Study On Acetylcholinesterase Of Drosophila Melanogaster Displayed On The Spore Surface Of Bacillus Subtilis

Pesticides are closely related to people's lives.In order to ensure the production of crops,people use a large amount of pesticides in the fields,causing the accumulation of pesticides in the soil,which may threaten the environment and food safety.Organophosphorus pesticides are one of the most used pesticides and cause chronic poisoning(such as nervous system disorders,organ toxicity,cancer,birth defects,reproductive toxicity,etc.).Between 2010 and 2018,there have been many food poisoning incidents caused by eating vegetables with excessive levels of organophosphorus pesticide residues.Gas chromatographic methods specified by national standards require expensive equipment and are not suitable for daily rapid on-site inspection.The organophosphorus pesticide can specifically irreversibly inhibit the activity of acetylcholinesterase(AChE)in the animal nervous system,and the enzyme inhibition method is a method for determining the residual amount of organophosphorus pesticides according to the degree of inhibition of AChE activity.It has the characteristics of rapid detection,simple equipment and reagents,low cost,etc.It is suitable for the detection of organophosphorus pesticide residues in fruits and vegetables.At present,AChE is mainly extracted from insect or animal blood,with low yield,high cost and unstable enzyme activity,which seriously hinders the application of AChE in pesticide residue detection.In this paper,Drosophila melanogaster acetylcholinesterase(DmAChE)was obtained.The gene was ligated into the pHT43 plasmid and replicated and extracted in E.coli,and the extracted plasmid was transferred into Bacillus subtilis WB800 N to obtain recombinant strain pHT43-ache-WB800 N.The expression of DmAChE was induced by IPTG and passed western.The expression of Dm AChE was successfully expressed by blot.The enzymatic properties showed that the activity of Dm AChE was53 U/mg,the Km value was 0.058 mmol/L,the optimum enzyme reaction temperature was 30 ?,and the optimum pH was 7.Based on the above work,we realized the immobilization of DmAChE by sporesurface display technology and improved the environmental tolerance of the enzyme.The acetylcholinesterase gene of Drosophila melanogaster was co-ligated with six spore capsid proteins(CotB,CotC,CotG,CotX,CotY,CotZ)onto a pHT43 vector,which was transformed into Bacillus subtilis WB800 N and sporulated.By observing the difference in immunofluorescence between different spore surfaces,the optimal anchor protein was determined to be CotG.The enzymatic properties showed that the enzyme activity was 0.7U/mg,and then the relative enzymatic activity of G-AChE at20-70 ? and pH=4.0-9.0 was detected.It was found that it was still at 70 ?.It can store more than 60% of relative enzyme activity,and its tolerance to acid and alkali is greatly increased.Under the condition of pH 4,the relative enzyme activity can still reach more than 60%,which is much higher than DmAChE temperature tolerance and acidity.Alkali tolerance.In summary,the Bacillus subtilis spore surface display system as a novel anti-reverse enzyme immobilization technology can solve the problem of unstable AChE enzyme activity in the detection of organophosphorus pesticides by enzyme inhibition method.Rapid detection of products capable of stable detection of organophosphorus pesticides can be developed.