Modif-ication Of Leishmania Major Peroxidase And Study On Its Multi-Enzyme System

Leishmania major peroxidase（LmP）is a heme peroxidase that consumes H₂O₂ with cytochrome C as the substrate.In this study,a number of reconstructed vectors containing LmP gene fragments derived from Leishmania,major strain Friedlin were constructed.Firstly,the fusion protein expression vector containing ELP tag and small ubiquitin-related modified protein SUMO tag were constructed by molecular biology.The change of the properties of the fusion protein was explored.The purification of three proteins without ELP tag was carried out by Ni-NTA metal chelate chromatography to achieve the desired purity.6ELP-LmP and LmP-6ELP protein with 3M Nacl conditions with two or three times ITC method to achieve the desired effect,to reference the simple purification of the label difficult to reduce operating costs.After modification,the enzyme activity was not affected negatively,and the catalytic rate even increased.The protein was characterized by circular dichroism and fluorescence spectroscopy.After the addition of H₂O₂,the protein with the smallest change in structure was ELP tagged.The Ksv values of the Stern-Volmer equation were measured by fluorescence spectroscopy,and the ELP tag was used to prove the stability of the LmP protein.The role of SUMO label is to help increase the solubility of proteins.The same conditions to express and purify LmP-SUMO and LmP,the former concentration is 10 times the latter,SUMO label solubilization effect is strong.The LmP-DAAO complex enzyme system was then established by fusion of LmP and D-amino acid oxidase.This way has improved the DAAO enzyme activity,which has a certain reference value in the industrial application of the enzyme.Finally,LmP and 5-hydroxymethylfurfural oxidase reaction system were directly mixed in vitro to improve the yield of product 2,5-furan dicarboxylic acid FDCA,which was of reference value in industry.