Intermolecular Interaction And Catalysis Of Laccase-natural Mediator Noncovalent Complex System

Lignin is a potential source of value-added chemicals of aromatic compounds.Laccase(LAC)is widely used in the modification and separation of lignin.LAC-mediator system(LMS)can be used in the oxidation of various compounds.Although a large number of studies have been carried out on lignin treated by LMS,the formation mechanism of LMS and the transformation pathway of lignin are still unclear,and the process of LMS modified lignin structure is not fully understood.In this paper,G-type lignin(GL)with strong affinity was used to form LMS with LAC.The internal interaction of LMS was investigated by fluorescence spectrometry.The mechanism of binding and mutation of Lac protein structure was clarified.The binding mode of LMS at molecular level was studied in depth to explore the effect of different GL mediators on the formation of LAC catalytic products.It is a supplement to the mechanism of enzyme catalysis and a reference for lignin biotransformation.It is speculated that the degradation pathway will provide the possibility for the conversion of lignin into valuable special chemicals used in various industries,consumption and pharmaceutical applications.The main research contents are as follows:(1)The binding model of LAC-sinapyl alcohol(SA)and the oxidation pathway of the mediator were studied.The changes of SA in the process of enzymatic was evaluated by molecular simulation,GC-MS,NMR and HPLC.These methods can explain the conformational change of LAC by SA binding;determine the binding model of LAC-SA;after identifying the main product 2,6-dimethoxy-1,4-benzoquinone(DMQ),the mechanism of the phenolic mediator SA participating in the oxidation reaction was determined;the effect of the initial concentration of SA on the DMQ yield was studied.The research shows how mediator can combine with lac to form a high active intermediate,which provides important information for further understanding of biodegradation process.(2)Preparation of LMS,fluorescence spectroscopy(FS),infrared,electrochemical analysis combined with theoretical modeling to study the intermolecular effects of guaiacol/ 4-ethylguaiacol/ vanillin-LAC non-covalent system.The binding parameters,electron transfer,and protein secondary structure changes between LAC-GL mediator non-covalent composite system(LGS)after GL binding LAC.The results show that the GL and same cavity of LAC is closely combined to form a composite system;the existence of GL increases the electron transfer;the LAC secondary structure spiral state and folded state are mutually transformed;molecular modeling shows that GL and LAC's HIS11,PRO346,LUE459 formed a precise non-covalent composite system through hydrogen bonding and hydrophobic interaction.(3)With syringyl alcohol(LA)as substrate,fluorescence spectrum(FS)and molecular dynamics simulation(MD)were used to study the binding of the substrate to LAC.The products and mechanism of LGS catalysis were studied by GC-MS and HPLC.The catalytic effect of GLS on dealkaline lignin(DL)was studied.The results show that the binding force between LA and LAC is weak,and the active cavity is more hydrophilic.The main product of LGS enzymatic hydrolysis lignin model substrate is the same as the product catalyzed by LAC,but the former has a catalytic rate twice that of LAC.It is inferred that the mechanism of LGS enzymatic hydrolysis is that the formation of non-covalent complex accelerates the electron transfer rate of LGS and LA,forming a subsequent reaction.For DL,LGS still showed higher catalytic efficiency than LAC.