Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33.
Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33.
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曲霉曲霉Z5的热稳定β-葡萄糖苷酶的表征及其在Pichia Pastoris X33中的功能表达。
DOI：
10.1186/1475-2859-11-25
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发表时间：
2012-02-17
影响因子：
6.4
通讯作者：
Shen Q
中科院分区：
文献类型：
--
作者：
Liu D;Zhang R;Yang X;Zhang Z;Song S;Miao Y;Shen Q
近年来，能源需求的增加强烈刺激了木质纤维素生物质转化为后续生产的还原糖的研究，β-葡萄糖苷酶因其在多种基础生物过程和有用β-葡萄糖苷的合成中发挥重要作用而成为研究的焦点。虽然研究了不同来源的β-葡萄糖苷酶，但β-葡萄糖苷酶的量不足以有效转化纤维素。本工作的目的是寻找热稳定、催化效率高的β-葡萄糖苷酶的新资源。本研究纯化了木质纤维素分解真菌烟曲霉Z5分泌的耐热天然β-葡萄糖苷酶（nBgl3），达到电泳均匀性。通过LC-MS/MS获得nBgl3的内部序列，并根据LC-MS/MS结果克隆其编码基因bgl3。bgl3包含2622 bp的开放阅读框（ORF），编码一个预测分子量为91.47 kDa的蛋白；氨基酸序列分析表明nBgl3是糖苷水解酶家族3的成员。在毕赤酵母X33中功能表达重组β-葡萄糖苷酶（rBgl3）。对纯化后的nBgl3和rBgl3的生化性能进行了测试，结果表明，两种酶在pH 6.0和60℃时均表现出最佳活性，在pH 4 ~ 7和温度50 ~ 70℃范围内均保持稳定。nBgl3和rBgl3对4-Nitrophenyl-β-D-glucopyranoside （pNPG）的比活性最高，分别为103.5±7.1和101.7±5.2 U mg-1。然而，这些酶对羧甲基纤维素、乳糖和木聚糖无活性。从烟曲霉Z5粗提物中纯化了一种天然β-葡萄糖苷酶nBgl3，达到电泳均匀性。根据LC-MS/MS结果获得的nBgl3内部序列克隆出bgl3基因，bgl3基因在毕赤酵母X33中表达。两种酶的比活性、pH稳定性、热稳定性和动力学性能（Km和Vmax）等生化性能均无显著差异。
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. In 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. An 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.
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影响因子：
1.6
作者：
Liu, Dongyang;Zhang, Ruifu;Shen, Qirong
通讯作者：
Shen, Qirong
影响因子：
1.9
作者：
Helal GA
通讯作者：
Helal GA
DOI：
10.1016/0922-338x(92)90138-k
发表时间：
1992-01-01
期刊：
JOURNAL OF FERMENTATION AND BIOENGINEERING
影响因子：
--
作者：
KIM, DW;KIM, TS;LEE, JK
通讯作者：
LEE, JK
影响因子：
5
作者：
Hong, Jiong;Tamaki, Hisanori;Kumagai, Hidehiko
通讯作者：
Kumagai, Hidehiko
DOI：
10.1016/j.ibiod.2011.04.005
发表时间：
2011-08-01
影响因子：
4.8
作者：
Liu, Dongyang;Zhang, Ruifu;Shen, Qirong
通讯作者：
Shen, Qirong