Purification and biochemical properties of a glucose-stimulated β-D-glucosidase produced by <Emphasis Type="Italic">Humicola grisea</Emphasis> var. <Emphasis Type="Italic">thermoidea</Emphasis> grown on sugarcane bagasse |
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Authors: | Cesar Vanderlei Nascimento Flávio Henrique Moreira Souza Douglas Chodi Masui Francisco Assis Leone Rosane Marina Peralta João Atílio Jorge Rosa Prazeres Melo Furriel |
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Institution: | 1.Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeir?o Preto,University of S?o Paulo,Ribeir?o Preto,Brasil;2.Department of Biochemistry,State University of Maringá,Maringá,Brasil;3.Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeir?o Preto,University of S?o Paulo,Ribeir?o Preto,Brasil |
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Abstract: | The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium,
but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction
medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme
was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single
band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57
and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively.
The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in
the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose
or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing
Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials. |
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