Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

Abstract

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.

Isolation, Screening, and Identification of Cellulolytic Bacteria from Natural Reserves in the Subtropical Region of China and Optimization of Cellulase Production byPaenibacillus terraeME27-1

“…However, maximum CMCase activity of bacteria at pH lower than 6.0 has been rarely observed, and the maximum CMCase activities of P. campinasensis BL11, P. polymyxa GS01, Paenibacillus sp. B39, and Bacillus mycoides S122C were observed at neutral or alkaline conditions [37, 38, 46, 47]. In the present study, the CMCase produced by the strain ME27-1 was stable at pH 5.0–9.5, and almost 85% residual activity was retained.…”

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

“…HSH-810 (Kim et al 2005) isolated from other environments, whose endo-1,4-β-glucanases also manifest optimal activity at pH 7.0 and notable activity at a wide pH ranging from 4.5 to 10. However, these characteristics may apparently constitute an exception, because the rule is that bacterial endo-1,4-β-glucanases work at an optimal pH, but do not show activity at wide intervals of pH, as has been documented in Sporocytophaga myxococcoides (Goksoyr 1988), Pseudomonas fluorescens (Yamane et al 1970); Ruminiclostridium thermocellum (Romaniec et al 1992), Aquifex aeolicus (Kim et al 2000), Ruminococcus albus (Ohara et al 2000), Cellulomonas biazotea (Rajoka et al 2004), Clostridium cellulovorans (Arai et al 2006), Bacillus mycoides (Balasubramanian et al 2012), and Bacillus sp. (Harshvardhan et al 2013).…”

Enhanced Production of Cellulase from Bacillus licheniformis K‐3 with Potential for Saccharification of Rice Straw

“…Among various options, bioethanol derived from lignocellulosic biomass has emerged as a strong alternative for transport fuel. Lignocellulosic biomass (LB), the most abundant renewable energy resource on earth, can be exploited for the production of several industrially important commodities including bioethanol biofuel 1. 2 However, the bioconversion of LB faces several problems including the need for severe pretreatment of LB, sugar loss and non‐availability of suitable enzyme system for efficient hydrolysis of LB to convert it into fermentable sugars 2.…”