Abstract
BackgroundRecently, 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.ResultsIn this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 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 bgl3 in Pichia pastoris 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.ConclusionsAn native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris 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.
Highlights
The increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and b-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful bglucosides
When a b-glucosidase was added to lignocellulosic materials, the release of phenolic compounds increased, indicating that cellulose-degrading enzymes may be involved in the breakdown of polymeric phenolic matrices [13]
After the microcrystalline cellulose (MC) column chromatography step, the nBgl3 enzyme was purified 85.1-fold with a specific activity of 127.7 U mg-1
Summary
The increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and b-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful bglucosides. Without appropriate treatment, a mass of agricultural, industrial and municipal cellulosic wastes has accumulated, resulting in the risk of environmental pollution [6]. Various methods, such as composting, mechanical treatment and chemical treatment, have been applied to treat these cellulosic wastes [7,8]. The hydrolysis of cellulose primarily depends on at least three enzymes, including endoglucanases (EGs), cellobiohydrolases (CBHs) and b-glucosidases, which work synergistically to degrade the cellulose [10]. EGs and CBHs can degrade native cellulose synergistically to generate cellobiose, which is a product inhibitor for these enzymes [11]. When a b-glucosidase was added to lignocellulosic materials, the release of phenolic compounds increased, indicating that cellulose-degrading enzymes may be involved in the breakdown of polymeric phenolic matrices [13]
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