Study On The Activity,structure And Biological Function Of Fungal Lytic Polysaccharide Monooxygenase

Cellulose,as the most abundant renewable resource on Earth,holds significant importance due to its utilization and bioconversion potential.The enzymatic liquefaction of cellulose is a major scientific challenge in the field of cellulose degradation research.The cellulases in glycoside hydrolases degrade cellulose by hydrolysis and play a key role in the liquefaction of cellulose into glucose.A recent advancement in enzymatic cellulose breakdown involves the identification of lytic polysaccharide monooxygenase（LPMO）.LPMO operates by oxidatively breaking down cellulose,resulting in a significant enhancement in the effectiveness of cellulase enzymes.This discovery holds significant scientific importance and also has potential value in the field of cellulolytic enzymes.LPMOs in the Carbohydrate-Active En Zymes database（CAZy）are classified as Auxiliary Activity（AA）oxidoreductases.There are a total of 17 families of AAs that have been found.Among these families,four specific families,namely AA9,AA10,AA15,and AA16,contain LPMOs capable of catalyzing the rupture of cellulose glycosidic bonds,of which the AA9 LPMO is derived from fungi.Currently there is a lack of accurate activity assays for AA9 LPMO,few structural resolution studies of the natural LPMO,and biological functions in vivo of AA9 LPMO are unclear.This study focuses on AA9 LPMO as the object of investigation,analyzing its oxidative activity,structure and enzyme activity,and its biological functions within the termite gut.The main results of the study are as follows:The protein matching AA9 LPMO from Thermoascus aurantiacus was purified using anion exchange ion chromatography and molecular sieve chromatography.Protein identification was performed using LC-MS/MS mass spectrometry as an AA9 LPMO（nTaAA9A）.The specific AA9 LPMO identified is denoted as nTaAA9A.nTaAA9A and the substrate PASC were subjected to a 48-hour reaction at 50℃,and the resulting products were analyzed using thin-layer chromatography（TLC）,matrix-assisted laser desorption ionization massspectrometry（MALDI-TOF-MS）,andhigh-performanceliquid chromatography-refractive index detector（HPLC-RID）.The results highlighted the enzyme’s effectiveness in breaking down cellulose using an oxidative process,which led to the creation of C1 and C4 oxidation byproducts.Additionally,TLC,MALDI-TOF-MS,and HPLC-RID examination of the reaction products between nTaAA9A and xylan substrate revealed that nTaAA9A has the ability to break down xylan and generate C1-and C4-oxidized xylo-oligosaccharides.Subsequent investigations demonstrated that nTaAA9A maintained84.3%,63.7%,and 35.3%of its functionality following incubation at temperatures of 60℃,70℃,and 80℃for a duration of 30 minutes,respectively.This suggests that nTaAA9A possesses remarkable resistance to high temperatures,indicating its considerable thermal stability.We have devised a technique to evaluate the activity of AA9 LPMOs of thermophilic fungi using HPLC-RID.The specific activities of nTaAA9A C1 and C4 oxidation were analyzed using the method and found to be 0.646 and 0.574 U/mg,respectively.We utilized this approach to assess the oxidative activities of the heterologously generated recombinant enzyme（rTaAA9A）at C1 and C4 positions,in order to confirm its effectiveness.The rTaAA9A enzyme exhibited C1 oxidation activity of 0.155 U/mg and C4 oxidation activity of0.153 U/mg.Overall,this method allows for the measurement of LPMO activity in thermophilic fungi and focuses on C1 and C4 oxidation.It also enables a comparison between the two,providing an efficient assay method for AA9 LPMO in thermophilic fungi.X-ray crystallographic diffraction imaging was used to further analyze the structure of nTaAA9A.nTaAA9A has aβ-sandwich fold comprising two twisted antiparallelβ-sheets linked by loops of different lengths and conformations.The active site is situated on a planar area of the molecule exposed to the solvent.A Cu²⁺ion participating in the catalytic process was detected at the N-terminal,confirming prior observations and indicating a distinct and strongly bound ion.The residue His1,involved in coordinating Cu²⁺,was discovered to be methylated.Comparison with the heterologously expressed recombinant enzyme（rTaAA9A）revealed that His1 of the rTaAA9A was not methylated.Also,in Asn138 owing to