Functional Expression Of Recombinant NHaseK And Its Molecular Modification

Nitrile Hydratase can catalyze 2-(4-chlorophenyl)-3-methylbutyronitrile (CPIN) to corresponding amide (CPIAm) that can be converted into the important synthetic precursor of fenvalerate. However, the activity catalyzing CPIN of reported NHase was very low, which tremendously limited the catalytic efficiency. Therefore, study of recombinant expression and molecular modification of Nitrile Hydratase to enhance its catalytic activity has important significance.NHaseK, the Nitrile Hydratase with the highest activity catalyzing CPIN as so far was researched in this study. First, the functional expression system in E.coli was constructed for NHaseK. Taking the advantage of two T7 promoters in pETDuet-1, eight recombinant plasmids were designed and constructed to express the three essential gene segments respectively encoding α-subunit, β-subunit and activator 17k of NHaseK. After transformed into E.coli BL21(DE3), pETDuet-NHaseK which put all the three gene segments in the first promoter expressed equivalent subunits and abundant activator with the highest activity of 0.78 U/mg protein. On this basis, different plasmid vectors and SD sequences were used to optimize the expression system. However, these factors didn’t show obvious effect. Considering the resistant gene of pRSFDuet-1 was more suitable for fermentation, pRSFDuet-1 was identified as the vector for expressing NHaseK with the activity of 222.6 U/LTo enhance NHaseK’s activity catalyzing CPIN, NHaseK-CPIN complex was constructed by homologous modeling and molecular docking. Then, according to the result of virtual saturation mutagenesis at 10 hotspots screened out by virtual alanine scanning, we chose αG119 to do saturation mutagenesis. However, all the mutants at αG119 decreased activity. According to the experimental result of alanine mutagenesis at the 10 hotspots, βD49 and αQ88 were chosen to do saturation mutagenesis and 14 mutants improved their catalytic activity. βD49G obtained the highest activity of 2.48 U/mg protein, which was 3.2 times of NHaseK. On the other hand, βF41 was chosen to do saturation mutagenesis because it’s near to the predicted channel. But we didn’t obtain positive mutant at βF41. Further, combination mutant was engineered based on positive mutants and the result was unsatisfied.Finally, the enzymatic property and catalytic processes of βD49G and NHaseK were studied. By the research of reaction kinetic, we found out Kcat of βD49G was increased to 4.4 times of NHaseK and that’s the key reason for enhanced activity. Furthermore, The half-life of βD49G in 35℃,40℃ and 45℃ was 114 h,76 h and 25 h respectively, which was 39%,72% and 56% longer than NHaseK in the same condition and meant βD49G got a better temperature stability. After tentative research of catalytic processes, the optimized operating condition of E.coli/pRSFDuet-βD49G was determined as 40℃, pH6.5 Na2HPO4-citric acid buffer and 5%(v/v) cosolvent methanol, which was the same as E.coli/pRSFDuet-NHaseK. In this condition,0.759 gDCW/L pD49G can convert 0.5 g/L CPIN to corresponding amide in an hour, while the same amount NHaseK need two hours to complete this reaction.