Engineered Thermobifida fusca cutinase with increased activity on polyester substrates
Engineered Thermobifida fusca cutinase with increased activity on polyester substrates
复制标题
DOI：
10.1002/biot.201000391
复制
发表时间：
2011-10-01
影响因子：
4.7
通讯作者：
Cavaco-Paulo, Artur
中科院分区：
文献类型：
--
作者：
Silva, Carla;Da, Shi;Cavaco-Paulo, Artur
来自 Thermobifida fusca 的细菌角质酶（名为 Tfu_0883）通过定点诱变进行了基因修饰，以增强其对聚对苯二甲酸乙二醇酯 (PET) 的活性。新的突变为 PET 定制了催化位点，增加了角质酶对这种疏水底物的亲和力以及水解它的能力。突变 I218A 被设计为创造空间，双突变 Q132A/T101A 被设计为既创造空间又增加疏水性。与野生型角质酶相比，双突变体对可溶性底物对硝基苯丁酸的活性增加了一倍，而对 PET，单突变体和双突变体都表现出相当高的水解效率。活性位点特定氨基酸的替换是提高Tfu_0883角质酶水解聚酯表面能力的有效途径。因此，这项研究为如何通过分子工程改善酶的功能和稳定性以使其在合成纤维生物转化中的应用提供了有价值的见解。
A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased twofold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.