Engineering Of Substrate Spectrum And Catalytic Performance Of Nitrile Hydratase Using Semi-rational Design Approach

Amide compounds are important synthetic building blocks in natural bioactive molecules and organic synthetic intermediates,and their corresponding products related to industrial,agricultural and pharmaceutical fields.Nitrile hydratase（NHase,EC 4.2.1.84）is a kind of metal enzyme that can catalyze the hydration of nitrile compounds to form amide compounds,which has been applied in the industrial production of acrylamide and nicotinamide.Compared with the traditional chemical catalytic method,the biocatalytic method based on nitrile hydratase has the advantages of mild reaction conditions and high atomic utilization.However,the reported NHases have still the problems of narrow substrate spectrum,low catalytic activity and poor stability,which seriously limits the efficient synthesis of high-value amide compounds.Therefore,mining new enzymes or engineering existing NHases is the key to solve the bottleneck of the catalytic performance of the enzyme.In this study,NHase was engineered by genetic engineering and protein engineering based on the semi-rational design strategy to broaden its substrate spectrum and improve its catalytic activity and stability.The main results are as follows:（1）The molecular engineering of NHase was carried out by the gating strategy of substrate tunnel entrance,which broadened the substrate spectrum of NHase.The thermophilic nitrile hydratase（Pt NHase）from Pseudonocardia thermophila JCM3095 was used as the research target.Through substrate channel prediction and sequence conservation analysis,five amino acid residue hotspots that potentially influence the catalytic performance of Pt NHase at the entrance of substrate channel were determined.Two dominant single point mutantsβM46R andβA129R were obtained by site-directed saturation mutagenesis.The catalytic activity ofβM46R andβA129R for toyocamycin was changed from nothing to something,and the catalytic activity for the other ten nitrile substrates was 1 to 14.6 times higher than that of the wild-type Pt NHase.The two-point combination mutation did not show higher catalytic activity.The crystal structures of two dominant mutantsβM46R andβA129R were obtained by X-Ray diffraction.Based on the static crystal structure and dynamic molecular dynamics simulation analysis,it was found that not only the surface entrance of the substrate channel but the cavity volume of binding pocket was larger than those of the wild type,which was conducive to the entry and binding of large-volume substrates,thereby improving the catalytic activity.（2）The catalytic activity of Pt NHase was further improved by reshaping substrate access tunnel and substrate binding pocket for efficient synthesis of high value-added macromolecular cinnamamide.Through molecular docking and substrate channel prediction,combined with multiple sequence alignment and conservation analysis methods,the amino acid residue sites between the internal bottleneck of the substrate channel and the substrate binding pocket that potentially influence the catalytic performance of Pt NHase were determined.A dominant combinatorial mutant Pt NHase（βF37P/L48P/F51N）was obtained by site-saturation mutagenesis and iterative mutagenesis,and its catalytic activity for cinnamonitrile was 14.9times than that of the wild-type Pt NHase.Two combination mutants obtained by combining with the above two dominant mutantsβM46R orβA129R did not show higher catalytic activity,it shows that the two mutations have no additivity.Structure analysis and dynamical cross-correlation matrix（DCCM）analysis based on molecular dynamics simulation revealed that the introduction of mutations enlarged the substrate access tunnel and binding pocket,enhanced overall anti-correlated movement of enzyme,which would promote product release during the dynamic process of catalysis.In a hydration process,the complete conversion of 5 m M cinnamonitrile was achieved by variantβF37P/L48P/F51N in a 50 m L reaction,with 100%cinnamamide yield and productivity of 0.736 g·L^-1·h^-1.（3）By engineering the key amino acid residues of the N-terminal Loop on the tetrameric Pt NHase interface,the mutantαL6T with enhanced thermal stability and acrylamide tolerance was obtained.Inter Prosurf was used to predict the interface residues of NHase tetramer.Combined with molecular dynamics simulation,it was determined that the N-terminal Loop region ofαsubunit affected the stability of NHase.Two potential residue sites were obtained by preliminary screening,and two dominant variants,αT2L andαL6T,were obtained by further screening with the site-directed saturation mutagenesis strategy.Its acrylamide tolerance was2.3 and 3.5 times that of the wild type,while the enzyme activity was 88.3%and 121.0%of the wild type,respectively.Two-point combination mutant does not show better performance.Through thermal stability test,it was found that the melting temperature（Tm）ofαL6T was79.3℃,which was 7.2℃higher than that of wild type.The half-life was extended from 17.3 to25.1 min.In addition,the catalytic activity ofαL6T for different nitrile substrates was also significantly improved.Crystal structure-guided analysis ofαL6T and molecular dynamics simulations revealed that increased enzyme surface hydration and the introduction of positive cross-correlation into the N-terminal loop of the tetramer interface caused the two loop regions to hook to each other,thus improving the resistance to high acrylamide concentrations.In a hydration reaction,the yield of acrylamide from acrylonitrile catalyzed by mutantαL6T reached720 g·L^-1,which was 43.5%higher than that of the wild type.