Screening,Enzymatic Characterization And Immobilization Of Key Enzymes For Degradation Of Chlorpyrifos In Skimmed Milk By Lactobacillus Bulgaricus

Chlorpyrifos is widely used as an insecticide to control various pests worldwide.Numerous studies have shown that chlorpyrifos and its main intermediate metabolite,3,5,6-trichloro-2-pyridine（TCP）could accumulate in non-target organisms through biomagnification,and have a strong toxic effect on non-target organisms,including humans.Bioremediation based on microbial metabolism is considered to be an environmentally friendly and efficient strategy for the removal of chlorpyrifos residues.In this study,gas chromatography（GC）was used to study the degradation of Lactobacillus bulgaricus against chlorpyrifos in the skimmed milk;Transcriptomics（RNA-Seq）was used to screen genes encoding key enzymes during the degradation and exploring their molecular mechanisms for detoxifying chlorpyrifos.Gas chromatography mass spectrometry（GC-MS/MS）was used to identify metabolic intermediates in the process of biodegrading.The intracellular phosphatase of L.bulgaricus was extracted using an ultrasonic crusher and its enzymatic properties were studied.The extracted phosphatase was immobilized by cross-linking sodium alginate and glutaraldehyde.（1）The results showed that L.bulgaricus was fermented in the skimmed milk containing chlorpyrifos（initial concentration was 1.00 mg/kg）,and the concentration of chlorpyrifos gradually decreased during the fermentation.At the 20^thh,the concentration of chlorpyrifos decreased to 0.59±0.03 mg/kg,and the degradation rate reached 40.06%.（2）By comparing the RNA-seq data of L.bulgaricus cultivated in the skimmed milk（containing 1.00 mg/kg chlorpyrifos）for 0 h and 20 h.A total of 383 genes were detected,among which 269 genes were significantly up-regulated and 114 genes were significantly down-regulated.The enzymes that may be related to chlorpyrifos degradation,including hydrolase,phosphatase,pyrophosphatase,oxidoreductase and dehydratase were screened.It is speculated that these enzymes may be involved in hydrolysis,desulfurization and oxidation,as well as dealkylation and sugar transfer in the degradation.At the same time,it was also found that the expression of membrane transporter genes was increasing.Three possible degradation intermediates in L.bulgaricus under chlorpyrifos stress were detected by GC-MS/MS was detected.（3）The method of ultrasonic fragmentation was used to extract intracellular phosphatase from L.bulgaricus.Through the optimization orthogonal experiment,the optimal p H value of this enzyme is 9.0,optimal temperature is 37℃,and it can be activated by Mg²⁺.The activity of phosphatase can reach 4.68±0.02 U/m L and it’s activity has increased by 1.3 times compared to the control group.The degradation rate of chlorpyrifos reached 75.40%within 2hours.The method of cross-linking sodium alginate with glutaraldehyde was used to immobilize the intracellular phosphatase of L.bulgaricus to obtain an immobilized enzyme.The optimal p H value of the immobilized enzyme is 10.0 and the optimal temperature is 57℃.Compared with the free enzyme,the immobilized enzyme can participate in the reaction at higher p H and temperature conditions.The degradation rate of chlorpyrifos by immobilized phosphatase was 77.63%.After three consecutive operations,the relative activity of immobilized enzyme remained above 60%.To sum up,gas chromatography was used to study the biodegradation of chlorpyrifos in the skimmed milk by L.bulgaricus on this study.The phosphatase that play an important role in the biodegradation of chlorpyrifos was selected by RNA-Seq.The intracellular phosphatase of L.bulgaricus was extracted by ultrasonic fragmentation,whose enzymatic properties and the biodegradation of chlorpyrifos in vitro was also studied.It provides new resources of gene and enzyme for biodegradation and remediation of chlorpyrifos,and also provides a new theoretical basis for the microbial degradation mechanism of organophosphorus pesticides.