Identification Of A Microcystins Degrading Bacterium X20and Its Enzymatic Pathway For MCLR Degradation

Microcystins(MCs) are a group of cyclic heptapeptide mainly produced by blooming cyanobacteria. The presence of MCs can be a potential threat to the ecosystem and human health. Microbial degradation of MCs is considered as a major elimination pathway for MCs in the environment. To date, more than40MC degrading bacteria had been isolated and the mechanism of biodegradation of MCs has been studied. Since different kinds of bacteria might degrade MCs through different pathways, it is necessary to explore the degradation mechanism for new isolate. In this study, we examined the position of MCs degrading enzymes in cell and the effects of environmental factors on enzymes activity after identifiying a newly isolated MC-degrading bacterium Strain X20. The use of classic protease inhibitors allowed the in vitro accumulation of transient products during the MCLR degradation. By identifying these products, the enzymatic pathway for MCLR biodegradation was deduced. The main research contents and results are as follows:(1) The identification and degradation ability of Strain X20Strain X20could degrade MCLR of5mg/L below the detection limit within10h at25℃. The colony of Strain X20on solid organic medium was yellow, round.1.0-1.5mm across, slippery, with regular edges and a moist surface. Strain X20is a gram-negative bacterium with ability of motility. The cells of Strain X20were short rod shaped, with a width of0.2-0.3μm and a length of0.4-1.3μm after cultivating for48h according to the SEM photo. Based on the analysis of colonial morphology, physiological and biochemical characteristics and the16S rDNA gene sequence, strain X20was identified as Sphingopyxis sp..(2) The position of MC degrading enzymes in strain X20and the effects of environmental factors on enzyme activityThe intracellular enzymes were mainly responsible for MC degradation process. Periplasmic enzymes presented low activity for MC degradation, while extracellular enzymes showed no degradation activity. The intracellular enzymes of strain X20degraded MCLR faster than its cells. The effects of pH, temperature and oxygen condition on the degradation of MCLR by intracellular enzymes were further studied. The results showed that the optimal pH value was7.5. the enzyme activity increased over the range of temperature from20to40℃and showed no difference under aerobic and anaerobic conditions.(3) The enzymatic pathway for MCLR degradation by Strain X20The results of enzyme and inhibitor studies showed that at least3kinds of intermediate degradation products were produced in the degradation of MCLR. They were linearized MCLR, tetrapeptides and Adda, respectively. From these results we could deduce that there were at least4enzymes involved in the MCLR degradation. Enzyme A, a metalloenzyme, was responsible for the ring opening of MCLR to get a linear MCLR. Enzyme B, a serine enzyme, was responsible for catalyzing the degradation of linear MCLR to a tetrapeptide degradation product. Enzyme C. a metalloenzyme. was responsible for decomposing tetrapeptide to Adda. Enzyme I) was responsible for degrading Adda to other substances.