Study On Fermentation Prosces Of Organic Phosphorus Hydrolase Produced By E.coli

The widespread use and even abuse of organophosphorus pesticides poses a serious threat to the ecological environment and human health.Microorganisms can catalyze the cleavage of phospholipid bonds of organophosphorus pesticides by self-produced organophosphorus hydrolase to produce low toxicity products,which have the advantages of high degradation efficiency,mild conditions and low price,showing good application prospects.In this paper,the fermentation process of organophosphorus hydrolase produced by E.coli BL-21(DE3)carrying plasmid pETDuet-opd-bl was studied.The composition and culture conditions of the medium were optimized by the response surface analysis method at the shake flask level.On the basis of this,the enzymatic properties of the organophosphorus hydrolase were studied.Finally,the enrichment fermentation of E.coli produced organic phosphorus hydrolase was studied,and the effect of optimized fermentation process on reducing COD of fermentation waste liquid was explored.Its main research contents and results are:(1)The single factor optimization experiment of organophosphorus hydrolase produced by E.coli BL-21(DE3)was carried out by selecting 8 factors(carbon source,nitrogen source,phosphate,lactose concentration,lactose addition time,initial pH,inoculum size,metal ion)which affect the enzyme activity of organophosphorus hydrolase.Through Plackett-Burman(PB)test screening,it was found that yeast powder concentration,lactose concentration and cobalt ion concentration were significant factors affecting the activity of organophosphorus hydrolase.The final fermentation medium was determined by the steepest climbing test and the response surface optimization based on the Box-Behnke test:yeast powder concentration 17.35 g/L,cobalt ion concentration 0.24 mmol/L.lactose concentration 11.30 g/L,lactose addition time 8 h,maltose concentration 6 g/L,phosphate concentration 0.1 mol/L,pH 7.0,inoculum 4%.The optimization of the composition and conditions of the medium increased the enzyme activity of the organophosphorus hydrolase from 10000 U/mL to 21237 U/mL.(2)Several enzymatic properties of organophosphorus hydrolase were studied.The results showed that the Michaelis constant Km of organophosphorus hydrolase to methyl parathion was 0.0207 mmol/L,Vmax was 23674 ?mol/min mL,and the enzyme activity of crude enzyme is 320 U/mg.The optimal reaction conditions of organophosphorus hydrolase were obtained by studying the enzymatic properties:the optimum pH was 8.5,the relative activity of the enzyme was above 70%in the range of pH 7.5-9.5,and the enzyme activity was relatively stable and the activity loss was less under alkaline conditions;the optimum reaction temperature is 30?,the high temperature tolerance of the enzyme is relatively poor,the temperature should not exceed 40?;the Co2+,Cu2+metal ions have an activation effect on the hydrolase,add 0.1 mmol/L cobalt copper metal ions respectively increased the activity of the enzyme by 20%and 10%,respectively;Ca2+and Mg2+strongly inhibited the activity of the hydrolase,and the activity of adding these ions was greatly reduced.For the long-term preservation of the enzyme sample,the preserved organophosphorus hydrolase has the best stability at 4? and still retains 73%of the initial enzyme activity for one month.(3)Amplified fermentation test of 7 L and 500 L fermentors was carried out,and the final optimized condition of 7 L fermenter was 30? for pre-induction culture,25? induced enzyme production,aeration of 3 L/min,stirring speed 200 r/min.Under these conditions,the growth rate of the cells OD600 can reach 7.76,and the enzyme activity can reach about 33000 U/mL.Amplification verification of the 500 L fermenter level was carried out,and the growth rate of the OD600 was 15.87,and the enzyme activity reached 33200 U/mL.In all the fermentation batches,the COD content of the fermentation waste liquid obtained by the 7 L pilot fermentation under the optimal conditions was 7500 mg/L,which was 4.87 times lower than that of the blank control.Under the optimal conditions,the COD content of the fermentation waste liquid obtained by 500 L pilot fermentation was 11700 mg/L,which was 3.63 times lower than that of the blank control.While ensuring high enzyme activity,the COD content is reduced,the pressure of subsequent fermentation waste liquid is reduced,and the further industrial application of organophosphorus hydrolase is strongly supported.