Purification and biochemical characterization of a novel thermo-stable carboxymethyl cellulase from Azorean isolate Bacillus mycoides S122C.
Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76 %, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62 kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by β-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50 °C and pH 7.0, respectively. The purified CMcellulase was stable with about 60 % activity in broad pH ranges from 5 to 10 and temperature of 40 to 60 °C. However, purified CMcellulase was stable at about 70 % at 70 °C and also stable overall at 78 % for surfactants. CMcellulase activity was inhibited by ions such as HgCl(2), followed by CuSo(4), FeCl(2), and MnCl(2), while CoCl(2) activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.
CIRN and Department of Biology, University of Azores, 9500-801, Ponta Delgada, Portugal. natesanbala@uac.pt
Natesan
N
Balasubramanian
Duarte
D
Toubarro
Mário
M
Teixeira
Nelson
N
Simõs
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018533:Biomass; D002480:Cellulase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D004791:Enzyme Inhibitors; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D013501:Surface-Active Agents; D002482:Cellulose; D002480:Cellulase; C019826:carboxymethylcellulase