4‐cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high‐value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4‐cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a “small and accurate” mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg−1, which was 3.8 times higher than that of the wild‐type AfNIT. Using the whole‐cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L−1 to produce 4‐cyanobenzoic acid with a final yield of 170.3 g L−1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4‐cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI‐assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies.

中文翻译：

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基于计算机筛选的腈水解酶修饰及4-氰基苯甲酸的高效生物合成

4-氰基苯甲酸是合成各种高价值有机化合物的重要中间体。利用腈水解酶酶解对苯二甲腈生产4-氰基苯甲酸具有反应途径简单、环境友好、产物易于分离等优点。为了高效开发满足工业化生产要求的腈水解酶，本研究采用的虚拟筛选方法已经建立并成熟。腈水解酶AfNIT具有对苯二甲腈水解活性，从总共371个氨基酸中，鉴定出三个候选位点（F168、S192和T201），并构建了“小而准确”的突变体库。从这个小突变体库中筛选出三重突变体F168V/T201N/S192F，比活性为227.3 U mg−1，比野生型 AfNIT 高 3.8 倍。使用含有突变体F168V/T201N/S192F的全细胞生物催化剂，成功水解了浓度为150 g·L的对苯二甲腈−1生产4-氰基苯甲酸，最终产量为170.3 g·L−1转化率为98.7%。本研究获得的腈水解酶突变体F168V/T201N/S192F可有效应用于以对苯二甲腈为底物的4-氰基苯甲酸的生物制造中。此外，结果还表明，通过最新的人工智能辅助计算机模拟方法，预测准确性得到了显着提高。该方法为辅助酶工程研究提供了一条有前途且可行的新技术途径，为其他相关研究奠定了理论基础。