Isolation of β-1,4-glucanase producing soil bacterium and genetic modification via in vitro mutagenesis

In this study, Bacillus sp. strains were isolated from soil samples at pH 5.0-6.0 and at two different temperatures (37 ℃ and 50℃). Among these strains, β-1,4-glucanase (CMCase) produces were determined. Bacillus sp. AZH strains were selected for further studies unwanted genes such as α-amylase, xylanase, protease were inactivated using Ethidium bromide (EtBr) and Ultraviolet ray (UV). β-1,4-glucanase enzymes of Bacillus sp. AZH (wild type strain) and it's mutant variants (Bacillus sp. AZH-mEB and Bacillus sp. AZH-mUV) were partially characterized. Optimum pH values for AZH-mUV, AZH-mEB and wild type strains were 7.0, 6.0 and 9.0, respectively. Optimum temperature values were, 60, 50 and 50°C in the same order. β-1,4-glucanase activities were compared with each other and calculated as 100, 100, and,71,5% relatively for wild type strain, AZH-mEB and AZH-mUV respectively. Bacillus sp. AZH-mUV cellulase maintained its residual activity after incubation at 30℃ for 15 min. while Bacillus sp. AZH-mEB and wild type strain cellulases maintained their rsidual activities after 15 minutes incubation at 40℃. The effects of metal ions on the enzyme activities were also investigated. CaCl2, MgCl2, KCl2, EDTA and urea were increased both wild type strain and Bacillus sp. AZH-mUV cellulase activities. Whereas for Bacillus sp. AZH-mEB urea and MgCl2inhibited cellulase activity in different rations, other metal ions increased its activity. Finally, the molecular weights of the cellulase enzymes of all three bacterial strains were determined by SDS-PAGE and zymogram analysis to be 40 kDa.