Isolation Of Carbendazim-degrading Bacterium And The Characterization Of Degrading Enzyme

Carbendazim?MBC?is one of the most active compounds of benzimidazole fungicides,wich has both protective and curative activities against a wide range of fungal diseases in fruits and vegetables.MBC is stable in the soil,and has slow rate of being degraded.Thus,growing concerns about the non-target toxicity of MBC has led to the requirement for identifying new methods to eliminate MBC contamination in environmental safely and efficiently.In this study,four carbendazim degrading bacteria were isolated from carbendazim-contaminated soil.The growth characteristics,degradation characteristics and degradation products of four strains were studied.And,the carbendazim hydrolyzing enzyme gene was cloned and heterologously expressed in Escherichia coli BL21?DE3?from the newly isolated bacterium.The catalytic mechanism of the enzyme was clarified by generating sulfhydryl blocking,amino acid mutations experiment and the hydrolysate analysis.The utilization potential of this enzyme for enzymatic bioremediation in soil and vegetables were investigated for further.The result which might lay a basis for the further study on the structure analysis of carbendazim hydrolyzing enzyme and have an important value for the development of new enzyme agent of carbendazim hydrolyzing enzyme and its application.The results for five experiments were shown as follows:1.Four carbendazim-degrading bacteria capable of utilizing carbendazim as the sole source of carbon were isolated from carbendazim-treated soil were preliminarily identified as Pseudomonas putida sp.djl^-1B,Pseudomonas sp.djl-5,Microbacterium sp.djl-6F and Agrobacterium sp.djl-8B respectively,according to its phenotypic features,biochemical characteristics,16 S rRNA gene sequencing and phylogenetic analysis.They all grow well when the temperature and pH value vary from 25 °C to 37 °C and 6.0 to 8.0.djl-6F has the fastest growth rate in growth stage whereas djl-8B has the lowest growth rate in growth stage on the four strains.djl^-1B behave like djl-5B in all growth stage.All the four strains grow well when the NaCl concentration vary from1% to 2%,NaCl concentration can affect the growth of four strains when lower than 1% or higher than 3%.The results of antibiotic resistance experiments showed that,twelve kinds of antibiotic including cefuroxime sodium,ceftazidime,amoxicillin and clavulanate potassium,maleic acid,azithromycin,clindamycin phosphate,cefoperazone sulbactam,ceftriaxone,ampicillin sodium,sodium penicillin,cefotaxime sodium,levofloxacin lactate and gentamicin sulfate shows varying inhibition effects for four strains.2.The detection result for the degradation properties and degradation products of four strains showed that,djl-6F has the most ability for degradation among four strains,the djl-8F is the weakest instead.The optimal degradation temperature is 30 °C for strain djl^-1B,djl-5B and djl-8B,and the optimal degradation temperature is 35 °C for strain djl-6F with the degradation ration of MBC is 99.5% after incubated for 96 h.The optimal degradation pH is 7.0 for strain djl-6F and djl-8B,and the optimal degradation pH is 7.5 for strain djl^-1B and djl-5B.The degradation ration presented some inhibition to the MBC when the pH < 6.5 or pH > 8.0.When the concentration of MBC was less than 150 mg/L,hydrolytic degradation rate of MBC for the strain djl^-1B,djl-5B and djl-6F were relatively fast.whereas,the hydrolytic degradation rate of MBC were significantly inhibited when the concentration of MBC was higher than 200 mg/L.The djl-8B was more susceptible to the concentration of MBC,for the degradation rate will decrease when the concentration higher than 100 mg/L.HPLC-MS analysis showed that the presence of 2-aminobenzimidazole?2-AB?and 2-hydroxybenzimidazole?2-HB?for all the four bacteria,and only benzimidazole?BZ?appeared in the degradation of djl-5B.pyrocatechol was not detected.3.The enzyme distribution experiment showed that the enzymes relative MBC degradation were inducible endoenzyme in strian djl^-1B