Metabolism Of Acetamiprid And Its Nitrile Hydratase By Streptomyces Canus CGMCC 13662

Acetamiprid(acetamiprid,ACE)is a kind of neonicotinoid insecticides widely used in the prevention and control of crop pests.In recent years,the long-term excessive use of ACE has brought many ecological and environmental problems.Microbial transformation is one of the most environmentally friendly,economical and efficient ways to remove ACE residues in the environment.The author's laboratory has been engaged in the study of the microbial biotransformation of nicotine pesticides for a long time,and screened some strains that can biotransform neonicotinoid insecticides.Among them,there is a strain that can efficiently transform ACE,Streptomyces canus CGMCC 13662,then the author conducted the study of ACE biotransformed by S.canus CGMCC 13662,analyzed its metabolic pathway and mechanism,cloned and expressed related metabolic enzymes,and determined enzymatic properties of the metabolic enzyme.It has also studied the structural characteristics and maturation mechanism of the metabolic enzyme.(1)The pathway of ACE metabolism in S.canus CGMCC 13662 was analyzed and the kinetics of ACE biotransformation by growing cells and resting cells was determined.High performance liquid chromatography(HPLC)analysis showed that the resting cells of S.canus CGMCC 13662 transformed ACE into a product peak with a retention time of 3.5-3.7 min after 2 d.The peak was analyzed by liquid chromatography-mass spectrometry(LC-MS).The product was identified as the amide product IM-1-2 of ACE.After 2 d,the growing cells of S.canus CGMCC 13662 in the ISP4 medium transformed ACE to two product peaks with retention times of 3.5 min and 2.7 min.LC-MS analysis showed that these two products were IM-1-2 and IM-1-4 respectively.The p H of ISP4 liquid medium becomes acidic after culturing the bacteria,and IM-1-2will spontaneously degrade to IM-1-4 under acidic conditions.The kinetics showed that resting cells of S.canus CGMCC 13662 biotransformed70.00%of ACE(the initial ACE concentration at 0.90 mmol/L)within 2 d,and transformed 0.79 mmol ACE to IM-1-2(0.65 mmol)in 4 d,the molar conversion rate is 82.28%,indicating that the ACE mainly via the hydration pathway produce IM-1-2.The degradation half-life of ACE is 27.70 h.The growing cells of S.canus CGMCC13662 biotransformed 16.16%of ACE in 2 d.After 12 d,S.canus CGMCC 13662degraded 90.32%of ACE residue(4.03 mg/kg soil)in sterilized soil.(2)The S.canus CGMCC 13662 nitrile hydratase gene cluster was cloned and successfully expressed in Escherichia coli.The target protein was purified and its enzymatic properties were analyzed.It was proved that ACE was converted to IM-1-2 by NHase.The S.canus CGMCC 13662 nitrile hydratase(Sc NHase)gene cluster(1136 bp)was successfully cloned from S.canus CGMCC 13662 genome.The Sc NHase gene cluster contained three genes,namely a?subunit gene(anh A,585 bp)and two?subunit genes(anh D,282 bp;anh E,288 bp),there were overlapping sequences between adjacent genes.Studies at home and abroad have shown that the NHase gene cluster is usually composed of one anh A and one?subunit gene(anh B),and contains the activation protein gene(anh C)which plays a key role in its activity.However,no active protein gene was found in the upstream and downstream of Sc NHase gene.E.coli-Q0,a heterologous recombinant expression strain of the Sc NHase gene,was constructed,HPLC analysis showed that the recombinant strain biotransformed ACE into IM-1-2,while E.coli with the control vector(without Sc NHase gene)did not.Therefore,the key metabolic enzyme of S.canus CGMCC 13662 for transforming ACE to IM-1-2 is NHase.Bioinformatics analysis and cobalt ion concentration gradient test results show that Sc NHase belongs to cobalt ion type NHase.SDS-PAGE results showed that Sc NHase had three subunits.Western blotting and MALDI-TOF MS analysis showed that the three subunits were anh D,anh E and anh A.These results indicate that Sc NHase is indeed a novel NHase composed of three subunits.The enzymatic characteristics showed that Sc NHase exhibited thermal stability in the range of 20-50?and acid-base tolerance in the p H range of 4-9.Enzymatic kinetic parameters K_m and V_max of Sc NHase for ACE were 5.85 mmol/L and15.99 U/mg respectively.The results of substrate specificity showed that Sc NHase could also transform other substances containing cyanide(C?N),for example,thiacloprid(THI),2-cyanopyridine and benzonitrile.In conclusion,S.canus CGMCC13662 not only can be used to repair ACE polluted environment,but also has the potential to transform nicotinoid insecticides thiacloprid and other toxic nitrile substances.