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

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Title	*Cloning, expression of β-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**
Authors	Revathi Masilamani¹, Om Prakash Sharma², Suresh Kumar Muthuvel² & Sakthivel Natarajan¹*
Affiliation	¹Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India; ²Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry 605014, India
Email	puns2005@gmail.com; *Corresponding author
Article Type	Hypothesis
Date	Received November 01, 2013; Revised November 05, 2013; Accepted November 06, 2013; Published December 06, 2013
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.
Citation	Masilamani et al. Bioinformation 9(19): 958-962 (2013)
Edited by	P Kangueane
ISSN	0973-2063
Publisher	Biomedical Informatics
License	This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License.