Isolation And Characterization Of Carbendazim-degrading Strain,Identification Of The Key Amino Acid Sites Of Its Carbendazim Hydrolase(Mhel)

Two bacterial strains designated as SD-4 and Y2 were isolated from the soil subjected to the long-term application of carbendazim.According to their phenotypic features and phylogenetic analysis(16S rRNA),they were identified preliminarily as Mycobacterium sp.(GenBank accession no.KX668473)and Luteimonas sp.(GenBank accession no.KP684142),respectively.Strain SD-4 was Gram-positive,rod-shaped,non-motile and non-spore-forming.Its colonies on LB ager were orange-pigmented.Strain SD-4 grew optimally at pH 7.0,30?and in the presence of 1%(w/v)NaCl.Strain SD-4 could utilize carbendazim(MBC)as the sole carbon and nitrogen sources for growth and degrade 50 mg·L-1 MBC at the average degradation rate of 0.63 mg·L-1·h-1 within 72 h.Strain SD-4 degraded MBC through the typical pathway,in which carbendazim was first hydrolyzed to 2-aminobenzimidazole(2-AB)and then converted to 2-hydroxybenzimidazole(2-HB),the latter was further subjected to ring cleavage and mineralization.The carbendazim hydrolase encoding gene mhel was cloned from strain SD-4 by polymerase chain reaction(PCR),its GenBank accession number was KX698097.The result of sequencing revealed that it showed 100%identity to the gene mheI from Nocardioides sp.strain SG-4G(GenBank accession no.GQ454794)and 99.7%identity from Rhodococcus erythropolis djl-11(GenBank accession no.HQ874282),respectively,indicating that the gene sequence of mheI were highly conserved in these strains.The mhel gene was successfully expressed in Escherichia coli BL21(DE3)by codon optimization,the optimized mhel designated as mhel',was deposited in GenBank under the accession number of KX668474.The sulfhydryl-blocking assay revealed that the activity of Mhel was closely related to cysteine,and the site-directed mutation experiment showed that Cys16 and Cys222 played important roles during the hydrolysis of carbendazim by Mhel.Therefore they affected its activity directly and were defined as the key amino acid sites.Strain Y2 was Gram-negative rod-shaped,non-motile and non-spore-forming.Its colonies on LB ager were yellow-pigmented.Strain Y2 grew optimally at pH 7.0,30? and in the presence of 2%(w/v)NaCl.The major fatty acids(>5%)were iso-C15:0,iso-C17:0,summed feature 9(C16:0 10-methyl and/or iso-C17?1?9c),iso-C11:0 3-OH and iso-C11:0.The major respiratory quinone was ubiquinone-8(Q-8),and the major polar lipids were phosphatidylethanolamine(PE),phosphatidylglycerol(PG),diphosphatidylglycerol(DPG),one unidentified aminolipids(AL)and four unidentified phospholipids(PL 1-4).Phylogenetic analysis of the 16S rRNA gene sequences showed that strain Y2 was most closely related to Luteimonas mephitis B1953/27.1T(99.1%),followed by Luteimonas lutimaris G3T(98.6%).The DNA G+C mol%content of strain Y2 was 68.9 mol%,and strain Y2 exhibited low DNA-DNA relatedness with Luteimonas mephitis B 1953/27.1T(43.6 ± 0.5%)and Luteimonas lutimaris G3T(43.9 ± 2.1%).According to results of polyphasic taxonomy methods,strain Y2 was identified a novel species of the genus Luteimonas,for which the name Luteimonas soli sp.nov.(= ACCC 19799T KCTC 42441T)was proposed.