腈水合酶
热稳定性
化学
基质(水族馆)
腈
生物催化
蛋白质工程
定向进化
催化作用
酶
组合化学
底物特异性
有机化学
生物化学
突变体
基因
反应机理
生物
生态学
作者
Junling Guo,Julia Berdychowska,Qianpeng Lai,Yiwei Meng,Zhongyi Cheng,Łukasz Pepłowski,Zhemin Zhou
标识
DOI:10.1016/j.ijbiomac.2022.09.071
摘要
Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus (S.t NHase) was successfully overexpressed in Escherichia coli. The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant βL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into βLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a "toolbox" containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.
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