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

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.

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…β-Glucosidase therefore has been purified from different sources including fungi, bacteria and yeast and various strategies have been employed for β-glucosidase purifications [65,66,67]. The pre-purification step usually involves protein precipitation/fractionation from microbial culture using ammonium sulfate at 75% [68], 80% [69,70,71,72], and 90% saturation [55,73]. Other workers have used ultrafiltration [66,74], acetone precipitation [75], and ethanol precipitation [76].…”

Predominance of Trichoderma and Penicillium in cellulolytic aerobic filamentous fungi from subtropical and tropical forests in China, and their use in finding highly efficient β-glucosidase

“…The pH stability of purified enzyme was assessed by incubating 500 μl of purified enzyme at 4°C for 24 hours in various buffers adjusted to different pH values, followed by checking the residual activity [17]. To determine the thermostability of β-glucosidase activity, the purified enzyme was incubated at different temperatures (30,35,40,45,50,55,60, and 65°C) in the absence of substrate.…”

“…Catalytic constants (K cat ) and catalytic efficiency ratios (K cat /K m ) were determined from the obtained kinetic parameter values [17].…”

“…In addition, β-glucosidase activities are generally measured using artificial substrates such as p-nitrophenyl-β-D-glycopyranoside (pNPG) or 4-methylumbelliferyl-β-D-glucuronide. When using these artificial substrates, most enzymes show higher catalytic activity toward artificial substrates than toward cellobiose [16][17][18][19][20]. However, UeBgl3A from Ustilago esculenta and a recombinant β-glucosidase from Caldicellulosiruptor saccharolyticus are exceptions, with higher catalytic activity toward cellobiose than toward artificial substrate [21,22].…”

Cellulolytic Bacillus May or May Not Produce β -Glucosidase Due to Their Environmental Origin – A Case Study

“…It is generally responsible for the regulation of the whole cellulolytic process as it not only produces glucose from cellobiose, but also reduces cellobiose inhibition, allowing endocellulase and exocellulase enzymes to function more efficiently [8]. Thus, β-glucosidases allow the cellulolytic enzymes to function more efficiently by producing glucose from cellobiose and reducing cellobiose inhibition [9].…”