Molecular Mechanism Of Manganese Oxidation By Catalase-Peroxidase In Endophytic Bacteria F01 Of Wetland Plants

The trace element manganese（Mn）is widely distributed in oceans and terrestrial soils around the world,and switches between three main valence states,Mn（II）,Mn（III）and Mn（IV）,constituting the manganese cycle in nature.Bacterial enzymatic reaction is the main factor for the production of manganese oxides in nature.The bacterial manganese oxides produced are highly oxidizing and adsorbent,and mediate the degradation of organic pollutants and the cycle of nutrient elements.They have broad research value and application potential in environmental restoration,energy development,carbon capture and release,geological evolution,and marine science.The accumulation of manganese oxide precipitation on the root surface of wetland plants affects the rhizosphere process and the degradation and transformation of rhizosphere organic matter.Endophytic bacteria can promote the formation of root surface manganese oxides,but there is a lack of research data on the physiological characteristics,structural basis,and degradation of organic matter of plant endophytic bacterial manganese oxidase.Although manganese oxidase from endophytic bacteria of wetland plants has not been identified and reported,manganese oxidase from some marine bacteria has been reported,mainly including the copper oxidase Mnx GEF and heme peroxidase Mop A ofα-proteobacteria.We isolated two proteins with Mn（II）oxidation activity from extracellular secretion of the endophytic halophytic bacterium Salinicola tamaricis F01 isolated from the wetland plant tamarix in the Yellow River Delta with molecular weights of about 80 k Da and 85 k Da,both of which were identified as catalase-peroxidase,named StKatG1 and StKatG2.On this basis,this thesis integrates bioinformatics,molecular genetics,materials chemistry and enzymology techniques to further clarify the function and mechanism of catalase-peroxidase mediating bacterial manganese oxidation.The specific research content includes:（Ⅰ）Bioinformatics analysis of StKatG1 and StKatG2:（1）Amino acid sequence and structural domain analysis:Analysis of amino acid sequences through websites such as Phyre2and Inter Pro shows that StKatG1 and StKatG2 belong to the catalase-peroxidase family（EC1.11.1.21）.In addition,StKatG1 and StKatG2 both contain two domains,namely the N-terminal heme binding peroxidase domain and the C-terminal non-heme binding peroxidase domain.（2）Phylogenetic analysis and multiple sequence alignment:Through the construction of an evolutionary tree and homologous protein sequence alignment analysis,it is shown that StKatG1and StKatG2 have significant sequence similarities with other functionally described catalase-peroxidase,and it was suggested that they may have a common ancestor.In summary,the amino acid sequence characteristics,structural domain characteristics,and evolutionary relationships of StKatG1 and StKatG2 have been revealed through bioinformatics analysis,providing a theoretical basis for further studying the functional characteristics of StKatG1 and StKatG2 proteins.（Ⅱ）Functional verification of catalase-peroxidase StKatG1:（1）Gene knockout:Relying on the established genetic manipulation system of endophytic bacterium S.tamaricis F01,a mutant of the stkat G1 gene deletion was constructed by gene knockout.The results showed that the knockout of the stkat G1 gene cannot grow under normal culture conditions,indicating that stkat G1 gene may be an essential gene for the F01 strain.（2）Gene recombination expression,separation and purification,and enzymatic properties research:The stkat G1 gene was recombinantly expressed in E.coli BL21,and StKatG1 recombinant protein was obtained by separation and purification.Using Mn Cl₂ as the substrate,combined with the detection data of the LBB method,it was proved that catalase-peroxidase StKatG1 has manganese oxidation activity.The optimum temperature for StKatG1 manganese oxidation is 50℃,and the optimum p H is 7.5.According to the catalytic kinetic analysis data,the Km of StKatG1 recombinant protein oxidized Mn Cl₂ was 1.291 mmol/L,Vm was 10.30μmol/L/min,and Kcat was 2.78 min^-1.Its enzymatic properties are significantly different from those of the reported heme peroxide enzymes,and it is a new type of manganese oxidase.