Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii
Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii
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DOI：
10.1007/s00792-006-0033-2
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发表时间：
2007-03-01
期刊：
影响因子：
2.9
通讯作者：
Ishikawa, Kazuhiko
中科院分区：
文献类型：
--
作者：
Kang, Hee-Jin;Uegaki, Koichi;Ishikawa, Kazuhiko
来自超嗜热古菌Pyrococcus horikoshii的超嗜热β-1，4内切葡聚糖酶（EGPh）对结晶纤维素表现出强的水解活性。EGPh的特征是：（1）它似乎具有二硫键，这在厌氧超嗜热古菌蛋白中是罕见的，和（2）它缺乏碳水化合物结合结构域，这是有效水解纤维素所必需的。我们首先通过分析各种半胱氨酸突变来研究二硫键与催化活性之间的关系。对羧甲基纤维素（CMC）的突变酶的活动增加，而没有任何损失的热稳定性。其次，我们制备了一种融合酶，使来自激烈毕赤酵母的几丁质酶的热稳定几丁质结合结构域连接到EGPh及其变体的C-末端。这些融合酶表现出比野生型EGPh对CMC和结晶纤维素（Avicel）更强的活性。
A hyperthermophilic beta-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).