The Role And Molecular Mechanism Of Wetland Plants Endophytes Klebsiella Aerogenes S27 In Biodegradation Of Malachite Green

Malachite Green?MG?,a synthetic triphenylmethane industrial dye with stable molecular structure.It was extensively used in food,dyeing,and fish farming industry because of its low cost and high efficacy.Recently,some reports showed that MG has toxicological effects on humans or other animals and resistant to biodegradation to caused serious environmental pollution problems.MG was enriched in fish body which lived in water environment,causing serious environmental pollution problems.As a decolourization method with eco-friendly and eco-adaptable characteristics was received more and more attentions by researchers.Some researchs have shown that the bioremediation of plant-microbes combined mechanisms will be widely used in deal with dye wastewater to degrade soil or water quality of small molecules.Endophytes could metabolized organic pollutants and promoted plant growth,promoting the improvement of polluted environment through phytoremediation.To our knowledge,this work represents the first direct isolation and identification of endophytic bacteria with ability to degrade MG.The strain named S27,was identified as Klebsiella aerogenes species based on the analysis of 16s rRNA gene sequence.In this research,Klebsiella aerogenes S27 was investigated and found to the role and molecular mechanism in biodegradation of MG.Further cloning and expression of triphenylmethane reductase?TMR?gene which has the most active during decolorization.Phytotoxicity studies revealed the less-toxic nature of the degraded metabolites compared to the dye.This study can lay the foundation for the biological process research of plant-endophyte combined repair.The results of the reseach can be devided into the following parts:?1?Degradation of MG by endophytic bacteria K.aerogenes S27 and its potential mechanism:isolation and identification of an endophytic bacteria from the wetland plant Suaeda salsa with ability to degrade MG.The strain named S27?Genbank Accession No.M201745?,was identified as Klebsiella aerogenes species based on morphological,physiological,and the analysis of 16s rRNA gene sequence.The results showed that the degradation of MG(100 mg l^-1)by strain S27 could be completed in 8 h under the conditions of 30°C and pH 7.0.UV-Vis and FT-IR analysis of samples from degradation of MG confirmed that MG degradated into several metabolites.Strain S27 demonstrated high decolorizing capability?more than90%?at high concentrations of MG(100mg l^-1)within 24 h under condition of NaCl0-9%?w/v?,pH 7-10 and temperature 25-41°C,respectively.The results on degradation reactions of enzyme analysis indicated that TMR played an important role involved in MG degradation.?2?Enzymatic properties of degradation of MG by novel triphenylmethane reductase KaTMR:the primers were designed based on the gene sequence of TMR reported in NCBI.To amplify the target gene was using the DNA of strain S27 as a template by PCR.The protein was named KaTMR?Accession No.MH377117?,comprised of 849 bp encoding 282 amino acids and predicted molecular weight of31.02 kDa.KaTMR showed only 42.6-43.3%identities in amino acids compared with well-studied TMRs,and it phylogenetically formed a new branch in the family of TMRs.To obtain the target protein,the cloned gene was constructed into a recombinant plasmid with the pET-22b plasmid and heterologously expressed in E.coli BL21.The kinetic constant of KaTMR was determined by a Lineweaver-Burk plot and K_m,V_max values and k_cat/K_m values were 68.89?mol l^-1,10.76?mol l^-1 min^-1and 1.23 s^-1?mol^-1.?3?Chemical mechanism and product toxicity analysis of endophytic bacteria K.aerogenes S27 and KaTMR for degradation of MG:the intermediates of MG derived from the biodegradation of MG by strain S27 identified by the liquid chromatography-mass spectrometry?LC-MS?analysis included N,N-dimethylaniline?m/z 121?and desmethyl malachite green?m/z 315?.LC-MS analysis was performed after the complete decoloration of MG incubated with the purified enzyme showed four peaks corresponding to N,N-dimethylaniline?m/z 121?,?4-dimethylamino-phenyl?-phenyl-methanone?m/z 225?,leucomalachite green?m/z330,LMG?,and desmethyl malachite green?m/z 315?,respectively.The results showed differences from the presence of the reported degradation products.Phytotoxicity studies revealed the less-toxic nature of the degraded metabolites by strain S27 and KaTMR compared with MG solution.In summary,it was the first time to discover the function of endophytic bacteria to degrade malachite green and proved that Klebsiella aerogenes species have the function of degrading MG.The nature and molecular mechanism of K.aerogenes S27degradation of MG were also studied.The molecular mechanism of degradation of MG by a new triphenylmethane reductase?KaTMR?was revealed for the first time.This research not only increases the microbial resources and enzyme resources with degrading activity,but also helps to develop new functional enzymes and provides important data for the study of plant-endophytes in bioremediation.