Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase.
Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase.
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嗜温木聚糖酶与超嗜热葡聚糖酶的结构域交换
DOI：
10.1186/1472-6750-12-28
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发表时间：
2012-06-07
影响因子：
3.5
通讯作者：
Chen H
中科院分区：
文献类型：
--
作者：
Liu L;Wang L;Zhang Z;Guo X;Li X;Chen H
结构域融合仅限于酶的一端。通过将中温黑曲霉GH11木聚糖酶(XYN)与超嗜热性海藻葡聚糖酶(GLU)互换，构建了两个嵌合体XYN-Glu和Glu-XYN，目的是产生具有葡聚糖酶活性的耐热木聚糖酶。在大肠杆菌BL21(DE3)中表达的两个嵌合体具有木聚糖酶和葡聚糖酶的双功能活性。XYN-Glu XYN部分的最佳反应温度(TOPT)为50 °C，热失活半衰期(t1/2)为50 °C，持续时间为47.6min，而XYN为47 °C和17.6min。谷氨酸-XYN部分的TOPT与XYN相当，T1/2(5.2 分钟)比XYN短。两种嵌合体的热稳定性均高于谷氨酸，三种酶的Topt值均大于96 °C，谷氨酸的最适pH为5.8，而异型谷氨酸和谷氨酸的最适pH为3.8。两个嵌合体两部分在降解底物方面相互合作。域互换对不同部分属性的影响不同。C端融合Glu结构域提高了木聚糖酶的热稳定性，而N端融合Glu结构域降低了木聚糖酶的热稳定性。在两端融合XYN结构域提高了葡聚糖酶的热稳定性，而在C末端融合XYN结构域使葡聚糖酶的pH特性向碱性环境升高了2个单位。在C-末端融合一个结构域更有助于酶的催化活性，而在N-末端融合一个更大的结构域会扰乱酶底物的结合亲和力。
Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity. When expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50 °C and thermal in-activation half-life (t1/2) at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates. Domain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.
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影响因子：
3.4
作者：
An, JM;Kim, YK;Yun, HD
通讯作者：
Yun, HD
影响因子：
2.7
作者：
Hong, Su-Young;Lee, Jin-Suk;Yun, Han-Dae
通讯作者：
Yun, Han-Dae
影响因子：
64.8
作者：
Nelson, KE;Clayton, RA;Fraser, CM
通讯作者：
Fraser, CM
影响因子：
2.1
作者：
Lu, Ping;Feng, Ming-Guang;Hu, Chun-Xia
通讯作者：
Hu, Chun-Xia
影响因子：
2.7
作者：
Hong, Su Young;Lee, Jin Suk;Yun, Han Dae
通讯作者：
Yun, Han Dae