（3）Analysis of the catalytic product of the enzyme:the composition and structure of the manganese oxide product obtained by the oxidation of Mn Cl₂by the StKatG1 protein were characterized.The manganese oxidation product was observed by scanning electron microscope as a compact lamellar structure;XPS results showed that the manganese oxidation product was a mixed valence manganese compound,and further XRD showed that the mixed valence manganese oxidation product contained Mn O₂,Mn₂O₃,Mn₃O₄ and Mn C₂O₄.（4）Analysis of key amino acid sites:The three-dimensional structure of StKatG1 protein was predicted by SWISS-MODEL and Alphafold2,and two binding sites of StKatG1 and manganese were predicted by using MIB website.Site-directed mutagenesis of the key amino acid residues Asp-63,Asp-65,Phe-66,and Asp-67 in the catalytic center of the enzyme and the Mn²⁺binding site were mutated to investigate their roles in StKatG1’s manganese oxidation activity.Combined with biochemical analysis data,it was demonstrated that aspartic acid residues in the manganese ion binding site are essential for StKatG1’s manganese oxidation activity,suggesting that electrostatic interactions may play an important role in the binding of Mn²⁺.In summary,the enzymatic properties of a novel manganese oxidase StKatG1 were revealed,and its molecular mechanism and structural basis for manganese oxidation were elucidated.（Ⅲ）Functional verification of catalase-peroxidase StKatG2:（1）gene recombination expression,isolation and purification,and enzymatic properties research:The stkat G2 gene was recombinantly expressed in E.coli BL21,and StKatG2 recombinant protein was obtained by separation and purification.Using Mn Cl₂ as substrate,combined with the detection data of the LBB method,it was proved that catalase-peroxidase StKatG2 has manganese oxidation activity.The optimum temperature for StKatG2 manganese oxidation is 52℃,and the optimum p H is 7.5.According to the catalytic kinetic analysis data,the Km of StKatG2 recombinant protein oxidized Mn Cl₂was 1.291 mmol/L,Vm was 10.30μmol/L/min,and Kcat was 2.78 min^-1.Its enzymatic properties differ slightly from StKatG1,but are different from other heme peroxides that have been reported.（2）Analysis of the catalytic product of the enzyme:The manganese oxidation product was observed to have a compact spherical structure by scanning electron microscopy,different from the product morphology of StKatG1;XPS results showed that the manganese oxidation product was a mixed valence manganese compound,and further XRD showed that the manganese oxidation product contained Mn O₂,Mn₂O₃,Mn₃O₄ and Mn C₂O₄.（3）Structure prediction and analysis:The three-dimensional structure of StKatG2 protein was predicted by SWISS-MODEL and Alphafold2,and the structural of StKatG1 and StKatG2 was compared using Py MOL software.The results showed that the structure of catalase-peroxidase StKatG2was significantly similar to that of StKatG1,but the differences inβ-folding and N-terminal flexible regions may explain the differences in enzymatic properties between StKatG2 and StKatG1.In summary,the enzymatic properties and structural basis of StKatG2 manganese oxidation,as well as its similarities and differences with StKatG1 were elucidated.（Ⅳ）Potential application of StKatG1 and StKatG2 in the degradation of malachite green:（1）StKatG1 has a decolorization effect on malachite green.The maximum decolorization rate of StKatG1 for 20 mg/L malachite green is 63.91%,and the maximum decolorization rate for 50mg/L malachite green is 51.78%.（2）StKatG2 has a decolorization effect on malachite green.The maximum decolorization rate of StKatG2 for 20 mg/L malachite green is 73.38%,and for 50mg/L malachite green,the maximum decolorization rate is 60.08%.（3）By comparing the degradation efficiency of malachite green by StKatG1 and StKatG2,it was found that under the same enzyme concentration conditions,StKatG2 had a stronger decolorization ability for malachite green（20 mg/L and 50 mg/L）relative to StKatG1.In summary,it was demonstrated that the manganese oxidation process of StKatG1 and StKatG2 can be coupled with malachite green degradation,providing a theoretical basis for their application in the degradation of organic pollutants.To sum up,this thesis elucidated the manganese oxidation function and molecular mechanism of the novel manganese oxidase StKatG1 and StKatG2 in the endophytic bacteria F01,and revealed the potential application of this type of enzyme in malachite green degradation.The research results will help expand the current academic understanding of the mechanism of bacterial manganese oxidation,and provide important data for a comprehensive understanding of the production mechanism of manganese oxidation in the rhizosphere of wetland plants and its application in environmental protection and other fields.