Identificação e purificação de uma β-glicosidase extracelular e construção de vetores para expressão constitutiva de celulases em Kluyveromyces marxianus UFV-3

• Autor(a) principal: Lopes, Mariana Rocha
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2434
• topic: β-glicosidase extracelular; Kluyveromyces marxianus UFV-3; extracellular β-glucosidase; Kluyveromyces marxianus; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Kluyveromyces marxianus is a yeast with many beneficial properties for the development an efficient process for producing ethanol from simultaneous saccharification and fermentation of biomass: a high growth rate, the utilization of a wide range of sugar types, a efficient system for protein secretion and fermentation capacity at temperatures around 42 ºC. The heterologous expression of thermostable cellulases in K. marxianus have been the focus of many researches. The vector pKLAC1-adh2p, for constitutive expression of cellulases in K. marxianus, was constructed at Laboratory of Molecular Biotechnology (LBM/UFV). In this study, the gene of a β-glucosidase from Aspegillus niger was cloned and inserted into pKLAC1-adh2p. The expression cassette, pKLAC1-adh2p-bgl1 was used for transformation of K. marxianus UFV-3, but the selection process was not efficient, and during screening of transformants yeasts, we observed the ability of the yeast K. marxianus to secrete a β-glucosidase. Thus, while works on standardization of an efficient system for heterologous expression in K. marxianus have been developed in laboratory (LBM/UFV), this β-glucosidase extracellular from K. marxianus was characterized. Theresponse surface methodology was used to optimize the production process and determine the parameters pH and temperature for enzyme activity. The best conditions for production were glucose 4%, pH 5.5 and incubation at 35°C. The highest activity was obtained at pH 5.5 and 55°C. Thermostability tests determined a half-life of 4 hours for the enzyme, when incubated at 60 ° C. Analysis of substrate specificity showed that the enzyme was able to hydrolyze cellobiose, p-nitrophenyl-β-Dglucopyranoside, o-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-cellobioside and p-nitrophenyl-β-D-xylopyranoside. The enzyme was purified by non-denaturing electrophoresis in polyacrylamide gel (PAGE) and zymogram analysis. The SDS-PAGE analysis revealed two major bands around 45 KDa, which were submitted at in-gel trypsin digestions and analyzed by mass spectrometry (MS/MS). The protein was identified as a β-glucosidase. To finish this work, the β-glucosidase was purified by ultrafiltration and anion exchange chromatography. The purification process was monitored by enzymatic assay using p-nitrophenyl-β-D-glucopyranoside as substrate and SDS-PAGE. These results show for the first time a β-glucosidase secreted in a fermentative yeast, this opens the possibility to use this yeast for cellulosic ethanol production, since the costs with the use of cellulases makes the process uneconomical.