The Role And Its Mechanism On Lignin Degradation By Bacteria-derived Laccase And Dye-decoloring Peroxidase DyP35

Lignin is an amorphous biopolymer connected by phenylpropane structures through C-C and C-O-C bonds.It is the most abundant renewable aromatic compound resource in nature.The degradation,bioconversion and utilization of lignin are the challenges faced by papermaking industry,straw resource utilization,and bioremediation of environmental pollution.Lignin is difficult to degrade due to its complex and recalcitrant,high molecular weight,and insolubility in water,which limits its valorization.Compared with physical and chemical methods,the bioconversion of lignin into high value-added platform compounds has the advantages of low cost and less secondary pollution,so it has attracted more and more attention.Bacteria and their secreted enzymes have the characteristics of easy gene manipulation and protein expression.And bacteria have rich biodiversity and good environmental adaptability.Therefore,the study of bacteria and their lignin-degrading enzymes has become an international hot spot.In recent years,more and more studies show that laccase and dye-decoloring peroxidase(DyPs)of bacterial origin play a vital role in lignin biodegradation.However,the depolymerization reaction mechanism of these two enzymes on lignin has not been fully elucidated,which restricts its application in the depolymerization of lignin.Therefore,clarifying the biocatalytic mechanism of bacterial laccase and DyPs on lignin will provide an important theoretical basis for the industrial production of lignin biodegradation.In this dissertation,the laccase(Lacc)gene from Bacillus ligninphilus L1 and the dye-decoloring peroxidase(DyP35)gene from Comamonas serinivorans C35 were heterologously expressed.The enzymatic properties of Lacc,LMS(Lacc mediator system)and DyP35 were characterized,and their modification mechanisms to lignin were elucidated.The main research results are as follows:(1)Two mutant strains E.coli BL21(Lacc)and E.coli BL21(DyP35)which harboring Lacc and DyP35 genes were obtained.The molecular weight of the Lacc is about 57 kDa,and the maximum specific activity is 201.57±0.53 U/mg;the molecular weight of the DyP35 was about 36 kDa,and maximum specific activity reached 317.87±0.25 U/mg.(2)Laccase played a key role in the degradation of lignin by strain L1.Compared with wild strain L1,the degradation rate of lignin by mutant strain L1(Lacc inactivated)was significantly reduced.The removal rate of mutant strain L1 to alkali lignin from 38.05% reduced to 15.43%,and the decolorization rate from 33.05% reduced to 7.52%.Similar,the removal rate of mutant strain L1 to milled wood lignin from 40.01% reduced to 16.39%,and the decolorization rate from 30.04% reduced to 6.67%.(3)The enzymatic properties analysis showed that the optimal reaction pH of Lacc was 3.0 and temperature was 37°C with ABTS as substrate,and the optimal reaction pH of DyP35 was 4.0 and temperature was 37°C.Lacc and DyP35 both showed excellent heat resistance(20-70? and 20-50?,respectively)and pH stability(2.0-5.0).(4)The modification mechanisms of lignin by Lacc and LMS were summarized as follows:(a)C-C coupling,(b)introduction of hydroxyl group into benzene ring,(c)methoxylation reaction to form methoxy group,(d)demethylation and decarboxylation,(e)reduction,(f)cleavage of cross-linking bonds(such as 4-O-5',?-5' and 5-5')and the removal of G,S and H-lignin units,(g)oxidizing the hydroxyl groups on the C? of the lignin side chain and forming carbonyl groups,(h)aliphatic C-C bonds and lignin side chain cleavage.(5)The modification mechanisms of lignin by DyP35 were summarized as follows:(a)attack the side chain structure on the benzene ring(such as ?-O-4,C = O and C-C bond),(b)removal of methoxy groups increases the degree of hydroxylation of lignin,(c)removal of G,S and H-lignin units,(d)cleaves lignin into low-molecular aromatic compounds,and the side chains of low-molecular aromatic compounds are further oxidized and modified into p-hydroxycinnamic acid and p-hydroxyphenylpropionic acid,vanillic acid and ferulic acid and other simple aromatic organic acids.In conclusion,this study comprehensively clarified the modification mechanism of Lacc degradation of lignin,described the reaction mechanism of Lacc oxidation of lignin,which provided some new insights for the catalytic mechanism of laccase,and further laid a foundation for the application of laccase towards a value-added lignin product.At the same time,the preliminary modification mechanism of DyP35 to lignin was also revealed,which may suggest a potential unique pathway for the biological refining of lignin substrates.This investigation may indeed provide an important guidance or option applied in the bio-refinery industry of biomass.