The Screening Of Substrate For Nitrile Hydratase Transformation Based On Docking Model

Nitrile hydratase (NHase, EC 4.2.1.84) from Rhodococcus ruber CGMCC3090 showed broad substrate specificity which can hydrolyze nitriles with high substrate tolerance and excellent selectivity. In this study, the nitrile chemicals were screened based on the model-docking technology and the characteristics of the conversions were analysed which preliminary clarified the trends of biotransformation by NHase from R. ruber CGMCC3090. Further more, we simply established the production process of catalysing 2,6-difluorobenzonitrile and cinnamonitrile to corresponding amides. The main results are as follows:(1) Screening for substrates according to the model-docking technology. The trends analysis:the smaller docking distance, the higher specific conversion ratio of NHase bioconversion. The substrates with docking angle close to 90° or 180° can be well transformed by R. ruber CGMCC3090. In view of the model-docking screening rules, we used model-docking techniques to select nine suitable substrates.(2) The characteristics of substrates biotransformation was analysed. R. ruber CGMCC3O9O can catalyze the nine selected nitriles. The catalytic kinetic parameters were calculated using michaelis-menten equation.2,6-difluorobenzonitrile:Km=0.95 mol/L, vmax=6.29 mmol/(ming), cinnamonnitrile:Km=0.74 mol/L, vmax= 8.72 mmol/(ming), 4-amino-benzonitrile:Km=0.82 mol/L, vmax=15.0 mmol/(ming),4-bromoxynil:Km=0.62 mol/L, vmax=11.9 mmol/(ming),2-cyano-3-methylpyridine:Km=0.41 mol/L, vmax=15.3 mmol/(ming). R. ruber CGMCC3090 prefer to catalyze short-chain aliphatic nitriles, aromatic and heterocylic nitriles with electron-withdrawing substituents.(3) The bioconversion processes of 2,6-difluorobenzonitrile and cinnamonnitrile which has the high industrial value was established. The present strains of catalyzing 2,6-difluorobenzonitrile and cinnamonnitrile have the defects of by-products, continuous illumination, steric hindrance of fluorine atoms or low conversion rate which hampered the industrial production of the amide. In this study, R. ruber CGMCC3090 cells showed excellent strong substrate concentration tolerance which up to 3.5 mol L-1 with no by-products. The addition of 1%(v/v) Tween 80 resulting in 1.9 and 1.4 fold increase respectively compared with the lacking organic solvents. In the reaction system, the suitable concentration of 2,6-difluorobenzonitrile and cinnamonnitrile was 1.5 mol/L, the fitting concentration of the bacterial cell was 2.30 g (DCW)/L.