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Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation

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Názov: Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation
Autori: Baocheng CUI, Jiao HUANG, Jiaxin LI, Yi GUO, Li WANG, Changhai LIANG
Zdroj: Shipin gongye ke-ji, Vol 43, Iss 7, Pp 148-154 (2022)
Informácie o vydavateľovi: The editorial department of Science and Technology of Food Industry, 2022.
Rok vydania: 2022
Zbierka: LCC:Food processing and manufacture
Predmety: nitrile hydratase, nicotinonitrile, virtual amino acid mutation, substrate affinity, Food processing and manufacture, TP368-456
Popis: Objective: A semi-rational design was used to increase the affinity of the nitrile hydratase (ReNHase) derived from Rhodococcus erythropolis CCM2595 with the substrate nicotinonitrile. Methods: The 1AHJ protein with high homology was found through sequence comparison and evaluated by software Swiss-Model and iTASSER. The molecular docking of nicotinonitrile with 1AHJ was then performed with Discovery Studio 2016 (DS), which aimed to obtain the virtual amino acid mutations with significantly improved affinity. The mutant recombinant plasmid was then constructed and transformed into E. coli expression competent cells for heterologous expression. After the purification of mutant ReNHase from the recombined E. coli, the biotransformation of nicotinonitrile was detected and analyzed by high performance liquid chromatography. Results: According to predicting the calculate mutation energy (Binding) of nicotinonitrile with ReNHase， CYS113 and CYS115 of αsubunit were mutated to TYR (C113Y) and ASN (C115N), VAL52 of βsubunit was mutated to ARG (V52R). According to the kinetic parameters of the reaction by the purified ReNHase followed the Michaelis–Menten model, the Km value of mutant ReNHase C113Y /C115N/ V52R decreased from 16.78 mmol/L to 12.69 mmol/L when compared with wild ReNHase, the enzyme activity increased from 12.14 U/mL to 15.15 U/mL. Conclusion: Compared with wild ReNHase, the substrate affinity of nicotinitrile with mutant ReNHase C113Y /C115N/ V52R increased by 24.37%, the enzyme activity increased by 24.79%. The above results provided a new theoretical basis for the industrial application of nicotinonitrile.
Druh dokumentu: article
Popis súboru: electronic resource
Jazyk: Chinese
ISSN: 1002-0306
Relation: https://doaj.org/toc/1002-0306
DOI: 10.13386/j.issn1002-0306.2021080147
Prístupová URL adresa: https://doaj.org/article/fc9b530d4ae74c2b93a184532c17f897
Prístupové číslo: edsdoj.fc9b530d4ae74c2b93a184532c17f897
Databáza: Directory of Open Access Journals
Popis
Abstrakt:Objective: A semi-rational design was used to increase the affinity of the nitrile hydratase (ReNHase) derived from Rhodococcus erythropolis CCM2595 with the substrate nicotinonitrile. Methods: The 1AHJ protein with high homology was found through sequence comparison and evaluated by software Swiss-Model and iTASSER. The molecular docking of nicotinonitrile with 1AHJ was then performed with Discovery Studio 2016 (DS), which aimed to obtain the virtual amino acid mutations with significantly improved affinity. The mutant recombinant plasmid was then constructed and transformed into E. coli expression competent cells for heterologous expression. After the purification of mutant ReNHase from the recombined E. coli, the biotransformation of nicotinonitrile was detected and analyzed by high performance liquid chromatography. Results: According to predicting the calculate mutation energy (Binding) of nicotinonitrile with ReNHase， CYS113 and CYS115 of αsubunit were mutated to TYR (C113Y) and ASN (C115N), VAL52 of βsubunit was mutated to ARG (V52R). According to the kinetic parameters of the reaction by the purified ReNHase followed the Michaelis–Menten model, the Km value of mutant ReNHase C113Y /C115N/ V52R decreased from 16.78 mmol/L to 12.69 mmol/L when compared with wild ReNHase, the enzyme activity increased from 12.14 U/mL to 15.15 U/mL. Conclusion: Compared with wild ReNHase, the substrate affinity of nicotinitrile with mutant ReNHase C113Y /C115N/ V52R increased by 24.37%, the enzyme activity increased by 24.79%. The above results provided a new theoretical basis for the industrial application of nicotinonitrile.
ISSN:10020306
DOI:10.13386/j.issn1002-0306.2021080147