Optimization Of The Expression Of Microcystinase And Its Application In Cyanobacteria Bloom

In recent years,the eutrophication has led to the frequent outbreak of cyanobacteria blooms.Microcystins(MCs)are released into water bodies after the death of cyanobacteria and endanger human health.In the previous study,our group obtained a microcystinase(MlrA)that can efficiently degrade MCs.Basis on the previous investigation;we optimized the expression of MlrA to improve its purity and activity.We also found that MlrA can not only degrade MCs but also kill cyanobacteria at the same time.And we futhur assessed the safety of MlrA and the degradated MC-LR.Specific studies are as follows:1.Optimization of the expression of MlrA.MlrA was constructed on pET-21b,and purified by affinity chromatography to make it more pure and active.2.MlrA degrades MCs produced in water bloom.In order to apply MlrA to the degradation of MCs produced in cyanobacterial blooms,the available range of MlrA were studied.The results showed that MlrA can effectively degrade intracellular and extracellular MC-LR.More importantly,it can also effectively degrade MC-LR under simulated water bloom conditions.To analyze MCs qualitatively and quantitatively,a solvent extraction-solid phase extraction method was established to extract the intracellular and extracellular MCs of FACHB 905,and the MCs content was detected by high performance liquid chromatography(HPLC).3.Effects of MlrA on the growth of cyanobacteria.This investigation found that MlrA have a good inhibitory effect on the M.aeruginosa FACHB905,but not on Synechocystis sp.PCC6803.It indicated that there is a different inhibitory effect between toxin-producing algae and non-toxin-producing algae.Further studies revealed that MlrA could degrade intracellular and extracellular MCs of M.aeruginosa FACHB905.Meanwhile,the pH value of M.aeruginosa FACHB905 medium was regulated to be weakly alkaline or neutral from strong alkalinity.MlrA also inhibited photosynthesis and caused stress to algal cells.Since MCs and high pH value are important factors for the predominant growth of toxic algae,we speculated that MlrA could inhibit the growth of toxic algae by degrading MCs,maintaining the pH value of medium,inhibition photosynthesis and causing oxidative stress.4.The safety assessment of MlrA anddegradated MCs.At first,three kinds of cells that are sensitive to 10 ?g/mL MC-LR were selected from 12 human cells:human kidney cancer cell line ACHN,human hepatoma cell line Huh7,and human normal hepatocyte cell line LO2.The effects of MlrA and the degradated MC-LR on cell activity were studied.These results demonstrated that MC-LR could inhibit the activity of 30%cells at the concentration of 20?g/mL,while MlrA and degradated MC-LR were not toxic to cells.It shows that MlrA and degradated MC-LR have no obvious toxic effect on human cells.