Enzymatic properties of two β‐glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

• Published in: Journal of applied microbiology 2006-08, Vol.101 (2), p.480-486
• Main Authors: Magalhães, P.O., Ferraz, A., Milagres, A.F.M.
• Format: Article
• Language: English
• Subjects: Basidiomycota - enzymology; beta-Glucosidase - analysis; beta-Glucosidase - metabolism; Biodegradation, Environmental; Biological and medical sciences; Bioreactors; Cellulase - analysis; Cellulase - metabolism; cellulases; Cellulose - metabolism; Ceriporiopsis subvermispora; electrophoresis; Electrophoresis, Polyacrylamide Gel; Environmental Microbiology; Fundamental and applied biological sciences. Psychology; Glucose - biosynthesis; Hot Temperature; Hydrogen-Ion Concentration; Microbiology; Pinus taeda; purification; solid‐state cultivation; Substrate Specificity; Wood; β‐glucosidase
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2006.02946.x

Aims:  Ceriporiopsis subvermispora produces endoglucanase and β‐glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major β‐glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. Methods and Results:  Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid‐state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l−1, pH 5·4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S‐300 gel filtration. The retained fraction attained 76% of β‐glucosidase activity with 3·7‐fold purification. Two β‐glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the β‐glucosidase of 110 kDa. The optimum pH and temperature were 3·5 and 60°C, respectively. The Km and Vmax values were respectively 3·29 mmol l−1 and 0·113 μmol min−1 for the hydrolysis of p‐nitrophenyl‐β‐glucopyranoside (pNPG) and 2·63 mmol l−1 and 0·103 μmol min−1, towards cellobiose. β‐Glucosidase activity was strongly increased by Mn2+ and Fe3+, while Cu2+ severely inhibited it. Conclusions:  Ceriporiopsis subvermispora produces small amounts of β‐glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two β‐glucosidases with 110 and 53 kDa. The 110 kDa β‐glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical β‐glucosidase. Significance and Impact of the Study:  Ceriporiopsis subvermispora produces β‐glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of β‐glucosidases during biopulping.