从垂穗披碱草内生真菌中克隆和鉴定两种主要内切葡聚糖酶。
Molecular cloning and characterization of two major endoglucanases from Penicillium decumbens.
机构信息
State Key Laboratory of Microbial Technology,Shandong University, 27 Shanda South Road, Jinan, Shandong 250100, P. R. China.
出版信息
J Microbiol Biotechnol. 2010 Feb;20(2):265-70.
Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of cel7B gene in its chromosomal DNA. The expression level of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that two genes were coordinately expressed and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimal active at 60 C and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, 5-fold higher enzyme activity towards carboxymethyl cellulose, barely beta-glucan and PASC respectively in comparison with that of Cel5A. However, their activities toward pNPC and Avicel were minor difference. The result suggested that Cel7B is a strict endoglucanase, while Cel5A show processivity because of its relative higher ability to hydrolyze the crystal cellulose.
采用改良的热不对称交错聚合酶链式反应（TAIL-PCR）方法，从里氏木霉 114-2 中克隆得到两个主要内切葡聚糖酶基因（cel7B 和 cel5A）。Southern 印迹结果表明，里氏木霉的染色体 DNA 中仅有 cel5A 基因的单拷贝和 cel7B 基因的单拷贝。通过实时定量 PCR 测定 cel5A 和 cel7B 的表达水平，表明两个基因协同表达，受葡萄糖抑制，被纤维素诱导。两个内切葡聚糖酶基因均在酿酒酵母中表达，并对重组蛋白进行了纯化。重组 Cel7B 和 Cel5A 均在 60°C 和 pH 4.0 下具有最佳活性。与 Cel5A 相比，重组 Cel7B 对羧甲基纤维素、几乎β-葡聚糖和 PASC 的酶活性分别高出 8 倍、30 倍和 5 倍，但对 pNPC 和 Avicel 的活性差异较小。结果表明，Cel7B 是一种严格的内切葡聚糖酶，而 Cel5A 由于其相对较高的水解结晶纤维素的能力，表现出较强的连续性。
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