Cloning, expression, characterization and application of protease produced by Bacillus cereus PMW8

• Published in: RSC advances 2016-01, Vol.6 (45), p.38611-38616
• Main Authors: Esakkiraj, Palanichamy, Meleppat, Balraj, Lakra, Avinash Kant, Ayyanna, Repally, Arul, Venkatesan
• Format: Article
• Language: English
• Subjects: Bacillus; Bacteria; Enzymes; Genes; Molecular weight; Protease; Solvents
• ISSN: 2046-2069; 2046-2069
• DOI: 10.1039/c5ra27671c

A protease gene of Bacillus cereus PMW8 isolated from a cow milk sample was cloned in E. coli DH5 alpha . Sequence analysis confirmed 1700 bp of protease gene and further expression was carried out using E. coli BL21(DE3)PLysS cells. The optimum conditions for expression were noted as 0.1 mM IPTG for 3 h at 37 degree C, whereupon the resultant protease was observed in soluble form. Affinity chromatography purification followed by SDS-PAGE resulted in two bands. Of the two, the first one was a precursor protein with a molecular weight of 70 kDa, identical to the predicted protein size from the nucleotide sequence, and the other was a matured protease with a molecular weight of 28 kDa. The matured protease was found to be active in a broad spectrum of pH from 7.0 to 10.0, where the optimum activity was exhibited at pH 9.0. Also, it retained 80% activity at pH 7.0-11.0 even after 2 h of incubation. Being thermophilic, the enzyme displayed excellent activity in a temperature range of 40-70 degree C, with a maximum at 60 degree C, and it showed good stability after 2 h of incubation. Metal ions such as CaCl sub(2), MgSO sub(4), MgCl sub(2), ZnSO sub(4), CuSO sub(4) and BaCl sub(2) were found to improve the enzyme activity compared to a control. The protease was active in the presence of most surfactants and the activity was best in SDS. Our protease was observed to be a thiol dependent enzyme, whose activity was greatly enhanced by beta -mercaptoethanol and DTT. Exposure to organic solvents didn't affect the protease and it retained 80% activity in all the solvents. Anti-biofilm activity revealed by the cloned B. cereus protease emphasized its role in hindering the biofilm formation of pathogenic bacteria such as Listeria monocytogenes, E. coli and Salmonella typhi.