Study On The Oxidation Modes And Synergistic Effects Of The AA9 Family Of Polysaccharide Monooxygenases From Thermoascus Aurantiacus

As a renewable energy source that can replace fossil fuels,cellulose has great potential to alleviate energy stress and environmental pollution.Traditional methods of degrading cellulose are difficult to achieve efficient conversion of biomass.The conventional method of degrading cellulose is accomplished by hydrolyzing the crystalline region of cellulose using glycoside hydrolases,but this method is difficult to achieve efficient conversion of biomass.In recent years,the discovery of Polysaccharide monooxygenase(PMO),a copper ion-dependent enzyme that degrades cellulose by oxidation,has provided a completely new idea for efficient cellulose degradation.At the same time,it opens up opportunities for crop protection and food security because it can act as a virulence factor for pathogenic oomycetes.In this study,we used the AA9 family PMO enzymes of Thermoascus aurantiacus to identify the enzyme oxidation products,determine the oxidation mode,the effect of aromatic amino acids and active central amino acids in the substrate binding plane on the TaPMO enzyme,the synergistic effect of TaPMO enzyme and mutant enzyme with cellulase.The target gene Tapmo was successfully cloned from Thermoascus aurantiacus and the TaPMO enzyme was successfully expressed in Pichia pastoris.The TaPMO enzyme was reacted with the substrate phosphate expanded cellulose(PASC)at 50?and p H 5.0 for 48 h.The TLC results showed that the amount of product production was proportional to the reaction time and the reaction products were mainly cello-oligosaccharides with a degree of polymerization from DP2 to DP5.MALDI-TOF-MS results showed that not only cello-oligosaccharides were present in the oxidation products of TaPMO enzyme,but also C1-oxidized and C4-oxidized oligosaccharides.After the hydrolysis of the reaction product by trifluoroacetic acid(TFA),the results using HPLC-RID indicated the presence of C1-oxidized oligosaccharides in the oxidized product of TaPMO enzyme;reaction products after reduction by Na BH₄and hydrolysis by TFA,the results using HPLC-RID indicated the presence of C4-oxidized oligosaccharides in the oxidized product of TaPMO enzyme.The above results indicate that TaPMO enzyme functions as a C1 and C4 positions oxidation of substrate.The effect of aromatic amino acids on the substrate binding plane of TaPMO enzyme on the enzyme oxidation product and oxidation mode was investigated by targeted mutation of Tyr24,Phe43 and Tyr212 in the substrate binding plane.The results of TLC showed that Tyr24,Phe43 and Tyr212 had different effects on the enzyme activity,with Phe43 and Tyr212having a greater effect on the enzyme activity.The enzymatic products of Tyr24,Phe43 and Tyr212 all contained oxidation of C1 and C4 positions.The results of HPLC-RID analysis of the mutase reaction products showed that C1 and C4 oxidation products were present in the oxidation products of Tyr24,Phe43 and Tyr212.The above results indicated that the mutation of Tyr24,Phe43 and Tyr212 did not have a significant effect on the oxidation mode of TaPMO enzyme,but had a significant effect on the enzyme activity.After targeted mutation of the active central amino acids His1,His86 and His175,TLC analysis showed that no product production was detected,indicating that these three sites are essential amino acids for enzyme activity.Three cellulases were selected to determine the synergistic effect of TaPMO enzyme and mutant enzyme with cellulase.The results showed that TaPMO increased the degradation efficiency of EG2,CBH and BGL by 1.16,0.77 and 0.52 times,respectively,Y24A increased the degradation efficiency of EG2,CBH and BGL by 0.35,0.20 and 0.17 times,respectively,F43A increased the degradation efficiency of EG2,CBH and BGL by 1.57,1.09 and 0.95times,and the degradation efficiency of Y212A with EG2,CBH and BGL was increased by0.13,0.10 and 0.13 times,respectively.The results indicated that the degradation efficiency of TaPMO enzymes and mutant enzymes to different cellulases were improved to different degrees,and the synergistic effect with endocellulase EG2 was particularly prominent.Meanwhile,the improvement of cellulase degradation efficiency by mutase F43A was more obvious compared with TaPMO enzyme,and it can be tentatively concluded that the enzymatic activity of F43A was higher than that of TaPMO enzyme.