillus polyfermenticus B4 in recombinant Bacillus subtilis via the T7 promoter, which reached a high activity of 473 ± 20 units/mL.…”

Beyond the Visible: Bioinspired Infrared Adaptive Materials

“…systematically investigated eight different passive radiative cooling modes for buildings, such as a roof pond, evaporative cooling roof, and thermal insulation on the roof. [ 81 ] A white‐colored roof exhibited superior energy‐saving performance. Furthermore, the “cool roof” has been considered one of the most popular radiative cooling technologies for use in future energy‐efficient green buildings.…”