and djl-5B,noninducible ectoenzyme in strain djl-6F and noninducible endoenzyme in strian djl-8B.The MBC hydrolyzing enzyme?MheI?gene?792bp?was cloned correctly from strain djl-6F,and it's His-tag fusion?MheI-6F?was heterologously expressed in E.coli BL21?DE3?,and purified through cobalt affinity chromatography.The detection result for kinetic parameters showed that,the Km value of MheI-6F was measured as 6.69 ± 2.1 ?M and the kcat was 160.88 ± 3.3 min^-1.Enzymatic characteristics of MheI-6F showed that,MheI-6F was identified stable when the temperature was ranging from 10 °C to 45 °C,80% of residual activity still remained after 2 h incubation.The maximum activity was found at 45 °C,and MheI-6F had lost most of its activity,for only 40.17% activity remained,at 70 °C.As the pH value increased,both the stability and activity of MheI-6F gradually rose,and peaked at pH=7.0.More than 80.0% of the total activity was retained after 2 h reaction at the pH ranging from 5.0 to 8.0.MheI-6F was found relative stabler under lower p H condition?4.0⁷.0?than it was under higher pH condition(8.0^-10.0).The enzymatic activity of MheI-6F was generally lower when metal ion was present in the reaction.Among the metal ions tested in our study,the addition of Fe3+ showed strongest effects on inhibiting the enzyme activity,with only 77.66% activity remained comparing with the control,whereas over 80.0% enzyme activity of MheI-6F remained when added with K+,Mg²⁺,Zn²⁺,Li+,Mn²⁺ and Ca²⁺.Glycerol,sodium azide and EDTA didn't show significant effect on MheI-6F activity.?-mercaptoethanol and Tween-20 showed slightly inhibitory effect on the enzyme activity.Surfactant SDS disrupted MheI-6F activity significantly with only 46.21% of activity remained comparing with the control,which had no chemicals presented in the reaction.HPLC-MS analysis revealed that MheI-6F catalyzes direct hydrolysis of MBC into 2-AB m/z 134 [M+H]+ without any cofactors.The sulfhydryl blocking experiment indicated that MheI-6F drastically lost 100% of its activity after incubated with sodium iodoacetate.The data of cysteine mutation showed that mutations at the site of 62,140 and 156 did not show significant effect on enzyme activity.MheI-6F C62 T,MheI-6F C140 T and MheI-6F C165 T remained more than 90% activity comparing with wild type MheI-6F.While mutations at the sites of 16 and 222 compromised the MheI-6F activity significantly.MheI-6F C16 T only had 45.06% activity,and MheI-6F C222 T had 64.51% activity comparing with wild type MheI-6F.The double mutated MheI-6F C16T&C222T was found to have no hydrolyzing activity over MBC.These result indicated that sulfhydryl is the active group,and two of the total five cysteines at the position of 16 and 222 were identified as the active site that the most contributes to the hydrolyzing activity of MheI-6F over MBC.4.The inoculation of Mhe-6F to MBC-contaminated cropland soil,orchard soil and greenhouse soil shortens the degradation half-life of MBC significantly from 8.15 d,8.93 d and 7.52 d to 56.80 min,63.58 min and 50.22 min.The inoculation of Mhe-6F to MBC-contaminated celery,spinach and rape shortens the degradation half-life of MBC significantly from 5.06 d,4.41 d and 4.26 d to 40.76 min,36.86 min and 31.50 min.These results indicate that Mhe-6F has a great prospect for environmental rehabilitation based on its extensive biodegradability on carbendazim.5.The methods of immobilizing MheI-6F free enzyme and the degradation-related characteristics were investigated.The optimal immobilization of the enzyme was achieved in 3% SA,4% CaCl₂ and immobilized for 4h.Immobilization time had a dominant impact for hydrolytic activity based on the orthogonal experiment.The optimal temperature for immobilized enzyme increased 5 °C than the free enzyme,and pH changed obviously in alkaline.And the immobilized enzyme had a higher capacity to withstand a broader range of temperatures and pH conditions than the free enzyme.The results provides basic information and technical support for future utilization of carbendazim-degrading enzyme as a bioremediation agent.