Hossein Barzegar - Recombinant Proteins Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
Mohammadreza Nassiri - Recombinant Proteins Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
Khadijeh Nasiri - Department of Exercise Physiology, Faculty of Sport Science, University of Mazandaran, Babolsar, Iran
Sara Mousavi - Department of Biology-Biochemistry, Payame Noor University, Mashhad Campus, Mashhad, Iran

Introduction: Endo-β-۱,۴-glucanase is the first enzyme in the conversion of cellulose to fermentable sugars. The objectives of this study were to clone and characterize a thermostable Endo-β-۱,۴-glucanase enzyme of Bacillus subtilis DR-۸۸۰۶ obtained from water samples from Dig Rostam, a hot mineral spring in Kerman, Iran. Materials and Methods: Endo-β-۱,۴-glucanase gene from a thermostable Bacillus subtilis bacterium was cloned and expressed in Escherichia coli. The recombinant proteins of the expression cell were tested by western blotting analysis. The enzymatic activity of the recombinant endoglucanase was measured using dinitrosalicylic acid method and carboxymethyl cellulose as substrate. Bioinformatics analysis was done to characterize domain organization and protein family through Pfam search server and PROSITE. Results: Based on ۱۶S ribosomal RNA sequence analysis, Bacillus is characterized and named as Bacillus subtilis DR-۸۸۰۶. Western blot analysis verified the recombinant endoglucanase by detecting a specific band of ~۵۵kDa. Amino acid homology analysis of the protein showed ۹۹% homology with that of endoglucanase from Bacillus subtilis. The optimum temperature for enzyme reaction was attained at a temperature of ۵۵°C. The cellulolytic activity of Endo-β-۱,۴-glucanase protein determined ۸.۵ IU ml-۱. It showed that endoglucanase amino acid sequence contains a glycosyl hydrolase family ۵, linker domain, and a cellulose-binding type ۳ domain. The GH۵ domain also contained a glycosyl hydrolase catalytic core. Conclusions: It is possible to consider the purified Endo-β-۱,۴-glucanase of B. Subtilis DR-۸۸۰۶ as an efficient cellulose producer. Further research is required to examine the industrial applications of this study.

Endoglucanase Enzyme, Cloning, Expression, Bacillus subtilis