5-cyanopentamide (5-CVAM) is an important chemical intermediate of chemical raw materials,1,21. E. C. Hann et al., Bioorg. Med. Chem. 7, 2239 (1999). https://doi.org/10.1016/S0968-0896(99)00157-12. H.-M. Weitz, R. Fischer, and F. J. Broecker, US Pat. 4119665A (1978). which can be used to synthesize a new herbicide, azirazinone, with low toxicity, high efficiency, and little adverse effects on the environment.33. R. Shapiro, R. DiCosimo, S. M. Hennessey, B. Stieglitz, O. Campopiano, and G. C. Chiang, Org. Process Res. Dev. 5, 593 (2001). https://doi.org/10.1021/op9901994 5-CVAM is generally synthesized from adiponitrile under high temperatures and pressures.33. R. Shapiro, R. DiCosimo, S. M. Hennessey, B. Stieglitz, O. Campopiano, and G. C. Chiang, Org. Process Res. Dev. 5, 593 (2001). https://doi.org/10.1021/op9901994 However, this chemical method has poor selectivity for dicarbonitrile and produces massive by-products during the production process, thus reducing the yield and purity of 5-CVAM and increasing the production cost.Biocatalysis is constantly evolving and has been widely adopted in the chemical industry.44. U. T. Bornscheuer, Philos. Trans. R. Soc. London A 376, 20170063 (2018). https://doi.org/10.1098/rsta.2017.0063 Nitrile hydratase (NHase; EC 4.2.1.84) is a key enzyme in nitrile metabolism that catalyze the hydration of nitriles to their corresponding amides.5,65. L. Martinkova and V. Křen, Biocatal. Biotransform. 20, 73 (2002). https://doi.org/10.1080/102424202900180696. S. Prasad and T. C. Bhalla, Biotechnol. Adv. 28, 725 (2010). https://doi.org/10.1016/j.biotechadv.2010.05.020 A variety of NHases has been used in industrial production due to their mild catalytic conditions and broad substrate acceptance. Among the known NHases, only a small part of them can catalyze adiponitrile to produce 5-CVAM, which are from Pseudomonas chlororaphis B23, Pseudomonas sp. SY031, Rhodococcus erythropolis CCM2595, and Rhodococcus ruber CGMCC3090, respectively. Despite the considerable advantages of NHases in catalyzing the generation of 5-CVAM from adiponitrile, the wide application of NHases in the industrial-scale production of 5-CVAM is still limited due to their poor tolerance to extreme environments, low substrate affinity, and low catalytic efficiency.77. J. Guo, Z. Cheng, J. Berdychowska, X. Zhu, L. Wang, L. Peplowski, and Z. Zhou, Int. J. Biol. Macromol. 181, 444 (2021). https://doi.org/10.1016/j.ijbiomac.2021.03.103 To enhance the catalytic performance of NHases, attention has been directed to the artificial modification of NHases. There have been many approaches to improve the stability of NHases such as protein engineering, enzyme immobilization, and medium engineering.88. L. Q. Ren, T. T. Chang, D. P. Ren, Y. Zhou, and B. C. Ye, Enzyme Microb. Technol. 131, 109331 (2019). https://doi.org/10.1016/j.enzmictec.2019.04.005 The current trends in protein engineering mainly include directed evolution, rational design, and semirational design. Directed evolution remains the predominant strategy for enzyme modification yet.99. Á Sánchez, J. C. Vila, C.-Y. Chang, J. Diaz-Colunga, S. Estrela, and M. Rebolleda-Gomez, Annu. Rev. Biophys. 50, 323 (2021). https://doi.org/10.1146/annurev-biophys-101220-072829 By virtue of the rapid development of bioinformatics tools and the available crystal structure of many enzymes in the Protein Data Bank, semirational protein engineering shows certain advantages and benefits compared with directed evolution and rational design.1010. Z. Cheng, W. Cui, Y. Xia, L. Peplowski, M. Kobayashi, and Z. Zhou, ChemCatChem 10, 449 (2018). https://doi.org/10.1002/cctc.201701170 The semirational design approach requires detailed knowledge about the structure and function of the enzyme, and the mutant with improved catalytic performance is designed by a computer.11–1311. Q. Chen, Y. Xiao, E. I. Shakhnovich, W. Zhang, and W. Mu, Int. J. Biol. Macromol. 154, 1356 (2020). https://doi.org/10.1016/j.ijbiomac.2019.11.01512. U. Markel, K. D. Essani, V. Besirlioglu, J. Schiffels, W. R. Streit, and U. Schwaneberg, Chem. Soc. Rev. 49, 233 (2020). https://doi.org/10.1039/C8CS00981C13. P. Xu, Z.-F. Ni, M.-H. Zong, X.-Y. Ou, J.-G. Yang, and W.-Y. Lou, Int. J. Biol. Macromol. 150, 9 (2020). https://doi.org/10.1016/j.ijbiomac.2020.02.033 This approach can reduce the mutant library, modify the enzyme protein purposefully, minimize the difficulty of identifying mutant catalytic activity, and reduce workload.1414. R. A. Sheldon and P. C. Pereira, Chem. Soc. Rev. 46, 2678 (2017). https://doi.org/10.1039/C6CS00854B There have been many successful cases of artificially modified enzymes. For example, two β-mannanase mutants were successfully obtained through the semirational strategy that combined structure-based computational analysis and site-directed mutagenesis, and their affinity to guar gum was increased by 34.3 and 44.8%, respectively.1515. J. Li, X. Wei, C. Tang, J. Wang, M. Zhao, Q. Pang, and M. Wu, J. Ind. Microbiol. Biotechnol. 41, 693 (2014). https://doi.org/10.1007/s10295-014-1406-7 In addition, the computer-aided semirational design enhanced the thermal stability of β-glucosidase mutants by 46.3%.1616. Z. Zong, L. Gao, W. Cai, L. Yu, C. Cui, S. Chen, and D. Zhang, BioEnergy Res. 8, 1384 (2015). https://doi.org/10.1007/s12155-015-9603-4Previous studies have identified an NHase from Rhodococcus erythropolis CCM2595 (ReNHase), showing significant regioselectivity and bioconversion to adiponitrile.17,1817. W. Du, J. Huang, B. Cui, Y. Guo, L. Wang, and C. Liang, Biotechnol. Biotechnol. Equip. 35, 1127 (2021). https://doi.org/10.1080/13102818.2021.194125318. L. Wang, S. Liu, W. Du, T. Dou, and C. Liang, ACS Omega 5, 18397 (2020). https://doi.org/10.1021/acsomega.0c02188 To further improve the substrate affinity of ReNHase to adiponitrile, semirational design strategies were adopted to predict mutation sites, including homology modeling, molecular docking, and calculation of binding free energy. On the basis of prediction, the mutant plasmid was constructed by site-directed mutagenesis and expressed in E. coil ArcticExpression (DE3). Then, the enzymatic properties and kinetic constants of the mutant NHase were analyzed.