title: Cloning, expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis.
abstract: A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.
authors.affiliation: Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India.
authors.forename: Revathi
authors.initials: R
authors.lastname: Masilamani
authors.affiliation: Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India.
authors.forename: Om Prakash
authors.initials: OP
authors.lastname: Sharma
authors.affiliation: Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India.
authors.forename: Suresh Kumar
authors.initials: SK
authors.lastname: Muthuvel
authors.affiliation: Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India.
authors.forename: Sakthivel
authors.initials: S
authors.lastname: Natarajan