Document Type : Original Article
Recombinant Proteins Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
Department of Exercise Physiology, Faculty of Sport Science, University of Mazandaran, Babolsar, Iran
Department of Biology-Biochemistry, Payame Noor University, Mashhad Campus, Mashhad, Iran
Introduction: Endo-β-1,4-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-1,4-glucanase enzyme of Bacillus subtilis DR-8806 obtained from water samples from Dig Rostam, a hot mineral spring in Kerman, Iran.
Materials and Methods: Endo-β-1,4-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 16S ribosomal RNA sequence analysis, Bacillus is characterized and named as Bacillus subtilis DR-8806. Western blot analysis verified the recombinant endoglucanase by detecting a specific band of ~55kDa. Amino acid homology analysis of the protein showed 99% homology with that of endoglucanase from Bacillus subtilis. The optimum temperature for enzyme reaction was attained at a temperature of 55°C. The cellulolytic activity of Endo-β-1,4-glucanase protein determined 8.5 IU ml-1. It showed that endoglucanase amino acid sequence contains a glycosyl hydrolase family 5, linker domain, and a cellulose-binding type 3 domain. The GH5 domain also contained a glycosyl hydrolase catalytic core.
Conclusions: It is possible to consider the purified Endo-β-1,4-glucanase of B. Subtilis DR-8806 as an efficient cellulose producer. Further research is required to examine the industrial applications of this study.