Study On ARTP Mutation Breeding And Cell Immobilization Of Pseudomonas Putida Producing Nitrilase

Nicotinic acid is one of the 13 enssential vitamins for the human body.It can be used as the vitamin drugs,food and drug additives,pharmaceutical or chemical synthesis intermediates,etc.The market demand is extremely high.However,the existing production processes of the domestic nicotinic acid industry are relatively backward and can cause great damage to the environment.The scale of production is small,so it is difficult to meet the demand for nicotinic acid.Nitrialse can catalyze 3-cyanopyridine to generate nicotinic acid in one step,which has advantages of mild reaction conditions,low by-products and environmental friendliness.However,the wild nitrilase has poor tolerance to substrates and poor thermal stability,and it is difficult to meet industrial application requirements.ARTP mutagenesis as a highly efficient mutagenesis technique has been widely used to increase microbial performance.Cell immobilization technology can further improve the stability of biocatalysts and improve the recycling rate.This research mainly focuses on the research of ARTP mutagenesis and cell immobilization of P.putida CGMCC3830 in order to to increase the production of nicotinic acid and meet the requirements of industrial applications.The study contents are as follows:?1?Construction of a high-throughput method for screening nitrilases:OPA-TCA micro-reaction method?based on 96-well plate?.The reagent concentrations were optimized:20 mM OPA reagent?final concentration?,60 mM TCA reagent?final concentration?,100%DMSO.The reacton conditons were confirmed:10 m L of reaction supernatant and 100?L of OPA reagent were added into a 96-well plate with a muti-well pipette.For reaction,the 96well plate was placed in an incubator at 20°C and 300 rpm for 20 min.Another 100?L of DMSO was added for dissolution,and 50?L of TCA reagent was added for coloring.Then absorbance of the blue-black compound can be measured at 600 nm.The sensitivity of adjusted OPA-TCA micro-reaction is better?y=0.0327x+0.0062,R²=0.9991?and can be used as a high-throught screening method for libraries of nitrialse-producing mutants.?2?Selection of nitrilase-producing bacteria with high substrate tolerance based on ARTP mutagenesis.The mutagenesis conditions were as follows:power?100 W?,air flow?10 slm?,distance?2 mm?,mutagenesis time?10 s?.The mutant D3,which was 2.51 times more enzymatically than the wild-type strain and had good genetic stability after 20 passages,was obtained by first screening and multiple rescreening with shake flasks.The tolerance to high concentration of 3-cyanopyridine was significantly increased.The optimal batch of feed concentration can be increased from 100 mM to 150 mM,and 189 g?L^-1 nicotinic acid was accumulated.The thermal stability of mut-D3 was significantly increased.After incubation at30°C for 24 h,75.72%of the intial enzyme activity was maintained while wild type maintained 66.68%of that.Mut-D3 enhanced the catalytic ability of nitrile compounds in the substrate spectrum,and its catalytic abilities to 4-cyanopyridine,benzonitrile,sussinonitrile,iminodiacetonitrile,and glycyanide were 2.24 times,2.68 times,3.36 times,6.21 times and2.64 times than that of wild type,reapectively.?3?SA-PVA composite immobilization of P.putida mutant.Immobilizaiton procedure was confirmed:SA and PVA were mixed in a ratio of 9:1 as a fixing material.Then the prepated bacterial suspension was mixed in a ratio of 1:1 with the fixing material and stirred well.The mixture was slowly dropped into a precooled curing solution?the saturated boric acid with CaCl₂?at 4°C with a syringe.The solidification time was 5 h and the operation was performed in an ice bath.The concentrations of added organic materials were optimized:8%PVA,2.5%SA,0.6%CaCl₂.The thermal stability of SA-PVA immobilized cells was futher increased.After incubation at 30°C for 24 h,the SA-PVA immobilized cells still retained86.11%of the intial enzyme activity.After storage for 30 days in a refrigerator at 4°C,SA-PVA immobilized cells also retained 80%of the enzymatic activity while free cells had only 40%of the enzyme activity.And the tolerance to the substrate was further improved.The batch feed concentration was increased from 150 mM to 250 mM.12 batches of3-cyanopyridine could be transformed into 346 g?L^-1 nicotinic acid.The yield was increased by 83%compared to mut-D3 free cells.?4?Addition of inorganic materials to enhance SA-PVA immobilized cell performance.The concentrations of inorganic materials were optimized:2.0%SiO₂ and 0.6%CaCO₃.The thermal stability of SA-PVA immobilized cells after addition of inorganic materials was further increased.After incubation at 30°C for 24 h,the SA-PVA immobilized cells can retain89.74%of the intial enzyme activity.The storage stability was slightly increased compared to SA-PVA immobilized cells at 10-50 days,after storage for 40 days in a refrigerator at 4°C,SA-PVA immobilized cells with inorganic materials also retained 72%of the enzymatic activity while the SA-PVA immobilized cells had only 60%of the enzymatic activity.The batch feed concentration was still 250 mM,but it was able to convert 14 batches and accumulate 418 g?L^-1 nicotinic acid,the yield was increased by approximately 21%.