| Biomass is a kind of production material and energy which could replace fossil resources.Due to its diversity and structural complexity,the degradation of plant biomass faces great challenges.In order to effectively degrade plant cell walls,many strategies,such as enzyme strategies,have been derived by microbes.A major breakthrough in this area was the discovery of a polysaccharide monooxygenase,which can catalyze the oxidative cleavage of stubborn polysaccharides and allow classical hydrolases to degrade biomass more efficiently.PMOs are a class of copper ion-dependent enzymes,which was originally classified as the GH61 family of sluggish glycoside hydrolases in fungi or the CBM33 family of carbohydrate binding modules that play a non-catalytic role in bacteria..In 2010,they were reclassified as AA9 or AA10 of the co-active enzyme family because of its ability to oxidize cellulose and chitin,and were collectively referred to as lytic polysaccharide monooxygenase or polysaccharide monooxygenase.Currently,PMOs are classified as the coactive enzymes in the carbohydrate active enzyme CAZy database,which form the AA9,AA10,AA11,AA13,AA14,AA15 and AA16 families.This study selected PMO enzymes from the AA9(Auxiliary Activity family 9)of Humicola insolens as the research objects.The Hi PMO1 gene was cloned from the AA9 family of Humicola insolens,and expressed in Pichia pastoris successfully.Then Hi PMO1 pure enzyme was obtained by nickel column affinity chromatography.The n-terminal sequencing of the enzyme identified that the first amino acid at the n-terminal was histidine,which was not modified by methylation.Using the methods of thin layer chromatography(TLC),matrix-assisted laser desorption ionization mass spectrometry(MALDI-TOF-MS),high performance liquid chromatography(HPAEC-PAD),bromination,enzymatic hydrolysis,and homology model,the composition and function of Hi PMO1 oxidation products have been studied intensively.With the presence of electron donor,Hi PMO1 enzyme reacted with insoluble substrate phosphoric acid-swollen cellulose(PASC)and soluble substrate xylodextran,respectively,at p H 5.0 and 50? for 48 h,and the soluble product of Hi PMO1 enzymewas analyzed.The TLC results showed that Hi PMO1 could degrade PASC and xyloglucan,and its ability to degrade xyloglucan is significantly lower than that of PASC degradation.In the process of oxidation cleavage of PASC,,the content of oxidation products increases with the increase of reaction time,and the products mainly consist of cellobiose to cellohexose.MALDI-TOF-MS analysis showed that the reaction products mainly consisted of non-oxidized cellooligosaccharides and oxidized cellooligosaccharides,and there were C1(m/z+16),C4 or C6(m/z-2)oxidized oligosaccharides.In addition,it has been found that there might be a C6-position oxidized to form a cellooligosaccharide containing glucuronic acid(m/z +12,+14,+28).In order to distinguish the C4 or C6 oxidation products,the(m/z-2)peak was analyzed by MALDI-TOF-MS/MS.The results showed that in addition to the fragment ion peaks of a large number of non-oxidized products,there were fragments ion peaks of C4 or C6 oxidation products and it is only possible to have oxidized fragment ions at the C6 position,indicating that C4 and C6 oxidation products may be present in the Hi PMO1 enzyme oxidation product.To accurately distinguish the C4 and C6 oxidation products,the oxidation products were analyzed after they were oxidized with bromine water by MALDI-TOF-MS.The results showed that there existed mass spectrum peak of(m/z+26),(m/z+28),(m/z+30)and(m/z+44),confirming the presence of C4 and C6 oxidation products at the same time.In summary,the Hi PMO1 enzyme has a function of oxidizing C1,C4 and C6 on the substrate PASC,and it may oxidize the C6 position to form a cellooligosaccharide containing glucuronic acid.The MALDI-TOF-MS results of the Hi PMO1 enzyme oxidation product(m/z +12,+14,+28)suggested that there might be a C6-position oxidized to form a cellooligosaccharide containing glucuronic acid.To this end,a new method was used to further analyze the soluble oxidation product.The soluble oxidation products were further digested with ?-glucuronidase and ?-glucosidase,and the enzymatic hydrolyzed products were subjected to LC-MS(full scan and single ion detection)and HPAEC-PAD detection.LC-MS results showed that there were mass spectrometry peaks of glucose,gluconic acid,glucuronic acid(glucuronolactone)and saccharic acid(gluconolactone).On this base,the mass spectrometry peaks of saccharic acid lactone and glucuronic acid were analyzed by LC-MS/MS to further confirm the presence of glucuronic acid and saccharic acid.The HPAEC-PAD results also showed that the presence of ion chromatographic peaks of glucose,gluconic acid,glucuronic acid,and saccharic acid.The results of LC-MS and HPAEC-PAD fully demonstrated the fact that the Hi PMO1 enzyme oxidized the cellulose C6 position to form a cellooligosaccharide containing glucuronic acid.In order to evaluate the difference between different oxidative activities of Hi PMO1 enzyme,quantitative analysis of gluconic acid,glucuronic acid and saccharic acid was carried out by HPAEC-PAD,showing that the amount of the substance,from the largest to the smallest,is gluconic acid,glucose diacid,glucuronic acid.The results indicated that the Hi PMO1 enzyme had different oxidation activities for C1 and C6,and the oxidation activity of C1 is greater than that of C6.Since there is no standard for the C4 oxidation product,the C4 oxidation activity was not evaluated.In addition,parallel experiments were carried out on the Ct PMO1 enzyme which also has a C6 oxidizing function.The oxidation products of Ct PMO1 enzyme were further digested with ?-glucuronidase and ?-glucosidase,and the hydrolyzate products were subjected to LC-MS and HPAEC-PAD detection.The results were similar to those from the Hi PMO1 enzyme.To sum up,the PMOs of the AA9 family in thermophilic fungi may have ubiquitous C6 oxidative function.The PMO enzyme with C6 oxidative function can continuously oxidize the C6 position in cellulose(-OH ?-CHO ?-COOH)and form a cellooligosaccharide containing glucuronic acid,which is then degraded by ?-glucosidase and ?-glucuronidase to produce glucose,gluconic acid,glucuronic acid and saccharic acid.It is even possible to produce unsaturated cellooligosaccharides by ?-elimination cleavage.This study provides a new mechanism for degrading cellulose by oxidizing cellulose C6 sites and suggests a new way to produce high value-added substances saccharic acid. |
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