| Carbendazim(MBC) is a systemic benzimidazole fungicide, which is used widely to control a broad range of fungal diseases in agriculture and forestry. MBC is also the hydrolytic product and the active component of some other widely used fungicides such as benomyl and thiophenate methyl. MBC is quite stable in soil and water, which in turn can lead to the contamination of foodstuffs. And this is a concern because MBC can harm the liver and endocrine system and has mutagenic and teratogenic effects on animals, even at low concentrations. Microbial metabolism is the main mechanism responsible for degradation of MBC in natural enviroment. In this study, a MBC-degrading strain dj1-6-2 was identified and characterized; the degradation properties and metabolism pathway of MBC were also investigated. All these results would be helpful to the bioremediation of MBC contaminated environmentsThe optimal temperature and pH for the growth of strain dj1-6-2 was 30℃,7.0 respectivitly. The NaCl tolerance range was up to 7%. The diagnostic amino acid is meso-diaminopimelic acid, and ribose, arabinose and galactose were dominant in whole-cell hydrolysates. Phospholipids are DPG, PE1, PG, PE, PIM, PI, PME and CL. The predominant menaquinone is MK-8(H2). Mycolic acids are present. The major fatty acids are C14:0 (9.95%), C15:0 (1.25%), C16:o(36.77%), C17:1ω8c (1.44%), C18:1ω9c (27.29%), C18:0(4.89%), and 10-methyl C18:0 (tuberculostearic acid, TBSA) (3.70%); The DNA G+C content is 60.1 mol%. The levels of DNA-DNA relatedness of strain djl-6-2T to R. qingshengii djl-6T, R. baikonurensis DSM 44587T, R. erythropolis DSM 43066T and R. globerulus DSM 43954Twere determined to be 27.65%,19.29%,18.64% and 10.59%, respectively. Based on the genotypic and phenotypic analysis, strain djl-6-2T was assigned to the genus Rhodococcus as a novel species, for which Rhodococcus jialingiae sp. nov. is proposed.Strain djl-6-2 was capable of degrading 100 mg·L-1 MBC within 96h, and could use it as the sole carbon and nitrogen source for growth. The optimum condition for the degradation of MBC was 30℃, pH7. A rapid qualitative analysis method to assay the activity of MBC-degrading enzyme was established. The ultrasonic method was the best method in breaking the cells and extracting the enzyme of strain djl-6-2. The enzyme induction and distribution experiment showed that MBC-degrading enzyme was a constitutive endoenzyme. The optimal reaction condition for the enzyme was 37℃and pH7, while it was stable over a range of 15℃～45℃and pH 6.0～8.0. Metal ion Zn2+, Fe3+ and K+ could inhibit the enzyme activity. Tween 80, SDS and Triton X-100 could also inhibit enzyme activity. Phenylmethylsulfonyl fluoride completely inhibited it. The HPLC and MS/MS analysis showed that MBC was hydrolyzed to 2-aminobenzimidazole (2-AB) and then converted to 2-hydroxybenzimidazole (2-HB).The influences on the degradation of 2- AB were studied. Only 3.7% of 2-AB was degraded in the mineral salt medium with 1.0 g·L-1 NH4NO3, while 97.3% of 2-AB was degraded in the medium without NH4NO3, and the degradation could be promoted by glucose. The optimum condition for 2-AB degradation in medium without NH4NO3 was 30℃, pH7. The degradation of 2-AB by strain djl-6-2 was inhibited by the ion Fe3+, Hg2+ and Al3+, while promoted by Mn2+, Fe2+. Accumulation of 2-HB happened during the degradation of 2-AB (100 mg-L"’)but not happened when the concentration of 2-AB was 10 mg·L-1.The inhibitor 3-FC could reduce the degradation speed of 2-HB. MBC,2-AB and 2-HB could induce the dioxygenase activities of the cells. The MS/MS analysis on intermediates during the degradation of 2-AB found the appearance of 2-HB and benzimidazole, and three other intermediates which were proposed to be dihydroxybenzimidazole, trihydroxybenzimidazole and a benzenic ring cleavage product.An extradiol dioxygenase, encoding the gene edo that was cloned from strain djl-6-2 by PCR, could catalyze catechol and 2,3-DHBP to open the benzenic ring. And then it was expressed in E. coli BL21 (DE3), and it could promote the crude enzyme of strain djl-6-2 to degrade 2-HB. Therefore, extradiol dioxygenase was supposed to take part in the benzenic ring opening of 2-HB. |
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