Isolation And Identification Of Carbendazim-Degrading Strain,Cloning And Expressionof A Novel Carbendazim Hydrolase Gene

Carbendazim（MBC）is a benzimidazole fungicide with broad spectrum.It is also the metabolite and active center of many other benzimidazole fungicides.Because of its widespread use throughout the world,the residues of carbendazim can be detected in soil,water and crops.Therefore,the elimination of carbendazim residues in soil and agricultural products has become an urgent problem to be solved.Microbial degradation is the main method to degrade pollutants in the environment and is an effective,safe,cheap method without secondary pollution.In this study,two carbendazim-degrading strains designated as D-J and djl-8 and another strain DH-5 capable of utilizing 2-hydroxybenzimidazole（2-HB）,the metabolite of carbendazim,as sole carbon source for growth,were isolated from the soil subjected to the long-term application of carbendazim and identified.The growth and degradation characteristics of strain D-J were studied due to its high degrading efficiency against carbendazim.The carbendazim hydrolase gene mhel was cloned from strain D-J and its horizontal transfer mechanism was analyzed by comparative genomics.A novel carbendazim hydrolase gene cbma was cloned from the carbendazim degradation strain djl-6-2,and its heterologous expression and enzymatic characteristics were also studied.The effects of the addition of carbendazim on the community structure of fungi and bacteria in yellow brown soils at different concentrations（2 and 8 mg·kg-1 dry soil）were studied by using MiSeq high-throughput sequencing technique.At the same time,the inoculation strain djl-6 on the degradation of carbendazim in the soil was also evaluated.In this thesis,the microbial degradation mechanism of carbendazim was studied from strain,gene and enzyme level.1 Isolation of carbendazim-degrading strains and investigation on their growth and degradation characteristicsTwo strains named Mycobacterium sp.D-J and Nocardioides sp.Djl-8,which could grow with carbendazim as the sole carbon source,were isolated from the soil subjected to the long-term application of carbendazim by continuous enrichment culturing.They could degrade 94.3%and 71.4%of 50 mg·L-1 carbendazim within 48 h,respectively.At the same time,a strain Sphingobacterium sp.DH-5 which could utilize 2-hydroxybenzimidazole,the metabolite of carbend azim,as the sole carbon source for growth was also isolated.The growth and degradation characteristics of strain D-J were studied due to its high degradation efficiency than strain djl-8 obtained from this experiment.The optimum growth and degradation temperature for the strain was 30℃,and the optimum growth and degradation pH values were 7.Strain D-J harbored a typical carbendazim degradation pathway.First,carbendazim is hydrolyzed to 2-aminobenzimidazole（2AB）and then degraded to 2-hydroxybenzimidazole,followed by further ring-opening of 2-hydroxybenzimidazole.This pathway is consistent with the metabolic pathway of carbendazim in strain djl-6-2,which has reported by our laboratory.2 Cloning of carbendazim hydrolase gene mhel from strain D-J and preliminary investigation on its horizontal transfer mechanismThe carbendazim is a key enzyme gene for the degradation of carbendazim,which is responsible for the hydrolysis of carbendazim to 2-aminobenzimidazole and the detoxification of carbendazim.In order to clone the gene,the genomic framework map strain Mycobacterium sp.D-J isolated in this study was sequenced,and the genomic integrity map of the carbendazim degradation strain Rhodococcus qingshengii djl-6 and Rhodococcus jialingiae djl-6-2 were also sequenced.By comparison,it was found that strains D-J and djl-6 both had a gene sequence,which showed 99%similarity to the reported hydrolase gene mhel（GQ454794.1）.Although the carbendazim-degrading pathway of strain djl-6-2 was consistent with that of strain D-J,the mhel gene was not found in its genome,indicating that there was a new carbendazim hydrolase gene in strain djl-6-2.As carbendazim hydrolase gene mhel exists in both strains D-J and djl-6,it is necessary to analyze whether the gene has a horizontal transfer phenomenon.The results showed that there was a DNA 15532 bp long fragment in these two strains.The fragment had 13 ORFs,including the mhel gene flanked by transposase at both ends.This fragment was located on the linear plasmid of 288399 bp in strain djl-6.Compared with the published genome of Mycobacterium sp.djl-10,part of the sequence of the above-mentioned 15532 bp framgent was in the large plasmid of the strain djl-10.It was 5654 bp in length and the similarity was 99%.Besides the mhel gene,it included 6 ORFs.The similarity between the protein encoding orf 6 and Asparaginase（WP₀64073 806.1）was 99%;the similarity between the protein encoding orf 7 and MHI（ACV42482.1）was 99%;the protein encoding orf 8 was similar to that of Sulfonate ABC transporter ATP-binding protein（WP₀64073804.1）The similarity of the protein encoding orf 9 to ABC transporter permease（WP₀64073803.1）was 99%;the similarity of the protein encoding orf 10 to ABC transporter permease（WP₀64073802.1）was 99%;the protein encoding orf 11 was 99%similar to that of Histidine ammonia-lyase（WP₀64073801.1）.3 Research on Cloning and expressing a novel carbendazim hydrolase gene cbma in strain djl-6-2In order to clone the carbendazim hydrolase gene in strain djl-6-2,the carbendazim hydrolase sample which can be analyzed by MALDI-TOF-MS was obtained by four purification procedures including ammonium sulfate precipitation,DEAE-Sepharose FF chromatography,Q-Sepharose FF chromatography and Superdex-200 gel filtration.According to peptide mass fingerprinting identification and the genomic sequence of strain,it was found that the target gene