|820-1||Purification and biochemical characterization of a β-glucosidase from the exo-1 mutant of Neurospora crassa|
|Autores:||Sandra Lazari Gentil (USP - UNIVERSIDADE DE SÃO PAULO- Departamento de Biologia) ; Luana Parras Meleiro (USP - Universidade de São Paulo- Departamento de Química) ; Ana Lúcia Ribeiro Latorre Zimbardi (USP - Universidade de São Paulo- Departamento de Química) ; Douglas Chodi Masui (USP - UNIVERSIDADE DE SÃO PAULO- Departamento de Biologia) ; João Atílio Jorge (USP - UNIVERSIDADE DE SÃO PAULO- Departamento de Biologia) ; Rosa Prazeres Melo Furriel (USP - Universidade de São Paulo- Departamento de Química) |
Currently, there is an increasing interest in the use of cellulosic residues for the production of ethanol, considered one of the best alternatives to fossil fuels. The enzymatic degradation of cellulose involves the action of endoglucanases, exoglucanases and β-glucosidases. Endo and exoglucanases act on the cellulose chain, respectively catalyzing the random cleavage of internal bonds and the release of cellobiose from reducing and non-reducing ends, whereas β-glucosidases act on short cellooligosaccharides and cellobiose, releasing glucose. This study aimed the purification and biochemical characterization of a β-glucosidase produced by the hipersecretory mutant exo-1 of Neurospora crassa under solid state fermentation. The fungus was cultured in wheat bran and water (1:2, m:v) for 240h at 25ºC, producing around 70 U/g solid substrate. Enzymatic activity was routinely assayed at 55°C, in 50 mM sodium acetate buffer, pH 5.0, using 0.4 mM p-nitrophenyl-β-D-glucopyranoside (pNP-Glu) as substrate. Enzyme purification involved precipitation with ammonium sulfate, followed by chromatography on DEAE-Fractogel and Phenyl Sepharose. The enzyme was purified 4.9 fold with 7% yield and specific activity of 16.3 U/mg. The homogeneity of the preparation was attested by the single protein band in 6% PAGE, coincident with an activity band using esculin as substrate. A single protein band was detected in 10% SDS-PAGE (apparent molecular mass 63.3 kDa). Optimal temperature and pH of activity were 55-60° C and 4.5-5.0, respectively. The purified enzyme was stable for 6h at 50° and 4h at 55°C and for 24h at 4ºC from pH 3.0-10.0. The enzyme was insensitive to Ni2+, Mn2+, Mg2+, Pb2+, Co2+, Ca2+, and EDTA, but inhibited 40 and 25%,respectively, by 1mM, Cu2+ and Hg2+.Insensitive to most mono- and disaccharides, the enzyme maintained 53% of the original activity in the presence of 10 mM glucose, and the activity was 60% enhanced by ethanol at 10% (v/v) concentration. The purified enzyme hydrolyzed pNP-Glu with Vm= 12.75 U/mg and KM= 0.043 mM, while for cellobiose Vm= 12.47 U/mg and KM= 1.05 mM. The good production levels in a cheap medium, the high thermal and pH stabilities and the insensitivity to metal ions of the β-glucosidase from exo-1 are interesting properties for biotechnological applications.
Palavras-chave: β-glucosidase, exo-1 mutant, Neurospora crassa, Purification, thermostable