Modification of nitrilase based on computer screening and efficient biosynthesis of 4-cyanobenzoic acid.

Autor: Jin YZ; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, China.; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China.; Zhejiang Yongtai Technology Co., Ltd., Linhai, Zhejiang, China., Shen JD; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, China.; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China., Wang YX; Princeton International School of Mathematics and Science, Princeton, New Jersey, USA., Shao HM; Zhejiang Yongtai Technology Co., Ltd., Linhai, Zhejiang, China., Xia HJ; Zhejiang Yongtai Technology Co., Ltd., Linhai, Zhejiang, China., Lin JH; Zhejiang Yongtai Technology Co., Ltd., Linhai, Zhejiang, China., Liu ZQ; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, China.; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China., Zheng YG; The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, China.; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China.
Jazyk: angličtina
Zdroj: Biotechnology journal [Biotechnol J] 2024 Mar; Vol. 19 (3), pp. e2300706.
DOI: 10.1002/biot.202300706
Abstrakt: 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.
(© 2024 Wiley-VCH GmbH.)
Databáze: MEDLINE
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