Enzymatic Pathway For MCLR Degradation By Bacterium CJ5

Microbial degradation is one of the main removal pathways of microcystins(MC)in water bodies. To date, researchers have done a lot of work on the microbialdegradation process of MC, but the research contents were mainly focus on screeningthe efficient aerobic MC-degrading bacteria and enhancing their degradationefficiency, also the degradation mechanism and pathways of aerobic MC-degradingbacteria were studied. However, there was no research about the degradation pathwayof MC by anaerobic MC-degrading bacteria. An anaerobic MCLR-degradingbacterium CJ5（Acidaminobacter sp.）was isolated from the sediment of Lake Dianchiby our laboratory, this bacterium can degrade MCLR rapidly in anaerobic condition,however, the degradation mechanism was not clear. We deduced that the degradingenzymes and pathways of anaerobic MC-degrading bacteria may differ from aerobicMC-degrading bacteria. So it is necessary to study the degradation mechanism ofanaerobic MC-degrading bacteria, thus to get a further understanding of the MCdegradation mechanism, and provide theoretical foundation to MC pollution treatmentby the microorganism or the protease its produced.In this thesis, we studied the degradation pathway of MCLR by anaerobicMC-degrading bacterium CJ5from the prespective of enzymology, and the factorsthat affecting the activity of the degrading enzyme. Furthermore, the degradingenzyme was isolated and purified. The specific research contents and results are asfollows:(1) To locate the degrading enzyme in Strain CJ5, we extracted and tested theMCLR degrading ability of different enzymes in CJ5. Results showed that in aerobicand anaerobic conditions, the intracellular enzymes can degrade about90%and60%of MCLR in14h respectively. However, the extracellular enzyme showed nodegrading activity in90h. It demonstrated that enzymes of Strain CJ5wasconcentrated inside the cell, oxygen did not inhibit the activity of intracellularenzyme.(2) During the anaerobic degradation process of MCLR by intracellular enzyme,a product with Adda was produced and accumulated. Mass spectrometry identification showed that it is a linear MCLR(H-Leu-MeAsp-Arg-Adda-Glu-Mdha-Ala-OH). It canbe deduced that the enzymatic degradation pathway of MCLR by CJ5was differentfrom the known aerobic degradation pathway of MC: With the action of intracellularenzyme, the Ala-Leu bond in annular MCLR was broken to generate Product A; thenthe Arg-Adda and Adda-Glu bonds were broken to generate Adda.(3) To further understand the characters of the intracellular enzyme of CJ5, westudied the influences of temperature, pH value, the concentration of protein andmetal ion on the enzymatic activity. Results are as follows, the degradation of MCLRby intracellular enzyme in CJ5were greatly influenced by temperature and pH, theoptimal enzyme catalytic degradation temperature was30℃and the optimum pHwas8. The degradation rate of MCLR was greatly influenced by the initialconcentration of intracellular enzyme, the degradation rate of MCLR increased withthe increase of protein concentration. Zn2+, Ca2+, Mg2+, Al3+did not affect thedegradation process of MCLR.(4) The intracellular enzyme of CJ5was purified by Ammonium Sulfateprecipitation, dialysis desalting and ion exchange column chromatography. Resultsshowed that after salting out by Ammonium Sulfate with60%saturation and dialysisdesalting, the degrading rate of MCLR in3h was56.1%. Then after DEAE-52ionexchange column chromatography, the eluting intracellular enzyme with1mol/LNaCl showed strong MCLR-degrading activity, the degrading rate of MCLR in3hwas50%. SDS-PAGE analysis showed that two kinds of proteins were exist in thiseluting intracellular enzyme, the molecular weights are22and80KDa.