the different glycosylation in that residue.Asn138 was glycosylated with at least two NAG molecules as found in the crystal structure.In rTaAA9A,there is only one NAG molecule attached after deglycosylation of the expressed rTaAA9A.Close inspection revealed different orientation for the side chain of Asn138 and,consequently,the position of the glycan moieties.In nTaAA9A,the two NAG molecules can sit on in a shallow groove.In contrast,the NAG molecule in rTaAA9A is pointing outwards and is exposed to the solvent.Currently,most of the studies on AA9 LPMOs focus on the degradation of cellulose,and their biological functions in organisms are unknown.Termites are a wood-eating insect that harms many kinds of forest trees,fruit trees,crops and other plants,which seriously affects forestry and agricultural production,so termite was chosen as the research object to study the biological function of AA9 LPMO in organisms.We used TLC,MALDI-TOF-MS,and HPLC-RID methods to analyze soluble oligosaccharides and insoluble cellulose extracted from the gut of the termite C.declivis.Our analysis confirmed the presence of C1-and C4-oxidized cello-oligosaccharides in the termite’s gut,indicating the existence of a mechanism for oxidative degradation of cellulose in the termite C.declivis gut.Eleven species of fungi were isolated from the gut of C.declivis by isolation and purification,and they belonged to nine genera in the phylum Ascomycota and Stramenophora,one of which was an unspecified species and possibly a new species.These results suggest that fungi are symbiotic microorganisms in the termite gut,with Ascomycota fungi being the dominant fungal community.Through a comparison with the CAZy database,we identified the presence of AA9 LPMO in three fungal species:Fusarium solani,Pleurotus ostreatus,and Talaromyces verruculosus.These findings show that the gut of the termite C.declivis has a diverse range of fungi that are capable of oxidative degradation of cellulose.One of these fungi was frequently isolated and morphologically and molecularly identified as Talaromyces funiculosus,which was detailed investigation due to its extensive distribution and the fact that its genome has not yet been sequenced.The genome of T.funiculosus was sequenced and added to the NCBI database under the accession number PRJNA508439.Upon comparison with the CAZy database,it was discovered that T.funiculosus possesses a total of 380 CAZymes.Out of the total,38 genes are classified under the AA family,whereas only one gene falls into the AA9family.The protein produced by this gene has been designated as TfAA9A.RNA was isolated from the gut of the termite C.declivis and reverse transcription polymerase chain reaction（RT-PCR）was conducted.RT-PCR research demonstrated the expression of TfAA9A in the gut of the termite C.declivis,indicating that TfAA9A was involved in the oxidative cleavage of cellulose in the gut of C.declivis.TfAA9A was then heterologously expressed in Pichia pastoris,and the ability of the TfAA9A enzyme to oxidatively degrade PASC was demonstrated by TLC,MALDI-TOF-MS,and HPLC-RID methods,further supporting the presence of oxidative degradation of cellulose in the termite gut.Due to the limitations of the culture method,the fungi in the gut of C.declivis were further analysed by ITS sequencing,which showed that a variety of fungi were identified from the gut of C.declivis,and that the fungi of the phylum Ascomycota were the dominant fungi.We conducted a comparison between the data obtained from ITS sequencing and the CAZy database.The data presented demonstrates that the gut of the termite C.declivis contains a diverse range of fungi that carry AA9 LPMO capable of oxidatively cleavage cellulose.Additional transcriptome and proteomics analysis revealed the presence of expressed genes and proteins of fungus AA9LPMO in the gut of the termite C.declivis.The above results indicate the existence of a mechanism for oxidative cellulose degradation by AA9 LPMO in the gut of C.declivis,and also elucidate that AA9 LPMO has a biological function of cellulose degradation in the organism.In conclusion,this study demonstrated that AA9 LPMO can degrade cellulose through oxidation,both in vitro and within the gut of C.declivis.These findings lay the groundwork for further research into the biological functions of AA9 LPMO and offer insights for potential practical applications of LPMO.