Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.
Improvement of the catalytic efficiency of a hyperthermophilic xylanase from Bispora sp. MEY-1.
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双孢菌 MEY-1 超嗜热木聚糖酶催化效率的提高
DOI：
10.1371/journal.pone.0189806
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Luo H
中科院分区：
文献类型：
--
作者：
Wang X;Zheng F;Wang Y;Tu T;Ma R;Su X;You S;Yao B;Xie X;Luo H
嗜极木聚糖酶引起了科学和工业的极大兴趣。本研究从双孢菌MEY-1中克隆了GH10木聚糖酶编码基因Xyl10E，并在毕赤酵母GS115中表达。推测得到的Xyl10E与结构为4F8X的leycettanus Talaromyces和canescens青霉菌GH10木聚糖酶的同源性最高，分别为62%和57%。Xyl10E在93 ~ 95℃和pH 4.0时活性最高，分别在80℃和90℃加热30 min时保持75%和48%以上的初始活性，在pH 1.0 ~ 7.0时几乎没有失去活性，但被SDS完全抑制。位于环路4上的两个残基A160和A161在催化过程中起作用。突变体A160D/E对底物的亲和力较高，Km值较低，而突变体A161D/E主要表现为Vmax值升高。所有这些突变体的催化效率都有显著提高。分子动力学模拟表明，A160E突变能够影响重要底物结合位点Y204，进而提高底物亲和力，A161D突变能够与底物形成氢键，促进底物结合或加速产物释放。本研究介绍了一种高度嗜热的真菌木聚糖酶，揭示了环4对催化效率的重要性。
Extremophilic xylanases have attracted great scientific and industrial interest. In this study, a GH10 xylanase-encoding gene, Xyl10E, was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris GS115. Deduced Xyl10E shares the highest identities of 62% and 57% with characterized family GH10 xylanases from Talaromyces leycettanus and Penicillium canescens (structure 4F8X), respectively. Xyl10E was most active at 93 to 95°C and pH 4.0, retained more than 75% or 48% of the initial activity when heated at 80°C or 90°C for 30 min, respectively, and hardly lost activity at pH 1.0 to 7.0, but was completely inhibited by SDS. Two residues, A160 and A161, located on loop 4, were identified to play roles in catalysis. Mutants A160D/E demonstrated higher affinity to substrate with lower Km values, while mutants A161D/E mainly displayed elevated Vmax values. All of these mutants had significantly improved catalytic efficiency. According to the molecular dynamics simulation, the mutation of A160E was able to affect the important substrate binding site Y204 and then improve the substrate affinity, and the mutation of A161D was capable of forming a hydrogen bond with the substrate to promote the substrate binding or accelerate the product release. This study introduces a highly thermophilic fungal xylanase and reveals the importance of loop 4 for catalytic efficiency.
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影响因子：
6.4
作者：
Turner P;Mamo G;Karlsson EN
通讯作者：
Karlsson EN
影响因子：
2.9
作者：
Sunna, A;Bergquist, PL
通讯作者：
Bergquist, PL
DOI：
10.1007/s10295-003-0049-x
发表时间：
2003-05-01
影响因子：
3.4
作者：
Saha, BC
通讯作者：
Saha, BC
影响因子：
5.5
作者：
Roe, Daniel R.;Cheatham, Thomas E., III
通讯作者：
Cheatham, Thomas E., III
影响因子：
5.4
作者：
Hoffmam, Zaira B.;Zanphorlin, Leticia M.;Ruller, Roberto
通讯作者：
Ruller, Roberto