Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast
Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast
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DOI：
10.1007/s00253-003-1379-3
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发表时间：
2003-11-01
影响因子：
5
通讯作者：
Kumagai, H
中科院分区：
文献类型：
--
作者：
Hong, J;Tamaki, H;Kumagai, H
从橙色嗜热菌IFO 9748中分离到一个编码纤维二糖水解酶（CBH）的基因，命名为cbh 1。cbh 1编码的氨基酸序列与糖苷水解酶家族7的氨基酸序列具有高度的同源性。为了确认编码CBH的基因的序列，克隆的基因在酵母酿酒酵母中表达，其中未发现纤维素酶活性，纯化基因产物并进行酶促表征。通过对反应产物的分析证实重组酶为CBH，并将其命名为CBHI。重组CBHI在65 ℃孵育1 h后保留其初始活性的80%以上，并且在pH 3.0-9.0范围内稳定。该酶的最适反应温度为65 ℃，最适pH为6.0。重组酶被发现是高度糖基化的，这种糖基化被证明有助于酶的热稳定性。CBHI在T.橙黄色
A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 degreesC and was stable in the pH range 3.0-9.0. The optimal temperature for enzyme activity was about 65 degreesC and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus.