Bacillus pumilus S124A carboxymethyl cellulase; a thermo stable enzyme with a wide substrate spectrum utility

摘要

Bacillus pumilus S124A was identified as carboxymethyl cellulase producing bacteria from Azorean Bacillus collection (Lab collection), which was isolated in local soils. The bacterium was identified by 16S rRNA sequence and designated as Bacillus pumilus S124A. NCBI-blast analysis showed B. pumilus S124A; 16S rRNA sequence has high identity to other B. pumilus strains. Phylogenetic analysis showed B. pumilus S124A close to B. pumilus LZBP14 strain. CMcellulase was purified from cells-free supernatants and post mano-Qpurification; 5.39% protein folds, and 0.88% recoveries were obtained. SDS-PAGE analysis showed molecular weight of the purified CMcellulase was estimated ~40 kDa and composed of a single subunit. NonoLC ESI-MS/MS analysis was yielded four peptides, and protein has identity to other cellulases. Purified CMcellulase showed high activity to cellobiose followed by CMcellulose. Kinetic analysis showed Km, and V_(max) were determined as 2.12mg/ml, 239 μmol/min/mg, respectively. Optimum temperature and pH for the purified CMcellulase activity were found at 50℃ and pH 6.0, respectively. Purified CMcellulase was maintained about 75% activity in a pH range of 4-8 and 70% activity in a temperature range of 40-70 ℃. CMcellulase activity was highly reduced by HgCl_2, followed by EDTA, PMSF whereas CoCl2 was activated CMcellulase activity.