Study On Oxidation Properties And Functions Of Polysaccharide Monooxygenase From Chaetomium Thermophilum

As human society entered the era of industrialization,fossil fuels were pushed to the edge of exhaustion.Facing to this“stumbling block”that is hindering the progress of human society,we have to accelerate the transformation of the energy mix and find new renewable energy sources.Lignocellulosic is one of the most abundant renewable resources on earth,and cellulose is the most abundant component.Cellulose,as the main component of plant cell wall,provides a physical barrier for the invasion of plant pathogens.Facing this stubborn crystal structure,cellulase alone is difficult to achieve efficient degradation.Later,the discovery of polysaccharide monooxygenase（PMOs）captured the attention of many researchers in the processes of finding new auxiliary enzymes.PMOs oxidatively cleave glycosidic bonds,so as to destroy the crystal structure of polysaccharides.Therefore,the study of PMOs not only provides an important means of biomass degradation,but also provides an important basis for the study on pathogenic mechanism of pathogenic bacteria.The enzyme activity of polysaccharide monooxygenase CtPMO9A from Chaetomium thermophilum was detected by a series of methods like thin-layer chromatography（TLC）,high-performance liquid chromatography-refractive index detector（HPLC-RID）,matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry（MALDI-TOF-MS）,trifluoroacetic acid（TFA）hydrolysis,methylation,sodium borohydride（Na BH₄）reduction and site-directed mutation.And the synergism of CtPMO9A with cellulase（CBHI、BGLI and EGⅡ）was detected by 3,5-dinitrosalicylic acid（DNS）assay.In order to examine the ability of CtPMO9A to degrade phosphate expandable cellulose（PASC）,we set up a series of reaction temperatures（40,50,60,70℃）and p H（3.0,4.0,5.0,6.0,7.0）.We used TLC analysis to dicover the non-oxidized oligosaccharides in the products of enzymatic hydrolysis.And then in order to detect C1 oxidation product（gluconic acid）,we performed the method using trifluoroacetic acid（TFA）to hydrolyze CtPMO9A soluble reaction products followed by HPAEC-PAD analysis.The results showed that the optimal temperature and p H of CtPMO9A were 50℃and 5.0,respectively.Under the optimal reaction conditions,we detected the presence of C4 oxidation products（galactose）by HPLC-RID when soluble reaction products were reduced by sodium borohydride.Meanwhile,the oxidation oligosaccharides of C1（m/z+30）and C4（m/z-16）were detected after using MALDI-TOF-MS to analyze soluble reaction products treated by methylation.These results indicated that CtPMO9A can oxidize C1 and C4 position on cellulose.Furthermore,active-cite residues（His1,His83 and Tyr168）of wild-type CtPMO9A enzyme（WT）were mutated by site-directed mutagenesis to form three mutated enzymes,H1A,H83A and Y168A,and reacted with substrate PASC.The results were that no non-oxidized oligosaccharides were detected by TLC analysis of soluble reaction products,and no C1 and C4 oxidation products were detected by HPLC-RID.We concluded that the enzyme activity completely lost as CtPMO9A activity center sites（His1,His83 and Tyr168）were mutated.At the same time,we also mutated the 166th glutamine（Gln166）near the active center into Q166A and Q166E.TLC analysis showed that products of Q166A were no oxidized oligosaccharide,but products of Q166E had glucose to cellobiose.For the mutated enzyme Q166A,both HPLC-RID and MALDI-TOF-MS did not detect oxidized oligosaccharide.However,C1 oxidation products were detected in the products of mutant enzyme Q166E.So the analysis results showed that Q166A lost the oxidation activities of C1and C4,while Q166E retained part of C1 oxidation activities.In conclusion,amino acids at the active center play key roles in the enzymatic activity of CtPMO9A,and Gln166 near the active center not only participates in the bond breaking reaction but also is substitutable.The synergistic action of CtPMO9A with cellulase（EGⅡ,BGLI and CBHI）was investigated.It was found that pretreatment of phosphoric expandable cellulose（PASC）with CtPMO9A increased the reducing sugar yield by 2.10,2.08 and 2.16 times,with the extent of synergism being 1.022,0.799 and 0.875,respectively.When CMC-Na was treated with CtPMO9A and EGⅡ,BGLI and CBHI,the reducing sugar yield increased by 0.94,3.38 and1.55 times,respectively,and the extent of synergism were 0.751,0.932 and 0.804.However,the synergistic catalytic effect of CtPMO9A with BGLI was not good in corn cob treatment,but only the reducing sugar yield of CtPMO9A with EGⅡand CBHI treatment were increased by 0.64 and 0.21 times,and the extent of synergism were 1.009 and 0.612.In order to optimize the reaction ratio of the mixed enzyme,the synergistic effect of CtPMO9A with EGⅡon PASC were detected respectively by DNS assay and HPLC-RID.The results showed that when EGⅡaccounted for 0.4%of the mixed enzyme system,the reducing sugar yield increased 5.41 times compared with the substrate treated by cellulase alone,and the contents of gluconic acid were the highest.In general,pretreatment of synthetic cellulose（PASC,CMC-Na）and natural cellulose（alkali-treated corn cob）with CtPMO9A improved the degradation efficiency of cellulase to varying degrees,and the proportion of mixed enzymes also impacted the effect of PASC degradation.