Molecular Evolution To Improve The Degradation Activity Of PTE Against The Nerve Agent VX

Green and efficient decontamination of nerve agent VX has always been one of the difficulties and hot spots in military decontamination technology.Traditional chemical decontaminants can effectively degrade VX,but they also have a series of problems including the strong irritation to the skin,corrosion on the surface of contaminated equipments,large consumption,serious environmental pollution and toxic by-products.Therefore,it is necessary to develop neotype decontaminants with the high activity,low corrosion,non-toxic by-products,and pollution-free.Phosphotriesterase is a natural active component and has a strong ability to degrade a variety of environmental pollutants.However,if the natural PTE is directly used as the active ingredients to degrade VX,it is necessary to solve the natural defects of PTE with low soluble expression in engineering bacteria,and further improve its catalytic activity to VX and stability.In this project,sb-PTE from Sphingomonas spp.and 2OB3 from Pseudomonas parvum were used as the starting enzymes.The high-efficiency expression vector and E.coli.cell wall synthesis related genes(msb B)knockout strain were constructed to improve the extracellular secretion of the enzyme,and provide sufficient targets for screening mutation library.We further optimized the microfluidic chip structure,screening accuracy,operational stability and other parameters of the high-throughput screening system,and finally realized the ultra-high throughput microfluidic screening of single-cell microdroplets.The droplet generation flux was greater than10 kHz,and the screening flux was greater than 2 kHz,and the error rate was less than3%.On the basis of the above,the PTE mutant library were established by error-prone PCR,which was screened by the fluorescence droplet ultra-high throughput screening system,and the benign mutants with improved hydrolytic activity to VX were obtained.We further immobilized the benign mutants with living biofilm functional materials,which greatly improved the catalytic stability and environmental tolerance of the enzyme.The main research contents are as follows:1)Construction of high-level expression vector:sb-PTE gene from Sphingomonas sp.strain TCM1 and 2OB3 gene from Pseudomonas parvum were used as target genes.The high-level expression plasmid containing maltose solubilizing tag protein was successfully constructed.The recombinant plasmid was transfected into E.coli BL21(DE3)for heterologous expression.The crude enzyme solution was purified by affinity chromatography.10 mg for MBP-sb-PTE and 5 mg for MBP-2OB3 of purified target protein were obtained in 1 liter of bacterial culture medium,which will provide sufficient enzyme for the preparation of enzyme-based decontaminants.The initial hydrolytic activities of MBP-sb-PTE and MBP-2OB3 for methyl parathion and VX were studied.It was found that the two enzymes had the highest activity when the active center was Zn²⁺,but showed different hydrolytic preferences for organophosphorus compounds containing P-O bond and P-S bond.MBP-sb-PTE showed good catalytic activity for the hydrolysis of VX.The way of hydrolysis of VX was to break P-S bond,generating thesulfhydryl compounds with toxicity much lower than VX.When methyl parathion was used as substrate of MBP-2OB3,the optimum reaction temperature and pH of MBP-2OB3 were 40?and pH8.5,respectively.The enzyme showed weak tolerance to high temperature,strong acid and alkaline conditions,high concentration of DMSO and salt solution.2)Construction of exocrine expression strain:The Red homologous recombination technology was used to knock out the gene msb B related to lipopolysaccharide synthesis on the cell wall of E.coli BL21(DE3),and the mutant strain E.coli BL21(DE3)k-msb B was constructed.The recombinant plasmids of MBP-sb-PTE and MBP-2OB3 were transfeected into the knockout strain for expression.The growth curve of the E.coli BL21(DE3)k-msb B showed that the deletion of the gene had little effect on the growth status of the strain.Western blot analysis and extracellular enzyme activity detection showed that the extracellular secretion of the two enzymes was greatly increased,and the catalytic hydrolysis activity of the two enzymes was retained.3)Establish an ultra-high flux separation method for droplet microfluidic based on fluorescence separation:The micro droplet generation,observation and sorting chips were fabricated respectively.By generating different fluorescent dye droplets,the micro droplet generation flux of the platform was tested as high as 10 kHz,and the generation process was stable.Furthermore,the droplet fusion rate was low,and there was no breakage phenomenon.The error rate was only 2.487%and 2.741%for two dye droplets.According to the characteristics of Poisson distribution,the single bacterial droplet was obtained by adjusting the concentration of bacteria.It was found that the number of bacterial cells increased significantly and the GFP protein could be expressed normally in droplets.Using the GFP expressed by E.coli BL21(DE3)as the sorting signal,we have achieved a very good separation between bacterial contained droplets and empty droplets.4)Directional evolution of PTE based on microfluidic ultra-high throughput screening:a large number of mutations were obtained by the method of error prone PCR.Two mutation library screening methods were used to screen the mutatants library(96 well plate screening and microfluidic ultra-high flux sorting),and the screening efficiency of the two screening methods was compared.The traditional mutation library screening method is not only heavy workload,but also low rate of benign mutation.After two rounds of microfluidic ultra-high flux sorting,the probability of obtaining benign mutants was more than 96%by the 96-well plate check.Among them,G314D not only has higher enzyme activity,but also accquire higher acid and temperature resistance.5)Construction of biofilm:the G314D mutant was used to constructed biofilm living functional materials.We further characterized its physical properties.Compared with the unimmobilized enzyme,the biofilm showed more stable enzyme catalytic activity under extreme conditions.The biofilm could maintain more than 80%activity in relatively high temperature,acid or alkaline,salty or organic solvent systems,which is expected to greatly improve the practical performance of biological enzyme materials.The biofilm material can be used for many times and has a good cyclic catalytic ability.After five cycles of catalytic activity,the relative activity remains above 90%.