(3)Expression vectors of different subunit gene combinations were constructed and expressed in E.coli.The influence of different subunits on enzyme activity was determined by analyzing the activity of NHase.By constructing and analyzing the homologous structure model of Sc NHase,the key amino acid sites of Sc NHase were identified.A phylogenetic tree of 26 species of NHase derived from different species was constructed.The results showed that the genetic distance between NHase of Streptomyces and Monosiga brevicollis NHase(Mb NHase)was the closest.Respectively constructed expression anh A,anh D and anh A combination(protein Anh DA),anh E combined with anh A(protein Anh EA)recombinant plasmid,recombinant plasmids expressed in E.coli,however these recombinant strains with no NHase activity,the results show that Sc NHase with three subunits is whole and active,this is different from Streptomyces rimosus NHase,its separate?subunit have NHase activity.anh D and anh E were fused into a fusion gene(anh D+anh E),which was combined with anh A and linked with p ET-28a,and introduced into E.coli Rosetta(E.coli-Q4).HPLC results showed that the activity of E.coli-Q4 for ACE was only 18.01±1.58%of that of E.coli-Q0(protein Anh DEA).This result indicated that Anh D and Anh E were necessary to the activity of NHase.Homology models of Sc NHase showed that two important conserved amino acid sites(Anh D-Glu56 and Anh E-His21)on the?subunit played an important role in the catalytic efficiency of Sc NHase,which explained why the NHase activity of above expression strains only expressed?subunit or the two subunits was inactive.(4)Determine the S.canus CGMCC 13662 whole genome sequence,analyzed the genomes of nitriles metabolic enzymes(nitrile-converting enzyme)gene and Sc NHase transcription unit.The whole genome sequence results of S.canus CGMCC 13662 showed that the whole genome contained 1 NHase gene cluster,2 nitrile hydrolase genes,4 amidase genes and 1 isonitrile hydratase gene.Transcription unit analysis of Sc NHase gene cluster showed that there were no corresponding transcription factors upstream of Sc NHase gene cluster.(5)The maturation mechanism of Sc NHase and the function of two?subunits were studied by means of atomic absorption spectroscopy,UV-VIS spectroscopy and gel filtration chromatography.The induced expression of apoprotein(Apo)under the condition of no addition of cobalt ions,and holoprotein(Holo)was produced under the condition of adding cobalt ions.The NHase activity of R-apo-Anh DEA produced by Apo-Anh DEA in in vitro activation buffer(phosphate buffer containing 10?mol/L Co Cl₂ and 2 mmol/L DTT)for 12 h is 35.98±4.32%of Holo-Anh DEA;Atomic absorption spectroscopy detected that each mole of R-apo-Anh A can bind 0.58±0.11 mol cobalt ions;the above results indicate that cobalt ions can combine with Apo-Anh DEA in a reducing environment in vitro to form R-apo-Anh DEA with NHase activity.After incubation for 12 h with Apo-Anh DEA and Holo-Anh DA,the NHase activity reached 54.41±1.35%of Holo-Anh DEA.The above results indicate that Holo-Anh DA activated Apo-Anh DEA to produce R-apo-Anh DEA with NHase activity.The UV-Vis spectra showed that R-Apo-Anh DEA had a characteristic peak of active Co-type NHase in the region of 300-350 nm.The molecular weight of the protein was predicted by gel filtration chromatography.Under the active condition,the molecular weight of Sc NHase(Holo-Anh DEA)was 92.74±1.50 k Da,while that of Anh DA was51.75±2.60 k Da.Therefore,it is speculated that Holo-Anh DEA is Hexamer Anh D₂E₂A₂and Anh DA is heterotrimer Anh D₂A.Holo-Anh DA exchanged?subunit with Apo-Anh DEA to generate R-apo-Anh DEA with NHase activity.According to the above results,it is speculated that the posttranslational maturation process of Sc NHase is coordinated by two mechanisms,namely,the mechanism by which cobalt ions bind to Anh A in the reduction environment in vitro and the Anh D involved in self-subunit swapping mechanism.The Anh D subunit acted as metal ion chaperone and self-subunit swapping chaperone in the posttranslational maturation of Sc NHase.In summary,S.canus CGMCC 13662 metabolized ACE into corresponding amide products through Sc NHase.Sc NHase has a unique three-subunit structure without activator protein.The post-translational maturation of Sc NHase is mediated by two possible mechanisms,?-subunit Anh D has the dual function of metal ion chaperone and self-subunit swapping chaperone.This paper is helpful to further study the structure and function of NHase and its maturation mechanism,and to guide search and discovery of new microbial resources for degrading nitrile compounds.