Study On Breeding And Fermentation Condition Optimization Of Strains With High Nitrile Hydratase Activity And Tolerance To Substrate

Nitrile hydratase (Nitrile hydratase, EC4.2.1.84) is the important biologicalcatalysts with which acrylonitrile is synthesized into acrylamide. With the growingdemand of acrylamide, Preparation of high activity of nitrile hydratase become the coretechnology for the biocatalytic production of acrylamide, is also one of the currentresearch focus.In this thesis, Natural separation and Mutation Breeding pairs Nocardia(Nocardia.sp) from ChangJiu NongKe Chemical Industry Co.;Ltd. The protoplastfusion of the stain with high nitrile hydratase producing and the substrate resistancemutant was studied. Combined with resistance marker screening method, a fusion strainwith high nitrile hydratase activity and substrate tolerated have been screened, and then,the fermentation conditions of the fusion strain were preliminary optimized. Thedetailed contents were as follows:(1) Natural separation the original strain NK0, a strain NK10with activitystabilized at12million U/mL was screened out. The thermal and UV doublemutagenesis were used to mutant the original strain NK0, the optimal mutagen dosagewas determined as heat induced5min and uv mutagenesis induced80s, got a mutationsNK8tolerated4%of acrylonitrile. NK10and NK8were as the parents of protoplastfusion.(2) The drug-resistant marker of the protoplast fusion parents was screened.Thirteen kinds of drugs were chosen as the sign of resistance to antibiotics of NK10andNK8, respectively by the method of drug-resistant marker. The resistant markers ofthe two stains were obtained. One of the medical tags is lincomycin at a concentrationof5μg/mL when NK10is resistance and NK8non-resistance. The other one iskanamycin at the concentration of75μg/mLwhen NK8is resistance and NK10isnon-resistance.(3) The protoplast fusion of the parents and identification of fusant. The hydrolysistime were investigate by means of single factor experiment, the results show that thebest hydrolysis time of the strain NK10is30min, the best hydrolysis time of the strainsNK8is50min. The protoplast fusion of parents strains protoplasts was studied bychemical PEG fusion method. Twenty individual fusants have been got by photocopyingmethod. A fusant with the highest level of activity was selected from the sub-culturefermentation，then the fusant named NK18.(4) Preliminary optimited the fementation conditions of the NK18. Orthogonaldesign were used to screened out the best fermented liquid starting pH, fermentationperiod, quantity, pack material coefficient. On this basis, Plackett-Burman (PB) designmethod was used to evaluate eight kinds of influence of culture medium components ofthe enzyme production evaluation, and the results show that: glucose, urea, potassiumhydrogen phosphate, potassium dihydrogen phosphate content is the major factor of thestrain producing nitrile hydratase. The steepest climbing experiment and the centralcomposite design were used to adopt the optimal level of the major factors. theoptimum conditions of the strains producing nitrile hydratase: initial pH8.0, thefermentation cycle54h, the inoculum size12%, medium volume12%, glucose22.62g/L, urea9.76g/L, K2HPO41.22g/L, KH2PO41.268g/L,MSG1g/L, MgSO40.2g/L, the DXL1mL/L. Under such conditions, the concentration of nitrile hydratase was increased to12.80million U/mL, which was2.33times higher than originalfermentation conditions.