was orf 04383,named cbma（Carbendazim amidase）.ORF analysis showed that this gene was 1434 bp in length,encoding a 4 77amino acid protein.The corresponding protein was searched against the Swiss-Prot database,which revealed that CbmA shared 96%identity with acylamidase from Rhodococcus erythropolis,the similarity of the other protein was below 34%,and very low similarity with carbendazim hydrolase Mhel.It indicates that the CbmA is a novel carbendazim hydrolase.According to the codon bias of E.coli,the cbma gene sequence was optimized and synthesized.Then the expression vector pET-29a-cbma was constructed and transformed into E.coli BL21（DE3）for heterologous expression of cbma,and CbmA was purified by the nickel column and the activity was analyzed.CbmA could hydrolyze carbendazim to 2-aminobenzimidazole and the option temperature for this and the option tempreture for this enzyme was 20-37℃;the optimum pH value for CbmA activity was 7.0;CbmA could not be activated by Al3+,Ca2+,Fe3+,Fe2+,Li+,Mg2+,Mn2+and Ni2+ at 0.1 mM,respectively;Cbm A could be slightly inhibited by Zn2+;CbmA could be inhibited by Cu2+ by 78.3%;and CbmA could be strongly inhibited by Hg2+.The enzyme CbmA could not be activated by EDTA,1,10-Phenanthroline monohydrate,Tween 20 and Tween 80;and The enzyme CbmA could be inhibited by Triton X-100 and SDS.The degradation rate of CbmA was（4.7±0.4）μM in Km,（203.4±8.5）min-1 in kcat and（43.3±2.8）μM-1·min-1 in kcat/Km,which was higher than the reported carbendazim degradation hydrolase Mhel at（27.9±3.4）μM-1·min-1 in kcat/Km.4 Investigation on the taxonomy of strains djl-8 and DH-5The research found the low gene homology of 16S rRNA between strain djl-8 and other Nocardioides:similar to Nocardioides ginkgobilobae SYP-A7303T at 97.08%,to Nocardioides soli mbc-2T at 96.87%,to Nocardioides pyridinolyticus OS4T at 96.59%,and to Nocardioides maradonensis RP-B30T at 96.57%,so the taxonomy of djl8.Basing on Polyphasic Taxonomy method was further researched.djl-8 was identified as new species of Nocardioidaceae named as Nocardioides agrisoli sp.nov.djl-8T（=CCTCC AB 2017058T=ACCC 19964T=KCTC 39844T）.The low gene homology of 16S rRNA between strain djl-8 and other Sphingobacteriums was also found:similar to Sphingobacteriun gobiense H7T at 96.0%,and to Sphingobacterium arenae H-12T at 94.5%,thus the taxonomy of strain DH-5 was further researched.DH-5 was identified as a new species of Sphingobacterium,named Sphingobacterium chuzhouense sp.nov.DH-5T（=ACCC 198567=KCTC 427467）.5.Research on Degradation of carbendazim in soil by strain djl-6 and its ecological effectsApplying carbendazim at different concentration（2 and 8 mg·kg-1 dry soil）to the yellow-brown soil,using MiSeq high-throughput sequencing technology,the effects on the fungi and bacterial community structure of the samples taken on Day 0,7,14,21 respectively were studied and the effects of djl-6（CFU=108 cfu g-1 dry soil）on carbendazim degradation were evaluated simultaneously.The results showed that the natural degradation rate of carbendazim in 14 d was about 32.5%and 34.1%respectively in carbendazim soil added with 2 and 8 mg·kg-1 dry soil,while the inoculated strain djl-6 could significantly promote the removal of carbendazim in soil,and the removal efficiency of carbendazim in 14 d was 93.3%and 94.8%,respectively.The analysis result of Alpha diversity of bacteria and fungal in soil showed that,applying carbendazim at different concentration（2 and 8 mg·kg-1 dry soil）could significantly decrease the bacterial community richness and diversity in soil in 0-14 d,compared with the blank soil,and the stronger impact was showed when the concentration of carbendazim was 8 mg kg-1 dry soil.At 0-7 d,the richness of fungal communities in soil could be reduced.In 7-14 d,the richness of fungal community was gradually restored.The recovery degree of dry soil with carbendazim concentration of 2 mg·kg-1 was stronger than that of 8 mg·kg-1 dry soil,and there was no significant changes in the diversity of fungal community in the whole process.In soils only inoculated with djl-6 showed no significant changes in the richness and diversity of bacterial and fungal communities.In the soil with different concentration of carbendazim and inoculated with strain djl-6,the richness and diversity of bacterial communities were significantly reduced in 0-14 d;in 0-7 d,the richness of fungal communities in soil could be reduced;in 7-14 d,the richness of fungal community was gradually restored.And there were no significant changes in the diversity of fungal community in the whole process.The analysis result of Beta diversity of bacteria and fungal in soil showed that,compared with the blank soil,when carbendazim concentration was 2 mg·kg-1 dry soil,the bacterial community structure was less affected in 0-14 d;in 0-7 d,there was a great influence on the fungal community structure;and in 7-14 d the fungal community structure was gradually recovered.When carbendazim concentration was 8 mg·kg-1 dry soil,the increase of carbendazim concentration would increase its influence on bacteria and fungi community structure in soil which induced the formation of completely different microbial communities.The strain djl-6 had little effect on bacterial and fungal community structure in the soil inoculated only with strain djl-6.In the soil with different concentration of carbendazim and inoculated with strain djl-6,in 0-14 d,with the decrease of carbendazim concentration,the community structure of bacteria and fungi in soil showed different degrees of influence.The results indicated that the strain djl-6 played a key role in the remediation of carbendazim contaminated soil.The inoculation of strain djl-6 had little effect on the microbial flora in the soil,and the strain had the potential to develop soil remediation agent.