Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae
Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae
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米霉产生的纤维二糖水解酶 (MoCel6A) 的表征
DOI：
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发表时间：
2010
影响因子：
4.4
通讯作者：
T. Takeda
中科院分区：
文献类型：
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作者：
Machiko Takahashi;Hideyuki Takahashi;Yuki Nakano;T. Konishi;R. Terauchi;T. Takeda
根据稻瘟病菌基因组全序列，推测稻瘟病菌基因组中存在三种GH-6家族纤维二糖水解酶。在这里，我们展示了Magnaporthe的GH-6家族纤维二糖水解酶（MoCel 6A）的特性，动力学和底物特异性。此外，还研究了纤维二糖对MoCel 6A活性的影响。与组氨酸标签连续融合的MoCel 6A在M.纯化，并通过亲和色谱法纯化。MoCel 6A对磷酸溶胀纤维素（PSC）、β-葡聚糖和纤维低聚糖衍生物的水解活性高于对纤维素的水解活性，其中以纤维低聚糖为最佳底物。一个串联排列的纤维素结合结构域（CBD）的N端引起纤维素和PSC的活性增加，而删除的CBD（催化结构域）显示纤维素的活性降低。MoCel 6A水解纤维低聚糖和磺酰罗丹明缀合的纤维低聚糖不受外源添加高达438 mM的纤维二糖的抑制，相反，其增强了活性，而来自M.超过29 mM的纤维二糖严重抑制MoCel 7A。此外，我们评估了纤维二糖对使用MoCel 6A和里氏木霉纤维二糖水解酶（TrCel 6A）的水解活性的影响，这两种纤维二糖水解酶是在曲霉中制备的。MoCel 6A显示在pH 4.5和pH 6.0下在292 mM纤维二糖存在下用作底物的纤维五糖的水解增加，并且增强的活性在pH 9.0下消失。相比之下，TrCel 6A在pH 4.5下表现出略微增加的水解，并且在pH 9.0下水解被严重抑制。这些结果表明，纤维二糖对水解活性的增强或抑制取决于反应混合物的pH。
ABSTRACT Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acid-swollen cellulose (PSC), β-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.
影响因子：
3.5
作者：
Beckerman, JL;Ebbole, DJ
通讯作者：
Ebbole, DJ