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Food and Fermentation Industries    2022, Vol. 48 Issue (20) : 70-77     DOI: 10.13995/j.cnki.11-1802/ts.030432
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Substrate affinity design for the improvement of nitrilase Nit6803 activity
LIU Xinyue, HAN Laichuang, LIU Zhongmei*
(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
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Abstract  Nitrilase (EC 3.5.5.1) is an ideal biocatalyst for a variety of essential bulk chemicals and pharmaceutical intermediates, due to its capability to catalyze nitrile to carboxylic acid with high stereoselectivity under mild reaction conditions. However, the low activity and poor thermal stability of natural enzymes still limit its industrial application. In recent years, it has been a research hotspot to break through the bottleneck of ‘trade-off' between activity and stability through protein engineering. This study proposes a novel enzyme-substrate affinity design strategy. The nitrilase Nit6803 derived from Syechocystis sp. PCC6803 was improved in activity through the rational design combining the Cartesian_ddG method in Rosetta suite and the enzyme-substrate affinity calculation based on free energy perturbation. The single-point mutants F64Y, W170G, and combination mutant F64Y/W170G with significantly improved activity were obtained. Among them, the specific enzyme activity of F64Y/W170G reached (22.48±0.64) U/mg, which was 4.56 times that of the wild type, and the thermal stability maintained. Whole-cell catalysis by adding 3-cyanopyridine in batches showed that F64Y/W170G had stronger catalytic ability than wild type, and greatly shortened the catalysis time when reaching the same conversion rate. The results demonstrated that the engineering strategy proposed in this study can effectively enhance the enzyme activity without decreasing its stability, which provides a new idea for the rational design of enzymes.
Keywords nitrilase      enzyme-substrate affinity      rational design      enzyme activity     
Issue Date: 18 November 2022
URL:  
http://sf1970.cnif.cn/EN/10.13995/j.cnki.11-1802/ts.030432     OR     http://sf1970.cnif.cn/EN/Y2022/V48/I20/70
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