Isolation And Identification Of Kresoxim-methyl Degrading Bacteria MJ-1,Analysis Of Degradation Pathway And Cloning Of Hydrolase Gene

Kresoxim-methyl is the first strobilurin fungicide developed by BASF in Germany.It is mainly used to prevent fungal diseases of fruits,vegetables and other crops.Kresoxim-methyl residues have been detected in soil,water and agricultural products,which are potentially harmful to the ecological environment and human health.Therefore,it is of great significance to study the degradation of kresoxim-methyl by microorganism.Although several kresoxim-methyl-degrading bacteria have been reported,no investigation on the related genes and enzymes have been reported yet.Therefore,the molecular mechanism of the degradation of kresoxim-methyl by microorganisms needs to be elucidated.This study started from the isolation of kresoxim-methyl-degrading bacteria,then we elucidated the microbial metabolism mechanism of kresoxim-methyl from the levels of metabolic pathway,gene and enzyme,and the following results were obtained.1.Isolation,identification,growth and degradation characteristics of kresoxim-methyl degrading bacteriaBy enrichment culturing,a kresoxim-methyl-degrading bacterial strain was isolated and named Hyphomicrobium sp.MJ-1 according to its bacterial morphology,biochemical characteristics and 16S r RNA gene phylogenetic analysis.Optimal growth temperature and p H of strain MJ-1 were 30°C and p H 7.0,respectively.The growth of strain MJ-1 was affected by the liquid volume,and the growth of the strain became better with the decrease of the liquid volume.The optimal Na Cl concentration was0.5 g·L^-1.Strain MJ-1 could grow with methanol or ethanol as the sole carbon source,and methanol was the optimal one.Strain MJ-1 could use ammonium chloride,beef extract,yeast extract and peptone as the sole nitrogen source for growth,and yeast extract was the optimal one.The kresoxim-methyl-degrading characteristics of strain MJ-1 were studied,and the results showed that the optimum temperature and p H for strain MJ-1 to degrade kresoxim-methyl were 30°C and p H 8.0,respectively.The decrease of liquid volume would be beneficial for the degradation of kresoxim-methyl by strain MJ-1.When the concentration of Na Cl in the medium was 0.5 g·L^-1,strain MJ-1 showed the best kresoxim-methyl degrading performance.Under the environmental conditions of 30℃and p H of 8.0,strain MJ-1 was able to completely degrade 0.32 m M kresoximone within 14 hours.Through HPLC-MS/MS,the degrading product of kresoxim-methyl by strain MJ-1 was identified and the metabolic pathway was proposed:kresoxim-methyl could be hydrolyzed into kresoxim acid and methanol by strain MJ-1.2.Cloning and functional identification of the kresoxim-methyl hydrolase gene kreHStrain MJ-1 can generate hydrolysis circle on solid LB plates added with 0.32 m M kresoxim-methyl.Taking advantage of this feature,one positive clone was picked from about16,000 transformants in the gene library constructed by the shotgun method.Kresoxim-methyl could be converted into kresoxim-methyl acid by this positive clone.A hydrolase gene kreH was obtained from the positive clone by sequencing.The gene was 1683 bp and it encoded 560 amino acids.The amino acid sequence analysis of KreH showed that it belonged to theα/βhydrolase superfamily.KreH showed highest amino acid sequences similarity with those of the pyrethroid hydrolase Ces2e（Q8BK48）derived from Mus musculus,which is33%.KreH had the closest relationship with the members of the VII family of esterase in the phylogenetic tree,so KreH was identified as a new esterase in the VII family of esterase.3.Study on the enzymatic characteristics of KreHKreH gene was heterologously expressed in E.coli BL21（DE3）,and KreH was purified by Strep-Tactin agarose gel FF column.The product of kresoxim-methyl hydrolyzed by KreH was identified,and the results showed that it could break the ester bond in kresoxim-methyl and hydrolyze kresoxim-methyl to kresoxim-methyl acid.The optimum temperature and p H for KreH were 30°C and 7.0,respectively.;EDTA and metal ions（1 m M）were added to the reaction system to compare the enzyme activity,the activity of KreH was not affected by EDTA,indicating that it was not a metal-dependent enzyme.The addition of Zn²⁺,Mg²⁺,Co²⁺,Cu²⁺,Mn²⁺and Ca²⁺showed no significant effect on the activity of KreH.The addition of Hg²⁺and Ni²⁺showed a slight promotion effect on the activity of KreH.While Fe²⁺slightly inhibited the activity of KreH.The substrate spectrum of KreH was also studied,and the results showed that it could only degrade kresoxim-methyl and trifloxystrobin,and it can’t degrade azoxystrobin,picoxystrobin,pyraclostrobin and some sulfonylurea herbicides.The amino acid sequence alignment showed that the characteristic sequence tag GXSXG and the typical conservative catalytic site Ser-Asp/Glu-His of theα/βhydrolase superfamily also existed in KreH.The three key amino acids（Ser235,Glu357 and His457）were mutated to alanine（Ala）by overlap extension PCR,then the mutant protein was purified,the hydrolysis activity of the mutants against kresoxim-methyl was determined and the results showed that all the mutants had lost its hydrolysis activity on kresoxim-methyl.This result proved that the Ser235,Glu357 and His457 triad were the conserved catalytic sites of KreH.The K_m of KreH for kresoxim-methyl and trifloxystrobin were 27.53584±4.39502μM、19.45823±3.73251μM,V_max were 0.06791±0.00235μmol s^-1 mg^-1 and 0.04749±0.00191μmol s^-1 mg^-1,k_cat were 4.11263±0.14232 s^-1 and 2.87599±0.11567 s^-1,and its catalytic efficiency k_cat/K_m were 0.14936μmol^-1 s^-1 and 0.14780μmol^-1 s^-1,respectively.These results showed that the hydrolase KreH in strain MJ-1 has a slightly higher affinity for kresoxim-methyl than trifloxystrobin,and the maximum reaction rate is higher than trifloxystrobin.The results of toxicity experiments showed that kresoxim-methyl can inhibit the growth of Chlorella.ellipsoidea,and the addition of strain MJ-1 can relieve the growth of C.ellipsoidea,showing its detoxification effect on kresoxim-methyl.