Characterization of a thermostable β-glucosidase from <it>Aspergillus fumigatus </it>Z5, and its functional expression in <it>Pichia pastoris </it>X33

• Main Authors: Liu Dongyang, Zhang Ruifu, Yang Xingming, Zhang Zhenhua, Song Song, Miao Youzhi, Shen Qirong
• Format: Article
• Language: English
• Published: BMC 2012-02-01
• Series: Microbial Cell Factories
• Online Access: http://www.microbialcellfactories.com/content/11/1/25

<p>Abstract</p> <p>Background</p> <p>Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.</p> <p>Results</p> <p>In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus <it>Aspergillus fumigatus </it>Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, <it>bgl3</it>, was cloned based on the peptide sequences obtained from the LC-MS/MS results. <it>bgl</it>3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of <it>bgl</it>3 in <it>Pichia pastoris </it>X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg^-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.</p> <p>Conclusions</p> <p>An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of <it>A. fumigatus </it>Z5. The gene <it>bgl</it>3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene <it>bgl3 </it>was expressed in <it>Pichia pastoris </it>X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.</p>