Characterization and Mechanism of Linearized-Microcystinase Involved in Bacterial Degradation of Microcystins.

Microcystins (MCs) are extremely hazardous to the ecological environment and public health. How to control and remove MCs is an unsolved problem all over the world. Some microbes and their enzymes are thought to be effective in degrading MCs. Microcystinase can linearize microcystin-leucine-arginine (MC-LR) via a specific locus. However, linearized MC-LR is also very toxic and needs to be removed. How linearized MC-LR was metabolized by linearized-microcystinase, especially how linearized-microcystinase binds to linearized MC-LR, has not been defined. A combination of in vitro experiments and computer simulation was applied to explore the characterization and molecular mechanisms for linearized MC-LR degraded by linearized-microcystinase. The purified linearized-microcystinase was obtained by recombinant Escherichia coli overexpressing. The concentration of linearized MC-LR was detected by high-performance liquid chromatography, and linearized MC-LR degradation products were analyzed by the mass spectrometer. Homology modeling was used to predict the structure of the linearized-microcystinase. Molecular docking techniques on the computer were used to simulate the binding sites of linearized-microcystinase and linearized MC-LR. The purified linearized-microcystinase was obtained successfully. The linearized-microcystinase degraded linearized MC-LR to tetrapeptide efficiently. The second structure of linearized-microcystinase consisted of many alpha-helices, beta-strands, and colis. Linearized-microcystinase interacted the linearized MC-LR with hydrogen bond, hydrophobic interaction, electrostatic forces, and the Van der Waals force. This study firstly reveals the characterization and specific enzymatic mechanism of linearized-microcystinase for catalyzing linearized MC-LR. These findings encourage the application of MC-degrading engineering bacteria and build a great technique for MC-LR biodegradation in environmental engineering.

Identification and characterization of the dominant Microcystis sp. cyanobacteria detected in Lake Dong Ting, China.

The presence of cyanobacteria in drinking water, aquatic foods and bathing water has created a significant major problem to global public health as these toxins induce damage in various organ including liver, cardiovascular, intestinal and central nervous systems. Although the morphologic, phylogenetic and toxicogenetic characteristics of cyanobacteria were identified in several lakes in China, many freshwater sources such as Dong Ting Lake, Hunan Province, China remain to be determined. Since the presence of these cyanobacteria may potentially affect human health, the aim of this study was to isolate, identify and characterize the most frequent occurring bloom-forming cyanobacteria in Dong Ting Lake, Hunan Province, China, which can provide information on the safety of utilization of this water source for drinking water, agriculture and recreation. Samples collected from the surface water of Dong Ting Lake were subjected to serial dilution in the lab for morphological analysis. Data demonstrated the morphological features were 2-5 µm diameters with rounded shapes and green color resembling Microcystis sp. The isolated cyanobacterial strain obtained from surface water samples in Dong Ting Lake was termed Microcystis sp. YFM2. The MC concentration was detected by enzyme-linked immunosorbent assay (ELISA) and found to be 92.88 µg/107 cells in Microcystis sp. YFM2. By polymerase chain reaction (PCR) results indicated that Microcystis sp. YFM2 isolated from Dong Ting Lake contained synthetase genes (mcyA-C). Our findings indicated that the dominant cyanobacteria Microcystis sp. YFM2 isolated from the freshwater Dong Ting Lake demonstrated morphologic, phylogenetic and toxicogenetic properties resembling a toxin generating cyanobacterium. Based upon this knowledge, it is essential to monitor the use of this Lake for future domestic, agricultural and recreational purposes.

Anaerobic degradation of microcystin-LR by an indigenous bacterial Enterobacter sp. YF3.

Microcystin-LR (MC-LR), a known hepatotoxin present in drinking water, and contaminated food and algal dietary supplements poses a threat to environmental and public health and thus needs to be removed. Previously microbial aerobic degradation was considered the predominant catabolic process for MC-LR inactivation, but the potential role of anaerobic microbes still needs to be determined. In our study an anaerobic MC-degrading bacterium Enterobacter sp. YF3 was isolated and identified that was capable of degrading MC-LR. Under optimal conditions the anaerobic Enterobacter sp. YF3 displayed a MC-degrading rate of 0.34 µg/ml/day. This process was dependent on temperature, pH and MC-LR concentration. Further the extracellular secretion of metabolites of anaerobic bacterium degraded MC-LR at 0.22 µg/ml/day. The parent MC-LR as well as two MC-degrading products was identified by high performance liquid chromatography (HPLC). The anaerobic MC-degrading Enterobacter sp. bacterium metabolized MC-LR independent of MC-degrading genes mlrABCD. Data indicate that anaerobic Enterobacter sp. YF3 produces MC-degrading products via a pathway that acts independently of mlrABCD genes which may add to the arsenal of bacteria to degrade microcystins.