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Purification and characterization of acidic cellulase from Bacillus amyloliquefaciens SS35 for hydrolyzing Parthenium hysterophorus biomass
Author(s) -
Singh Shuchi,
Dikshit Pritam Kumar,
Moholkar Vijayanand S.,
Goyal Arun
Publication year - 2014
Publication title -
environmental progress and sustainable energy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.495
H-Index - 66
eISSN - 1944-7450
pISSN - 1944-7442
DOI - 10.1002/ep.12046
Subject(s) - cellulase , chemistry , bacillus amyloliquefaciens , hydrolysis , enzyme assay , chromatography , reducing sugar , bacillus licheniformis , metal ions in aqueous solution , enzyme , starch , nuclear chemistry , food science , sugar , biochemistry , metal , bacteria , organic chemistry , biology , bacillus subtilis , fermentation , genetics
This study reports purification and characterization of an acidic endoglucanase (carboxymethylcellulase, CMCase) produced by Bacillus amyloliquefaciens SS35. Purification of enzyme was done using ion exchange chromatography (Yield = 2.1%, Purification fold = 18.5). The molecular weight of the CMCase was determined as ∼37 kDa. The purified CMCase was able to hydrolyze carboxymethylcellulose (CMC), barley– β – d –Glucan, lichenan, hydroxyethylcellulose, starch, and xylan, which indicated endoglucanase activity. However, the enzyme could not hydrolyze avicel and p –nitrophenyl– β – d –glucopyranoside ( p NPG). These results were indications of absence of exoglucanase and β –glucosidase activity in the enzyme. Optimum temperature and pH for CMCase activity were determined as 55°C and 5.0, respectively. The enzyme also displayed high stability in temperature range of 20–40°C and pH range of 5.0–9.0 for more than 20 h with significant residual CMCase activity of 80%. Five metal ions, viz. Co 2+ , Ca 2+ , K + , Na + , Mn 2+ were found to be cofactors of the enzyme that enhanced its activity, while other metal ions such as Fe 3+ , Zn 2+ , Hg 2+ rendered inhibition effect on the enzyme. Using Lineweaver–Burk plot, the kinetic parameters K m and V max for CMCase were determined as 0.33 mg/mL and 4.19 μmol/mg/min, respectively. The asset of the enzyme was evaluated with hydrolysis of pretreated Parthenium hysterophorus . The total reducing sugar yield of 271 mg/g biomass was obtained. © 2014 American Institute of Chemical Engineers Environ Prog, 34: 810–818, 2015