Catalytic Performance Modification Of A Thermophilic Nitrile Hydrase Based On Substrate Access Tunnel Engineering And Its Application

Nitrile hydratase（NHase,EC 4.2.1.84）is one of the key enzymes in nitrile metabolism in microorganisms,which can catalyze the hydration of nitriles to corresponding amides.Compared with the traditional chemical synthesis method,its catalytic conditions are mild and the by-products are less,so it has been used in the production of nicotinamide,acrylamide,5-cyanopentamide in industry.Hydration is an exothermic reaction that requires high industrial nitrile hydratase stability.It was previously reported that nitrile hydratase（Cal.t NHase）from extremely thermophilic bacteria Caldalkalibacillus thermarum TA2.A1 had high thermal stability,but its catalytic activity for nitrile substrates still had room for improvement.In this paper,taking Cal.t NHase as the research object,the semi-rational transformation of wild-type Cal.t NHase based on substrate channel engineering was carried out to improve the catalytic performance of the enzyme,and the subsequent fermentation process and amide synthesis process were optimized.The main results are as follows:（1）The computer-aided semirational design was used to construct the three-dimensional model of Cal.t NHase,and eight hot amino acid residues of the substrate channel were calculated and analyzed.Through the representative amino acid mutation in the hot spot area,the conformation of the substrate channel was reshaped,and the catalytic activity was improved.The specific enzyme activity of nicotinitrile catalyzed by high activity mutant Cal.t NHase-βL48H was 820 U·mg^-1,which was 3.7 times higher than that of the wild enzyme,and the specific enzyme activity of 2-cyanopyrazine and hexonitrile was 691 U·mg^-1 and 17.5 U·mg^-1respectively,3.1 and 6.2 times higher than that of wild type.（2）Based on the dominant mutant Cal.t NHase-βL48H,the highly stable point mutationαA20V obtained in the previous study was introduced,and the optimal mutant Cal.t NHase-αA20V/βL48H with higher catalytic performance was obtained.the enzyme activity of nicotinitrile was increased to 902 U·mg^-1,the half-life of nicotinamide was 2 h at 65℃,and the half-inhibitory concentration of nicotinamide was 48.3%(w·v^-1).Through molecular dynamics simulation and substrate channel calculation,it is clear that the main reason for the improvement of catalytic performance of the mutant is the geometric conformation at the entrance of the substrate channel.（3）The recombinant Escherichia coli containing pET-24a（+）-Cal.t NHase-αA20V/βL48H plasmid was fermented with high density.The fermentation conditions of enzyme production and nicotinamide synthesis were optimized.The recombinant strain was fermented with high density in a 5 L fermentor.When the culture time was 24.5 h,the cell density reached the maximum OD₆₀₀=110.0,and the nicotinonitrile enzyme activity catalyzed by cell fluid was6805 U·m L^-1.Compared with Rhodococcus rhodochrous J1 fermentation,its fermentation period is short and the enzyme yield is high.The cell liquid of OD₆₀₀=5 with cell density was used to catalyze the reaction by feeding in batches.The concentration of nicotinamide reached706 g·L^-1 when the reaction reached 157 min,which was the highest yield reported.