Enzymatic Properties And Functions Of Lytic Polysaccharide Monooxygenases From Brown-rot Fungi

Brown-rot fungi are common decomposers in forest ecosystems and can oxidize lignocellulose using a non-enzymatic Fenton reaction(Fe²⁺+H₂O₂?·OH+OH^-+Fe³⁺).In recent years,it has been found that lytic polysaccharide monooxygenase(LPMO),which is widely present in wood rot fungi,can oxidize a series of cellulose,hemicellulose and other polysaccharides driven by external electron donors such as ascorbic acid and bisphenol,and promote the hydrolysis of polysaccharides,but the characteristics and function of brown-rot fungal LPMO is rarely reported.Therefore,the AA9 and AA14 family LPMO derived from brown-rot fungi were investigated in this study.Three LPMOs recombinant proteins obtained by heterologous expression were systematically compared.On the one hand,the oxidation characteristics and enzymatic synergistic effects of the three LPMOs on different polysaccharide substrates.O n the other hand,the relationship between Fenton reaction and LPMO oxidation reaction was preliminarily studied.The main results are as following:Three LPMO genes were obtained from the brown-rot fungi Serpula lacrymans and Laetiporus cremeiporus.Using the Escherichia coli expression system,three LPMOs recombinant proteins were obtained:SlLPMO9A,SlLPMO14A,LcLPMO14A.None of them contains C BM binding domains.Among them,SlLPMO9A is the LPMO of the AA9family,SlLPMO14A and LcLPMO14A are hypothetical proteins,and their structure and sequence have high homology with the LPMO of the AA14 family.Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF/TOF MS)was used to study the oxidation products of polysaccharides under the three LPMOs and evaluate the oxidation properties of LPMOs.The results show that SlLPMO9A belonging to the AA9 family LPMO can oxidize cellulose;SlLPMO14A and LcLPMO14A can oxidize xylan in poplar,but has no activity against free xylan,which is confirmed to be the AA14 family LPMO;when SlLPMO14A and LcLPMO14A oxidize masson pine,not only oxidation products of xylan cleavage were detected,and oxidation products suspected to be cleaved from mannan were found.SlLPMO9A,SlLPMO14A and LcLPMO14A can promote the hydrolysis of cellulose,glucose content increased by 46.70%,11.14%,11.71%.Using the biosynthetic cellulose-hemicellulose membrane as a substrate,xylan and mannan loaded on cellulose can be oxidized by SlLPMO14A.This is the first time to confirm that the AA14 family LPMO has the characteristics of oxidized mannan.The degradation products of lignin via Fenton reaction in vitro can be used as the electron donor of LPMO,driving LPMO to oxidize polysaccharides and enhance the hydrolysis of cellulose.GC-MS analysis showed that the Fenton reaction resulted in the production of hydroquinones such as 1,4-hydroquinone,2,5-dihydroxybenzaldehyde and 2,5-dihydroxyphenylacetic acid.Biochemical analysis proved that hydroquinones can reduce LPMO,and 2,6-dimethoxy-1,4-hydroquinone(DBQH₂)has the strongest reducing ability,which can drive AA9 and AA14 family LPMO to oxidize the corresponding polysaccharide substrate.Thereby the role of hydroquinones products in Fenton reaction-oxidized lignin in driving LPMO to oxidize polysaccharides was confirmed.Additionally,LPMO can promote the Fenton reaction mediated by hydroquinones in brown-rot fungi.Addition of SlLPMO9A,SlLPMO14A and LcLPMO14A into the DBQH₂-mediated Fenton reaction system,H₂O₂concentration increased by 2.48 times,1.86 times and 2.04 times,the reduction of Fe³⁺increased by 1.63 times,1.25 times and 1.5 times,and the·OH content increased by 1.85times,1.61 times and 1.56 times,respectively.In summary,the brown-rot fungal LPMO can oxidize a series of polysaccharide substrates,such as cellulose,xylan,mannan,etc.At the same time,the LPMO oxidation reaction is coupled with the extracellular Fenton reaction under the help of hydroquinone compounds.This study has important theoretical significance for the study of the characteristics and function of brown-rot fungal LPMO.