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. 2018 Oct 17;8(1):173.
doi: 10.1186/s13568-018-0696-y.

Production, Purification and Characterization of an Acid/Alkali and Thermo Tolerant Cellulase From Schizophyllum Commune NAIMCC-F-03379 and Its Application in Hydrolysis of Lignocellulosic Wastes

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Free PMC article

Production, Purification and Characterization of an Acid/Alkali and Thermo Tolerant Cellulase From Schizophyllum Commune NAIMCC-F-03379 and Its Application in Hydrolysis of Lignocellulosic Wastes

Bikash Kumar et al. AMB Express. .
Free PMC article

Abstract

A cellulase producing fungus Schizophyllum commune NAIMCC-F-03379 was isolated from decomposed leaf sample of Lantana camera. The nutritional components (wheat bran, magnesium sulphate and calcium chloride concentrations) and physical parameters (temperature and pH) were optimised by response surface methodology for enhanced cellulase production by S. commune NAIMCC-F-03379. The optimized medium contained: 1% (w/v) wheat bran, 0.3 g/L MgSO4, 0.8-1.0 g/L CaCl2, optimum temperature and pH were 25 °C and 5 respectively. Under optimum condition, 5.35-fold increase in CMCase and 6.62-fold increase in FPase activity was obtained as compared to un-optimized condition. Crude cellulase enzyme was subjected to different purification techniques and comparative evaluation of their efficiency was performed. The aqueous two-phase system using polyethylene glycol 8000/MnSO4 system showed maximum purification with 10.4-fold increase in activity, 79.5% yield and 0.5 partition coefficient. The cellulase enzyme obtained from S. commune NAIMCC-F-03379 has shown high stability i.e. more than 55% relative activity after 12 h of incubation over wide range of temperature (25-65 °C) and pH (3-10). The molecular weight of the cellulase enzyme was estimated as ~ 60 kDa by using sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and zymography. Km and Vmax value of cellulase on carboxy-methyl cellulose were obtained as 0.0909 mg/mL and 45.45 μmol/min mg respectively. Rice straw and wheat bran were subjected to hydrolysis using cellulase and cellulase-xylanase cocktail and analysed by thin layer chromatography and high performance liquid chromatography (HPLC). The HPLC analysis showed glucose concentration of 1.162 mg/mL after enzymatic hydrolysis of rice straw.

Keywords: Aqueous two-phase system; Cellulase; Hydrolysis; Response surface methodology; Schizophyllum commune.

Figures

Fig. 1
Fig. 1
Cellulase production using different media and agro-residues. a Cellulase production by Schizophyllum commune NAIMCC-F-03379 using different media (M1, M2, M3 and M4). b Selection of agro-residues for cellulase production by Schizophyllum commune NAIMCC-F-03379 in M4 Media
Fig. 2
Fig. 2
Response surface plot and contour plot for CMCase and FPase production from Schizophyllum commune NAIMCC-F-03379 showing interaction between different variable: For CMCase activity a pH and calcium chloride, b wheat bran and temperature; For FPase activity c temperature and magnesium sulphate, d wheat bran and calcium chloride
Fig. 3
Fig. 3
SDS-PAGE analysis and zymography: (1) Protein Ladder, (2) SDS-PAGE of crude cellulase, (3) zymography of crude cellulase, (4) SDS-PAGE of partially purified cellulase enzyme by ATPS with PEG 8000/MnSO4 system, (5) Zymography of the partially Purified cellulase enzyme by ATPS with PEG 8000/MnSO4 system. CMC was used as substrate in zymography
Fig. 4
Fig. 4
Stability and kinetic analysis of partially purified Schizophyllum commune NAIMCC-F-03379: a thermal stability of the partially purified cellulase enzyme at different temperature (25–65 °C) for different interval of time. b Stability of the partially purified cellulase enzyme produced at different pH (3–10) for different interval of time. c Kinetics (Michaelis–Menten and Lineweaver–Burk plots) of cellulase enzyme for CMC as substrate
Fig. 5
Fig. 5
Hydrolysis of rice straw and wheat bran. a Hydrolysis of rice straw and wheat bran for the detection of amount of sugar released with time by using commercial cellulase ONZUKAR, cellulase obtained from Schizophyllum commune NAIMCC-F-03379, xylanase obtained from A. oryzae and combination of cellulase and xylanase. b Analysis of hydrolysis product. Glucose as control (1), rice straw by commercial cellulase (2), rice straw by cellulase obtained from Schizophyllum commune NAIMCC-F-03379 (3), rice straw by xylanase obtained from Aspergillus oryzae (4), rice straw by combination of cellulase and xylanase (5), wheat bran by commercial cellulase (6), wheat bran by cellulase obtained from Schizophyllum commune NAIMCC-F-03379 (7), wheat bran by xylanase obtained from Aspergillus oryzae (8) and wheat bran by combination of cellulase and xylanase (9) on TLC plates

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