Catalytic Characterization and Molecular Analysis Endo1, 4-Beta Glucanase Gene of Bacillus subtilis Strain A14h Isolated from Rice Fields

Cellulases can hydrolysis cellulose into glucose units which act in a synergistic manner of three enzymes endo-beta -1، 4-glucanase، cellobiohydrolase and beta-glucosidase. Nowadays، cellulases are used in various industries including animal feeds، textile، sewage treatment، brewery industries and biofuel production. Demand for cellulolytic enzymes with unique catalytic properties is growing more rapidly than ever before. In this study، Bacillus subtilis A14h with cellulase production potential was identified using biochemical and molecular (16SrDNA gene) methods. The optimum conditions of B. subtilis A14h cellulase activity was evaluated in the presence of two type of substrates (carboxymethyl cellulose and beta-glucan)، temperatures range of 30-70°C،and pH 4. 6. Also، endo 1، 4-beta glucanase gene amplified using specific primers، sequencing outcome was recorded in the NCBI database. Sequence analysis and construction of phylogenetic trees was performed using vector NTI and Mega. 4 softwares. The results showed that the isolate belongs to the Bacillus subtilis species. The phylogenetic analysis and comparison of the amino acid sequence of endo -1، 4-beta glucanase gene showed that the enzyme catalytic region belongs to glycosyl hydrolases family 5 and the non catalytic region was placed in the CBM3 family. The B. subtilis A14h cellulase was most closely related to the cellulase of B. subtilis strains. The results showed that the highest enzyme activity equal to 1464. 25 U/ml was obtained in the presence of beta-glucan substrate at 55° C.