Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost
Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost
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DOI：
10.1016/j.pep.2011.06.008
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发表时间：
2011-10-01
影响因子：
1.6
通讯作者：
Shen, Qirong
中科院分区：
文献类型：
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作者：
Liu, Dongyang;Zhang, Ruifu;Shen, Qirong
从木质纤维素分解真菌烟曲霉Z5中克隆了两个编码内切葡聚糖酶的基因，命名为eg12和eg13，并在毕赤酵母X33中成功表达。推导的eg12和eg13编码的氨基酸序列与糖苷水解酶家族5的序列具有很强的相似性。SDS-PAGE和蛋白质印迹分析表明重组酶被分泌到培养基中，酶谱分析证实两种重组酶都具有内切葡聚糖酶活性。研究了两种重组酶的几种生化特性：Eg12 和 Eg13 分别在 pH 5.0 和 4.0、50 和 60 摄氏度下显示出最佳活性。 Eg12和Eg13在4-7范围内表现出良好的pH稳定性，并且两种酶在30至60℃范围内表现出良好的热稳定性。测定了以羧甲基纤维素（CMC，可溶性纤维素，由β-1，4-连接的葡萄糖残基聚合）为底物在最佳条件下的K（m）和V（max）值。研究了酶对多种纤维寡糖底物的活性，Eg12可以水解纤维四糖和纤维五糖，但不能水解纤维二糖和纤维三糖，而Eg13可以水解除纤维二糖之外的所有纤维寡糖。 (C) 2011 Elsevier Inc. 保留所有权利。
Two genes encoding endoglucanase, designated as eg12 and eg13, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by eg12 and eg13 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Eg12 and Eg13 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60 degrees C, respectively. Eg12 and Eg13 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60 degrees C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by beta-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Eg12 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Eg13 can hydrolyze all cello-oligosaccharides, except cellobiose. (C) 2011 Elsevier Inc. All rights reserved.