Cloning And Expression Of 2-aminobenzimidazole Hydrolase Gene From Carbendazim-degrading Strain Djl-6-2

Carbendazim is an endogenous benzimidazole fungicide.It is widely used all over the world due to its high efficiency and broad spectrum.The residues in soil,water and crops have not only destroyed the ecological environment,but also threatened the health of human.Thus,the elimination of carbendazim residues from agricultural products and soil has appealed to increasing attention of researchers.The capacious application prospects of microbial degradation methods can not be separated from their advantages of economy,environmental protection,safety and effectiveness.So far several carbendazim-degrading bacteria have been isolated,however,only the carbendazim hydrolase gene responsible for its first degradation has been reported.The carbendazim-degrading bacterium Rhodococcus jialingiae djl-6-2 was isolated in our laboratory which has the typical degradation pathway of carbendazim.The carbendazim was converted to 2-hydroxybenzimidazole(2-HB)via 2-aminobenzimidazole(2-AB)followed by the further ring-opening of 2-HB until its complete degradation.We have cloned the carbendazim hydrolase gene cbma responsible for the hydrolysis of carbendazim to 2-AB.In this study,we will identify the 2-AB hydrolase gene responsible for hydrolyzing 2-AB to 2-HB by this strain.Meanwhile,a Gram-negative bacterium YHM-9 with strong tolerance to carbendazim(1000 mg L-1)was isolated from long-term application of carbendazim in farmland soil,and its taxonomy was studied.The main results appear as shown below.1.Purification of 2-aminobenzimidazole hydrolase from carbendazim-degrading bacteria djl-6-2 The study of enzymatic properties of crude enzymes is the basis of protein purification.Therefore,we first studied the properties of the crude enzymes of 2-AB hydrolase from strain djl-6-2.The results showed that the optimum conditions for the hydrolysis were 30?35? and pH 7.0.EDTA(10 mM)had no marked effect on enzyme activity,indicating that the enzyme was not metal-dependent.Organic solvents inhibited the enzyme activity strongly,among which 10%acetone and 10 mM SDS reduced the activity to 10%of the original activity.1 mM Mn2+activate the enzyme activity,while 1 mM Ni2+,Ca2+,Mg2+and Fe2+inhibit the enzyme activity.The 2-AB hydrolase sample which can be analyzed by LC-MS/MS was obtained by four purification procedures including ammonium sulfate precipitation,HiTrap Q FF chromatography,Butyl-650M hydrophobic chromatography and Superdex-200 gel filtration.The results of mass spectrometry identification were compared with the genomic sequence of strain djl-6-2,combined with amino acid sequence alignment.Three probable hydrolase genes were preliminarily screened.The ORFs encoding the protein were orf 04172,orf 01752 and orf 00389,respectively.Among which the similarity between the protein encoding orf 04172 and Lactamase-like protein aptB(Q95Q18.1)was 33%;the similarity between the protein encoding orf 01752 and Porphobilinogen deaminase(Q5YP70.1)was 79%;and the protein encoding orf 00389 was 26%similar to that of 5-nitroanthranilic acid aminohydrolase(D3WZ85.1).Based on the results above,we speculate that all of them(orf 04172,orf 01752 and orf 00389)may be genes encoding 2-aminobenzimidazole hydrolase in strain djl-6-2.2.Cloning and expression of 2-AB hydrolase gene from strain djl-6-2According to the results of LC-MS/MS analysis,primers were designed for the three possible 2-AB hydrolase gene sequences.The expression vector was then constructed and expressed heterologously in E.coli BL21(DE3).The degradation activity of 2-AB by the recombinant strain E.coli/pET29a(+)-orf 00389 was preliminarily verified by crude enzyme after induced expression and ultrasonic fragmentation,while the recombinant strains with orf 04172 and orf 01752 had no 2-AB hydrolase activity.After purified by Ni-NTA column,the purified enzymes activity were tested and analyzed by HPLC and MS/MS,which further proved the ability of the orf 00389-encoded enzyme to hydrolyze 2-AB and then named it AbaH.The full length of the gene abaH is 1344 bp,encoding 448 amino acids.Nucleic acid and amino acid sequence analysis showed that abaH had the highest similarity(26%)with 5NAA deaminase from Bradyrhizobium sp.Strain JS329,suggesting that abaH might be a new gene.3.Investigation on the taxonomy of carbendazim-tolerant strain YHM-9In this study,strain YHM-9 with strong carbendazim tolerance(1000 mg L-1)was isolated from long-term application of carbendazim in farmland soil,and its multiphase taxonomy was studied.Strain YHM-9 was Gram-negative,rod-shaped,non-motile and non-spore-forming.The similarity with the type strain Pedobacter W-WS13T(KP641351)was 97.37%.Strain YHM-9 grew optimally at pH 7.0,30? and in the presence of 0%(w/v)NaCl.The DNA G+C content is 38.9 mol%.The major respiratory quinone was MK-7.The major polyamine was sym-homospermidine.The major polar lipids were phosphatidylethanolamine,minor amounts of two glycolipids,one unidentified phospholipid,one unidentified aminolipid and several unidentified polar lipids.The dominant fatty acids(>5%)are iso-C15:0,summed feature 3(C16:1?7c and/or C16:1?6c),iso-C17:0 3-OH,summed feature 4(iso-C17:1 I and/or anteiso-C17:1 B)and anteiso-C15:0.On the basis of the multiple taxonomic analysis,strain YHM-9T represents a novel species of the genus Pedobacter,for which the name Pedobacter agrisoli sp.nov.(=JCM 32093T=CCTCC AB 2017125T)is proposed.