"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.
"Toolbox" construction of an extremophilic nitrile hydratase from Streptomyces thermoautotrophicus for the promising industrial production of various amides.
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DOI：
10.1016/j.ijbiomac.2022.09.071
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发表时间：
2022-09
影响因子：
8.2
通讯作者：
Junling Guo;Julia Berdychowska;Qianpeng Lai;Yiwei Meng;Zhongyi Cheng;L. Peplowski;Zhemin Zhou
中科院分区：
文献类型：
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作者：
Junling Guo;Julia Berdychowska;Qianpeng Lai;Yiwei Meng;Zhongyi Cheng;L. Peplowski;Zhemin Zhou
腈水合酶（NHase; EC 4.2.1.84）被广泛用于从腈合成相应的酰胺，是最成功的绿色生物催化剂。然而，底物的可接受性有限以及在恶劣反应条件下的不稳定性阻碍了其广泛的工业应用。在这里，一个基因编码来自热自养链霉菌(S。tNHase)在大肠杆菌中成功过表达。该酶表现出优异的热稳定性，在65℃热处理252 min后，酶的残余活性保持了50%。为了进一步提高s的催化性能。最后，对其衬底通道进行了半合理的工程设计。突变体βL48D对3-氰吡啶的比活性为566.18±18.86 U/mg，是其亲本酶（73.80±5.76 U/mg）的7.7倍。分子动力学模拟表明，在βLeu48中引入天冬氨酸导致底物通道入口的开放更大、更频繁。在此基础上，进一步建立了包含底物通道上各种突变体的“工具箱”，其对各种腈底物的催化活性得到了广泛提高，显示出高效合成多种高价值酰胺的巨大潜力。
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 Streptomycesthermoautotrophicus(S.tNHase) was successfully overexpressed inEscherichia 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 ofS.tNHase, 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.