Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation
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-...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | zho |
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The editorial department of Science and Technology of Food Industry
2022-04-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021080147 |
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| author | Baocheng CUI Jiao HUANG Jiaxin LI Yi GUO Li WANG Changhai LIANG |
| author_facet | Baocheng CUI Jiao HUANG Jiaxin LI Yi GUO Li WANG Changhai LIANG |
| author_sort | Baocheng CUI |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T16:07:59Z |
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| institution | Directory Open Access Journal |
| issn | 1002-0306 |
| language | zho |
| last_indexed | 2024-04-11T16:07:59Z |
| publishDate | 2022-04-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
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| series | Shipin gongye ke-ji |
| spelling | doaj.art-fc9b530d4ae74c2b93a184532c17f8972022-12-22T04:14:47ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062022-04-0143714815410.13386/j.issn1002-0306.20210801472021080147-7Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot MutationBaocheng CUI0Jiao HUANG1Jiaxin LI2Yi GUO3Li WANG4Changhai LIANG5School of Life Science and Pharmacy, Dalian University of Technology, Panjin 124221, ChinaSchool of Life Science and Pharmacy, Dalian University of Technology, Panjin 124221, ChinaSchool of Chemical Engineering, Dalian University of Technology, Dalian 116024, ChinaSchool of Chemical Engineering, Dalian University of Technology, Dalian 116024, ChinaSchool of Life Science and Pharmacy, Dalian University of Technology, Panjin 124221, ChinaSchool of Chemical Engineering, Dalian University of Technology, Dalian 116024, ChinaObjective: 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.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021080147nitrile hydratasenicotinonitrilevirtual amino acid mutationsubstrate affinity |
| spellingShingle | Baocheng CUI Jiao HUANG Jiaxin LI Yi GUO Li WANG Changhai LIANG Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation Shipin gongye ke-ji nitrile hydratase nicotinonitrile virtual amino acid mutation substrate affinity |
| title | Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation |
| title_full | Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation |
| title_fullStr | Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation |
| title_full_unstemmed | Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation |
| title_short | Modification of Substrate Affinity of Nitrile Hydratase Based on Amino Acid Hotspot Mutation |
| title_sort | modification of substrate affinity of nitrile hydratase based on amino acid hotspot mutation |
| topic | nitrile hydratase nicotinonitrile virtual amino acid mutation substrate affinity |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2021080147 |
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