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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 60C, pH 8.0 and maintained 54% activity at 80C 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.