Identification Of Oxidative Products Of AA9 Family Polysaccharide Monooxygenase From Scytalidium Thermophilum

Cellulose and chitin are the two most abundant biomass resources on earth,they have great potential for application in converting into clean energy,and these are also of great significance in solving current energy shortage and environmental pollution.Traditional cleansing utilization of cellulose and chitin is accomplished by glycoside hydrolase degradation,but the catalytic efficiency of glycoside hydrolases for the crystalline regions of the substrate is very low,severely limiting the efficient use of biomass.Recently,scientists have discovered a new class of cellulose-degrading enzymes,a copper ion-dependent polysaccharide monooxygenase(PMO)and belongs to the auxiliary activity family 9(AA9).PMO can oxidize and cleave the polysaccharide chains of cellulose with vitamin C,and significantly increase hydrolysis efficiency,which has attracted widespread attention.In this study,we cloned a gene of auxiliary activity family 9(AA9)from Scytalidium thermophilum named pmo7651 and successfully expressed the enzyme PMO7651 in Pichia pastoris,with the methods of thin-layer chromatography(TLC),matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF-MS),bromine oxidation and site-directed mutagenesis,for intensive studying PMO7651 on enzyme activity determination,enzymatic products component identification,oxidation mode construction,effect of substrate-bound aromatic amino acids on enzyme activity and enzymatic product components.In addition,the synergistic effect of the PMO7651 enzyme on other cellulases was also determined.The PMO7651 enzyme was reacted with the insoluble substrate phosphoric acid-swollen cellulose(PASC)at pH 5.0,50°C and 200 rpm for 48 h.The TLC analysis showed that the amount of products formed gradually increased overtime,and mainly produced cello-oligosaccharides with a degree of polymerization(DP)from DP2 to DP5.MALDI-TOF-MS analysis showed that in addition to cellooligosaccharides,the PMO7651 enzymatic products also contained oxidized oligosaccharides,oxidized oligosaccharides containing oxidized oligosaccharides at the C1,C4 or C6 positions.However,since the C4 and C6 positions have the same mass spectral peak,they need to be further identified by other methods.In order to further determine the oxidation of the C4 or C6 position of PMO7651,the enzymatic hydrolysate was oxidized with saturated bromine water for MALDI-TOF-MS analysis,and m/z+14,m/z+28 and m/z+30 were detected.The peak value of the PMO7651 is determined to be simultaneously oxidized at the C1,C4,and C6 positions.In order to study the effects of aromatic amino acids Tyr40,His171 and Phe181 on the PMO7651 substrate binding plane on enzyme activity and its products,site-directed mutagenesis was performed on these three points to obtain three mutant enzymes.The mutant enzyme was subjected to enzymatic hydrolysis,and the TLC results showed that Tyr40 and Phe181 could detect the oligosaccharide produced by the reaction,while His171 did not detect the obvious products.MALDI-TOF-MS analysis showed Tyr40 retained part of the oxidation activity at the C1,C4/C6 positions,His171 lost the oxidation at the C4/C6 position,and Phe181 substantially retained the oxidation at the C1,C4/C6 positions.This indicates that the three aromatic amino acid site mutations on the substrate binding plane have different effects on the activity of the enzyme.In this experiment,three different cellulases were selected to determine the synergistic effect of PMO7651 on cellulases,the results showed that the degradation efficiency of EG1 by PMO7651 was increased by 1.03 times;Similarly,the degradation efficiency of CBH1 was increased by 0.42 times;the degradation efficiency of BGL1 was increased by 0.18 times,which indicated that the enzymatic hydrolysis efficiency was improved at different degrees with the enzyme of PMO7651.