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

CUI Baocheng; HUANG Jiao; LI Jiaxin; GUO Yi; WANG Li; LIANG Changhai

doi:10.13386/j.issn1002-0306.2021080147

Volume 43 Issue 7

Mar. 2022

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Science and Technology of Food Industry > 2022 > 43(7): 148-154. > DOI: 10.13386/j.issn1002-0306.2021080147

CUI Baocheng, HUANG Jiao, LI Jiaxin, et al. Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation[J]. Science and Technology of Food Industry, 2022, 43(7): 148−154. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021080147.

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

1.
School of Life Science and Pharmacy, Dalian University of Technology, Panjin 124221, China
2.
School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China

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Received Date: August 15, 2021
Available Online: February 09, 2022

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Abstract

Abstract

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.
- nitrile hydratase,
- nicotinonitrile,
- virtual amino acid mutation,
- substrate affinity

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