Resrarch On The Characteristics And Enzymatic Pathway For Biodegradation Microcystin-LR By Sphingopyxis Sp.USTB-05

The study investigated the characteristics of the degradation of MCs by the high efficient degradation of microcystins(MCs)Sphingopyxis sp.USTB-05 bacterium,Which previously isolated from Dianchi Lake sediment.Research on the characteristics of the USTB-05 bacteria degradation of MCs,Removal of microcystins using Carbon Nanotubes(CNTs)embedded with Sphingopyxis sp.USTB-05,Further improving the efficiency of the bacterial degradation of MCs.The molecular cloning of USTB-05 degradation of MCs gene,construction recombinant bacteria,purification of recombinant bacteria enzyme,detection of biodegradation producst by the recombinant bacteria enzyme,Investigation of the preliminary enzyme pathway of the MCs degradation by the USTB-05.The specific conclusion were as follows:(1)Initial 9.58 mg/L of microcystin-LR(MC-LR)could be completely eliminated within4 hours by USTB-05.Initial 11.6 mg/L of MC-LR could be completely eliminated within 8hours by the USTB-05 cell free extracts(CE)containing protein of 350 mg/L.The microcystins degradation efficiency by the Single-walled carbon nanotubes embedded with bacteria which was about 10 % more than USTB-05 degradation alone.The degradation rate of MC-LR by USTB-05 CE containing the same concentration protein: single-walled carbon nanotubes + USTB-05 CE > multi-walled carbon nanotubes + USTB-05 CE.(2)The MCs degradation gene was sucessed cloning by the primer design and PCR amplification genetic engineering technology.The constructed recombinant plasmids pT15/USTB-05-A,pET30a(+)/USTB-05-B and pET30a(+)/USTB-05-C were transfed to the E.coli BL21(DE3).Respectively,The recombinant bacteria A,B and C was obtained.(3)The recombinant A,B,C protein was purifyed by the Ni-IDA affinity columns.The protein concentration respectively measurement was 1 mg/mL,0.2 mg/mL,1.2 mg/mL by Bradford method.The purified protein purity was 95.4 %,91.1 % and 95.4 % by the R250 stain of sds-page gel Assessment.(4)The microcystins degradation efficiency by the carbon nanotubes embedded with recombinant bacteria was about 39 % more than recombinant bacteria degradation alone.Initial 11.6 mg/L of MC-LR could be completely eliminated within 2 hours by the recombinant bacteria CE containing protein of 350 mg/L.The degradation rate of MC-LR byrecombinant bacteria CE containing the same concentration protein: multi-walled carbon nanotubes + recombinant bacteria CE > single wall carbon nanotubes + recombinant bacteria CE.(5)The investigation on the enzyme pathway of the MC-LR or products degradation by three kinds of high efficient recombinant strains enzyme.The Microcystins degradation products was detected by high performance liquid chromatography(HPLC)technique.The results show that the first USTB-05 bacteria enzyme could quickly catalyze MC-LR,generating linear MC-LR.The second enzyme could quickly catalyze the linear MC-LR,generating Adda-Glu-Mdha-Ala.The third enzyme not only could rapidly catalyze the Adda-Glu-Mdha-Ala,Then generating Adda,but also could catalyze the linear MC-LR generated the Adda.