Isolation And Characterization Of Carbendazim-Degrading Strains, Cloning And Expression Of The MheI Gene

Two bacteria strains mbc-1 and mbc-2 capable of degrading carbendazim were isolated from the soil under the long-term application of carbendazim. Strain mbc-1and mbc-2 were preliminarily identified as Mycobacterium sp. (GenBank Accession No. HQ873672) and Nocardioides sp. (GenBank Accession No. JF937914) respectively, according to its physiological & biochemical characteristics and the analysis of its 16S rRNA gene sequence.Biological characteristics of strain mbc-1 and mbc-2 were investigated respectively. Strain mbc-1 and mbc-2 could grow well under the condition of pH 6.0-8.0, the optimal pH was 7.0. The optimal temperature were 30℃. The optimal carbon source for the growth of strain mbc-1 and mbc-2 were glucose, and the optimal organic nitrogen source were yeast extract.The optimal inorganic nitrogen source for the growth of mbc-1 and mbc-2 were niter and urea respectively.Strain mbc-1 and mbc-2 could utilize carbendazim as the sole carbon source for growth; The degradation rate against 50 mg-L-1 carbendazim of strain mbc-1 and mbc-2 was 97.9% and 86.1% respectively within 84h. The optimal degrading conditions for both of them were 30℃and pH 7.0. Based on the analysis of the metabolitesappeared during the degradation, The proposed metabolic pathway for both of them was as following:MBC was initially hydrolyzed to 2-aminobenzimidazole (2-AB) and then converted to 2-hydroxybenzimidazole (2-HB).The primers were designed according to the only reported sequences of the carbendazim, (Methyl-1H-Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase gene (mheI). The mhel detection of strain mbc-1 and mbc-2 was carried out by PCR and the mhel gene was obtained from both strain mbc-1 and mbc-2. Both sequences had 99% similarity to mhel gene (GenBank accession No. GQ454794.1) The mheI gene was overexpressed in E.coli BL21(DE3), the protein with the right molecular size (24.3KD) was produced; it showed the carbendazim-hydrolyzing esterase character and could hyddrolyze carbendazim to 2-aminobenzimidazole.Since the gene sequence of mheI was highly conserved in different MBC-degrading bacteria, we carried out the investigateion on the horizontal gene transferring affair in these strains, A rapid and efficient chromosome walking method (SEFA-PCR) was employed to clone the upstream and downstream fragments of the mhel gene. The obtaind sequences were spliced with the mhel gene, resulting a fragment in mbc-1 (5983bp in length) and fragment (4529bp in length) in mbc-2. The sequence analysis showed, for mbc-1, the 5985 bp fragement included orf1(maximal similiarity with a alcohol dehydrogenase,57%), orf2(maximal similiarity with a L-asparaginaseⅡ,92%), orf3(maximal similiarity with a binding-protein dependent transport system inner membrane protein 96%), orf4 (maximal similiarity with a transporter-like protein,95%); the 4529 bp fragment if mbc-2 included orfl (maximal similiarity with a L-asparaginaseⅡ,92%), orf2, orf3 (maximal similiarity with a alcohol dehydrogenase,34%), orf4 (maximal similiarity with a ABC transporter permease protein,79%) orf5 (maximal similiarity with a transporter-like protein,95%).The prsent study did not find the evidence for the gene-transferring affair of mheI, but it still needs further study.