Studies On Regioselectivity Of AA9 Family Polysaccharide Monooxygenase From Chaetomium Thermophilum

Plant biomass is extremely abundant renewable carbohydrates on earth,and enzymatic degradation of biomass to glucose has great potential for biofuel production.This enzymatic degradation has low hydrolysis efficiency and the high cost of cellulases that are major barriers to economical biofuel production.Recently,a new class of cellulose-degrading enzymes,named Cu²⁺-dependent polysaccharide monooxygenases?PMOs?have been discovered in bacteria and fungi.Because of their oxidative degradation of cellulose dramatically boosts cellulase activity in cellulose hydrolysis,PMOs are attracting increasing interest.In this study,we focused on an auxiliary activity family 9?AA9?PMO from Chaetomium thermophilum with the methods of thin-layer chromatography?TLC?,matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry?MALDI-TOF-MS?,high-performance anion exchange chromatography with pulsed amperometric detection?HPAEC-PAD?,iodine oxidation,sodium borohydride?NaBH₄?reduction,bromine oxidation and site-directed mutagenesis,for intensive studying this AA9 PMO on its regioselectivity.In this thesis,cDNA of complete ctpmo1 gene was amplified from C.thermophilum.we expressed CtPMO1 protein heterologously.We determined the N-terminal amino acid sequence indicating that CtPMO1 was correctly processed and the nature N-terminal histidine residue was not methylated.We incubated CtPMO1 with insoluble substrate phosphoric acid-swollen cellulose?PASC?in pH5.0 for 48 hours at 50°C.TLC analysis of soluble reaction products showed that product concentration increased over time,and mainly produced cello-oligosaccharides with a degree of polymerization?DP?from DP2 to DP6.MALDI-TOF-MS analysis showed that the treatment produced oxidized oligosaccharides.MS/MS fragmentation of the highest peak m/z 525 from MALDI-TOF-MS analysis,showed the presence of nonoxidized fragmentation ions and C6 or C4-oxidized fragmentation ions,especially the only C6-oxidized fragmentation ions,indicating C4 and C6 oxidized products existed in CtPMO1 soluble reaction products.¹H NMR spectrum of CtPMO1 soluble reaction products that dissolved in DMSO-d₆ displayed an aldehyde proton signal at?8.39,indicating the presence of C6 oxidized products.For C1 oxidized products,we performed the method using trifluoroacetic acid?TFA?to hydrolyze CtPMO1 soluble reaction products followed by HPAEC-PAD analysis.In this study,a simple and effective new method of using Br₂ to oxidize CtPMO1 soluble reaction products was developed to directly identify C4 and C6-oxidized products by MALDI-TOF-MS,and detected the ion peaks m/z+28 and m/z+30 which means C4 and C6 oxidized products respectively.In order to examine the effectiveness of new method,we performed the method using NaBH₄ to reduce CtPMO1 soluble reaction products followed by hydrolysis with TFA,to determine C4 oxidized products.In conclusion,CtPMO1 can oxidize C1,C4 and C6 position on cellulose.CtPMO1 insoluble reaction products?residual PASC?was hydrolyzed by endo-1,4-?-D-glucanase followed by the new method analysis.The result showed that C1 and C4 oxidized products presented in CtPMO1 insoluble reaction products,and C6 oxidized products are minor on CtPMO1 insoluble reaction products.We incubated CtPMO1 with cellopentaose and celluheptaose in pH5.0 for 48 hours at50°C.TLC analysis showed that CtPMO1 can degrade these two soluble cello-oligosaccharides,especially celluheptaose.MALDI-TOF-MS analysis showed that not only reduced oligosaccharides,oxidized oligosaccharides also existed in enzymatic reaction products,indicating that CtPMO1 oxidize C1 and C4 or C6 position on celloheptaose.After Br₂ oxidizing enzymatic solution products,MALDI-TOF-MS analysis showed that CtPMO1 oxidized C4 and C6 position on celloheptaose,demonstrating C6 oxidation were likely occurred on soluble oligosaccharides.Site-directed mutagenesis of four aromatic amino acid Tyr27,His64,His157 and Tyr206on the substrate binding flat plane was carried out to study regioselectivity of CtPMO1.TLC analysis showed that mutants have varied activities.His64 and His157 affected enzyme activity obviously,and then Tyr206,but Tyr27 had no significant influence to enzyme activity.MALDI-TOF-MS analysis showed that the construction of enzymatic reaction products also changed,demonstrating His157 is required for activity of CtPMO1 with its mutant lost all enzyme activity;His64 plays a key rule to C1 and C4 or C6 oxidation of CtPMO1;Tyr206affects enzyme activity with lose of partical activity;but Tyr27 had no significant influence to product construction.After Br₂ oxidizing mutant enzymatic solution products,MALDI-TOF-MS analysis showed that Tyr27 did not effect C1/C4/C6 oxidation of CtPMO1;His64 effected C4 and C6 oxidation;Tyr206 effected C6 oxidation.The analysis of mutants insoluble also supported the results of CtPMO1 regioselectivity.Furthermore,primary research of other residues on CtPMO1 plane surface were performed.TLC analysis showed that there was no products after mutants of His1,His83 and Tyr168incubating with substrate,which play an important role on CtPMO1 activity.Mutant of Gln166to alanine lost all activity,but to glutamic acid performed the different cleavage from wild type,suggesting this site was related to cleave pattern of CtPMO1.Sequence alignment and phylogenetic tree showed that C6 oxidation PMOs including CtPMO1 were clustered three clades,corresponding to the reported three PMO types respectively.Taken together with results above,we suggested that there should be C1/C6,C4/C6 and C1/C4/C6-oxidizing PMOs in AA9 family.