Design of Hyperthermophilic Lipase Chimeras by Key Motif‐Directed Recombination
Design of Hyperthermophilic Lipase Chimeras by Key Motif‐Directed Recombination
复制标题
DOI：
10.1002/cbic.201402456
复制
发表时间：
2015-02
期刊：
影响因子：
3.2
通讯作者：
Xiaoli Zhou;Le Gao;Guangyu Yang;Donglai Liu;A. Bai;Binchun Li;Z. Deng;Yan Feng
中科院分区：
文献类型：
--
作者：
Xiaoli Zhou;Le Gao;Guangyu Yang;Donglai Liu;A. Bai;Binchun Li;Z. Deng;Yan Feng
在一个超家族中，不同的自然进化结构域的分离为酶功能的飞跃提供了更大的机会。如何重组来自远亲的蛋白质模块，同时减少交叉界面的破坏是一个具有挑战性的问题。在这里，我们通过使用MEME服务器和PROMOTIF程序，在α/β-水解酶折叠超家族的结构基序α-环内确定了一个关键基序，即序列VVSVN（D）YR。为了获得热稳定的脂肪酶样酶，通过来自嗜温脂肪酶和具有小于21%的氨基酸同一性的超嗜热酯酶/肽酶的结构域的重组，在关键基序区域处工程化两个嵌合体。嵌合体保留了其嗜温亲本的理想底物偏好，并在50 °C下表现出超过100倍的热稳定性。通过定点突变，我们进一步将嵌合体的活性提高了4.6倍。这里提出的重组策略使得能够产生新型催化剂。
Recombination of diverse natural evolved domains within a superfamily offers greater opportunity for enzyme function leaps. How to recombine protein modules from distant parents with less disruption in cross‐interfaces is a challenging issue. Here, we identified the existence of a key motif, the sequence VVSVN(D)YR, within a structural motif ψ loop in the α/β‐hydrolase fold superfamily, by using a MEME server and the PROMOTIF program. To obtain thermostable lipase‐like enzymes, two chimeras were engineered at the key motif regions through recombination of domains from a mesophilic lipase and a hyperthermophilic esterase/peptidase with amino acid identity less than 21 %. The chimeras retained the desirable substrate preference of their mesophilic parent and exhibited more than 100‐fold increased thermostability at 50 °C. Through site‐directed mutation, we further improved activity of the chimera by 4.6‐fold. The recombination strategy presented here enables the creation of novel catalysts.