Study On The Construction Of Carboxylesterase Whole-cell Biocatalyst And Its Biodegradation To Pesticides

Pesticides are used to kill harmful insects and weeds in crops.However,with the extensive use of pesticides,pesticide residues continue to accumulate,causing toxic effects on non-target organisms,such as humans,animals and aquatic organisms.At the same time,they will seriously pollute the soil,water and air.Therefore,it is urgent to eliminate these pesticide residues from the environment.Carbamate and pyrethroid pesticides have been widely used successively.Because both pesticides contain ester bonds in their parent structures,esterases(hydrolases),especially carboxylesterase,can catalyze ester bond cleavage,and they play an important role in the initial steps of hydrolysis of the two pesticides.However,the application of purified carboxylesterase is limited by its high price,complicated purification and poor stability.Developing the whole-cell catalyst for carboxylesterase surface display can solve the above problems.Surface display technology refers to anchoring specific protein or short peptide(target protein)and outer membrane protein(anchoring protein)in microbial cell surface in the form of fusion protein through genetic engineering.The whole-cell catalyst prepared by this technology can be reused,and the stability of the enzyme is enhanced,the operation is simple and convenient,the cell does not need to be broken,and the enzyme directly reacts with the substrate.According to different chemical structures,pyrethroids can be divided into type I group without cyano group and type II group with ? cyano group.Because of the existence of ?-cyano,pyrethroids type II have more neurotoxic effects than pyrethroids type I.In this study,the soil sprayed with pyrethroid pesticides for a long time was used as the experimental material,lambda-cyhalothrin was used as the sole carbon source for strain screening,and the strain was named Bacillus velezensis sd by morphological analysis,16 S r RNA analysis and genome analysis.According to the whole genome sequencing and annotation analysis,two candidate carboxylesterase genes were selected from B.velezensis sd genome,named car Cby and car CB2 respectively.In this study,the whole-cell catalyst was constructed by heterologous expression and cell surface display technology,and the enzymatic properties and pesticide degradation functions of two carboxylesterase CarCby and CarCB2 and their whole-cell catalysts were studied.The main research progress is as follows:(1)Construction and verification of whole-cell catalystWith a plasmid pET-28a(+)with T7 promoter as the basic plasmid,the Nterminal INPN of ice nucleoprotein(inak)synthesized from Pseudomonas syringae KCTC1832 was used as the anchoring motif to display CarCby and CarCB2,respectively,and the recombinant plasmids pET-28a(+)/CarCby/INPN and pET-28a(+)/CarCB2/INPN were constructed.Using green fluorescent protein(gfp)as reporter gene,the whole-cell catalyst was successfully constructed and expressed by fluorescence microscope analysis,cell fractionation,SDS-PAGE analysis and protease K sensitivity analysis.(2)Study on the optimum substrate,temperature and p HCarboxylesterase car Cby is 1449 bp in length,encoding 482 amino acids,with theoretical molecular weight of 52.8 k Da and the isoelectric point of 4.90.The optimum substrates of purified carboxylesterase CarCby and its whole-cell catalyst were p-NPC4,the optimum temperature were 30?,and the optimum p H was 8.0 and7.5 respectively.Carboxylesterase car CB2 is 738 bp in length,encoding 246 amino acids,with theoretical molecular weight of 28.3 k Da and the isoelectric point of 5.23.The purified carboxylesterase CarCB2 and its whole-cell catalyst were all best substrates of p-NPC4,the best temperature were 30?,and the best p H was 7.0 and 7.5respectively.(3)Study on temperature and p H stabilityCompared with the purified carboxylesterase CarCby,the whole-cell catalyst has better temperature stability.CarCby kept 80% and 6% relative activity after incubation at 4? and 30? for 134 hours,and only 3% relative activity after incubation at 37? for 96 hours,while the whole-cell catalyst still had 92%,67% and53% relative activity after incubation for 35 days.In addition,the whole-cell catalyst has better p H stability.After incubation at 37? for 1 hour,CarCby was unstable at p H 7.5-12,with a relative activity of 32-100%,while the whole-cell catalyst still has a relative activity of > 50% at p H 6-12.Compared with the purified carboxylesterase CarCB2,the whole-cell catalyst also has better temperature stability.CarCB2 kept 49% and 2% relative activity after incubation at 4? and 30? for 120 hours,and only 1% relative activity after incubation at 37? for 72 hours,while the whole-cell catalyst still had 124%,61%and 52% relative activity after incubation for 35 days.After incubation at 37? for 1 hour,the relative activity of whole-cell catalyst between p H 6.5-9.5 and CarCB2 between p H 6.0-10 was > 50%.(4)The effects of metal ions and chemical reagentsCompared with CarCby,some metal ions and chemical reagents have little effect on the whole-cell catalyst: Fe²⁺,Zn²⁺,Co²⁺,Fe3+,Al3+,Ni²⁺,Cu²⁺ and SDS inhibited CarCby by 77-100% and whole-cell catalyst by whole-cell catalyst 63-100%,both of which showed strong inhibition.However,compared with the whole-cell catalyst,Mg²⁺,K+,Ag+,Ca²⁺,methanol,acetonitrile,CTAB and DTT have stronger inhibitory effects on CarCby(CarCby was inhibited by 20.2-97.3%,and whole-cell catalyst was inhibited by 0-56.3%).Compared with the whole-cell catalyst,Ca²⁺,Co²⁺,Mn²⁺,Zn²⁺,Fe²⁺,Fe3+,urea and Tween-80 have stronger inhibitory effects on CarCB2(the inhibition of CarCB2 was 68.4-99.3% and that of whole-cell catalyst was 4.3-86.2%).Al3+,Cu²⁺,Ag+,EDTA,SDS and CTAB all showed strong inhibition(the inhibition of CarCB2 was91.0-100% and that of whole-cell catalyst was 84.2-100%).(5)Study on pesticide degradation functionCarCby and its whole-cell catalyst can degrade carbaryl.Carbaryl(70 mg/L)was hydrolyzed by whole-cell catalyst(43%)and CarCby(15 mg/L)respectively within12 hours.The mass spectrometry(m/z)analysis showed that the hydrolysis products of carbaryl catalyzed by both of them were 1-naphthol.CarCB2 and its whole-cell catalyst can degrade Lambda-cyhalothrin(i.e.,betacyhalothrin),and the hydrolysis rates of Lambda-cyhalothrin(30 mg/L)in 150 min are 85%(whole-cell catalyst)and 79.6%(CarCB2),respectively.It was identified by mass spectrometry(m/z)that the final product of the hydrolysis of Lambdacyhalothrin by them was 3-phenoxybenzaldehyde(3-PBH).To sum up,two carboxylesterase CarCby and CarCB2 successfully constructed whole-cell catalysts by surface display technology.Compared with CarCby and CarCB2,the whole-cell catalyst has better temperature stability and tolerance to some metal ions and chemical reagents.Compared with CarCby,the displayed CarCby whole-cell catalyst also has better p H stability.In addition,the degradation efficiency of carbaryl and Lambda-cyhalothrin by the two whole-cell catalysts was better than that of the purified CarCby and CarCB2,respectively.The research results provide a theoretical basis for bioremediation.