Objectives

Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases.

Results

In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications.

Conclusions

Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

中文翻译：

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转换来自 Acidovorax facilis 72 W 的腈水解酶的二级和天然活性，以高效生产 2-吡啶甲酰胺

目标

催化混杂，或催化二次反应的能力，通过扩大生物催化反应的范围为工业生物催化提供了新的机会。一些腈水解酶将腈转化为酰胺，称为二级活性，显示出生产酰胺的巨大潜力。我们的目标是利用腈水解酶的酰胺形成潜力。

结果

在这项研究中，我们表征并改变了来自Acidovorax facilis 72 W (Nit72W)的腈水解酶对不同底物的二级活性。我们将 Nit72W 对 2-氰基吡啶的二级活性提高了 196 倍，并通过修改活性口袋创造了对苯甲腈和对硝基苯乙腈的活性。令人惊讶的是，最好的突变体 W188M 在 12 小时内将 250 mM 2-氰基吡啶完全转化为 98% 以上的 2-吡啶甲酰胺，比活性为 90 U/mg，并显示出工业应用的潜力。

结论

Nit72W 被修改以增加其对酰胺生产的次级活性，尤其是 2-吡啶甲酰胺。