Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution
Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution
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DOI：
10.1007/s00253-008-1789-3
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发表时间：
2009-03-01
影响因子：
5
通讯作者：
Liu, Ziduo
中科院分区：
文献类型：
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作者：
Lin, Ling;Meng, Xin;Liu, Ziduo
枯草芽孢杆菌内切-1,4-葡聚糖酶（Cel5A）通过裂解葡萄糖链的内部键来水解纤维素，随机产生新的末端。利用易出错聚合酶链反应（PCR）和DNA洗牌的定向进化技术，从包含71,000个菌落的突变文库中筛选了几个具有改进催化活性的Cel5A变体。与野生型酶相比，M44-11、S75和S78对羧甲基纤维素钠（CMC）的活性提高了2.03 ~ 2.68倍，M44-11对pH值的耐受性更强，热稳定性更高。M44-11、S75、S78和WT蛋白的结构模型显示，除了S75的突变V255A靠近酶催化中心的亲核试剂Glu257外，大部分取代位点都不在严格保守区域。此外，M44-11的V74A和D272G不在底物结合位点和催化中心，可能会提高稳定性和催化活性。这些结果为糖苷水解酶家族5 （GH5）酶的定向进化提供了有益的参考。
Bacillus subtilis endo-beta-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).