| Energy is the internal driving force for the normal operation of a country,which is directly related to the stability and development of the society,affects the economic lifeline and security of the country,and also affects the protection,development and utilization of the ecological environment.Plant biomass resources are regarded as a new type of carbon energy and renewable resources that can replace fossil energy because of their wide distribution,renewable and large reserves.Now,the research on plant biomass resources has yielded fruitful results,but due to the diversity of plant biomass and the precision and complexity of its structure,there are still many obstacles to the degradation of plant biomass.In recent years,a new type of enzyme system for cellulose degradation has been discovered in the field of enzymatic degradation of plant biomass: copper-dependent polysaccharide monooxygenases(PMOs),which can oxidatively split macromolecules including cellulose.Therefore,the advent of PMOs provides a new idea for the degradation of cellulose.PMOs are classes of enzymes that have been widely studied in recent years,and they are the first kind of oxidases that can be catalyzed by copper ions.The enzymes are widely distributed in fungi,bacteria and viruses that have the ability to degrade cellulose.Due to the low activity of degrading cellulose,once discovered,this enzyme has long been classified as glycoside hydrolase 61 family(GH61)and carbohydrate binding module 33 family(CBM33)family.In 2013,because of the new classification,the two families were divided into auxiliary active enzymes,which were classified as AA9 family and AA10 family,respectively.Up to now,there are a total of 8 families of PMOs have been found.PMOs are copper ion-dependent enzymes that break glycosidic bonds through hydroxylation at different carbon positions in the presence of reducing agents.Due to the special bond of breaking mechanism,the difficulty of separating cellulose chains from highly crystalline cellulose is reduced,thereby the degradation efficiency of cellulase can get improved.PMOs also play a crucial role in plant pathology.As the cell wall degrading enzymes,they can dagrade the cell walls when plant pathogenic microorganisms infect plants,eliminate plant defense barriers,and accelerate the invasion of pathogenic microorganisms.And PMOs can act as effectors participates in the pathogenic process of pathogenic microorganisms or cause the immune defense response of host plants.In addition,PMOs can also participate in the process of symbiosis between different species,such as nutrient transport and information transmission.PMOs are widely distributed among fungi,bacteria and viruses with cellulose-degrading activity.In this study,in order to explore the enzymatic hydrolysis characteristics of PMOs and the functions in animal digestive tract,firstly,the PMOs enzyme HiPMO1 among the AA9(Auxiliary Activity family 9)of Scytalidium thermophilum was used as the research object.And the activity,oxidation mode,product composition of the enzyme and its synergistic effect with cellulase were were deeply analyzed and identified.Secondly,we also explored the biological functions of PMOs enzymes of the AA9 family of thermophilic fungi in the horse gut,and analyzed and identified the colonization of thermophilic fungi,the expression of AA9 PMOs protein and the composition of the product.The results are as follows:First,the eukaryotic expression of the HiPMO1 enzyme was successfully carried out in Pichia pastoris,and HiPMO1 reacted with substrate PASC(phosphate expanded cellulose)at p H 5.0 and 50? for 48 hours in the presence of reducing agent.The results of TLC showed that HiPMO1 enzyme was active on the insoluble substrate PASC and could oxidatively cleave the substrate PASC,the amount of products were also increased with time went on,and the products ranged from cellobiose to cellopentose.Methylation of the product,then MALDI-TOF-MS analysis of the methylated sample showed that there were also some various types of oxidized oligosaccharides besides the non-oxidized fiber oligosaccharide in the reaction product.Oxidized cellooligosaccharides mainly include C1-oxidized oligosaccharides and C4-oxidized oligosaccharides.We also carried out trifluoroacetic acid hydrolysis about the 48 h enzymatic hydrolysis product of HiPMO1,and found that C1-oxidized monosaccharides,gluconic acids,existed in the HPLC-RID analysis of the acid hydrolysis product.Meanwhile,HPLC-RID analysis showed that there was also galactose formed by the reduction of C4 oxidized product by sodium borohydride.In addition,we also conducted preliminary studies on the synergy between HiPMO1 enzyme and cellulases.After PASC was treated with HiPMO1,the substrate was enzymatically hydrolyzed with three cellulases(EGII,CBHI,BGLI),the production was detected by 3,5-Dinitrosalicylic acid(DNS),and the amount of reducing sugars produced increased by 1.35 times,1.49 times and1.33 times,respectively;while using CMC-Na as the substrate,increased by 0.24 times,0.36 times and 0.55 times,respectively.In summary,there are two oxidation modes of C1 and C4 in the reaction products of HiPMO1 enzyme,besides this,different substances and different types of cellulase will affect the synergistic effect of HiPMO1 enzyme.Most importantly,we studied the biological functions of thermophilic fungi AA9 PMO in animal guts in this paper.The insoluble cellulose from fresh horse feces and horse stomach was separated and extracted.After treatment with endo-1,4-?-D-cellulase,the reaction product was modified by methylation and MALDI-TOF-MS analysis,LC-MS,HPAEC-PAD and HPLC-RID analysis after trifluoroacetic acid hydrolysis,the results showed that C1 oxidized cellulose is present in fresh horse feces and horse stomach insoluble cellulose.Moreover,the soluble cello-oligosaccharides in horse stomach were extracted and separated using the principle of ethanol extraction of oligosaccharides.The results of MALDI-TOF-MS and HPLC-RID analysis showed that C1 oxidized oligosaccharides and C4 oxidized cello-oligosaccharides existed in the soluble oligosaccharides in horse stomach oligosaccharides.In conclusion,the oxidative cleavage of cellulose existed in the horse gut,and the products mainly included C1 and C4 oxidized oligosaccharides.We successfully isolated three thermophilic fungi to identify the species of thermophilic fungi in the horse gut.The internal transcribed spacer(ITS)sequence identified the three thermophilic fungi as S.thermophilum,C.thermophilum and T.aurantiacus.When the three thermophilic fungi were transferred to cellulose-containing medium at 37°C,it was observed that they could grow normally and the AA9 family genes including Hipmol,Ctpmol and Ta AA9 A were cloned from the three thermophilic fungi.In order to clarify the expression characteristics of three thermophilic fungi PMOs in the stomach of horses,we extracted total RNA from the stomach and intestinal chyme of horses,and verified that Ctpmo1 and Ta AA9 A,two AA9 s family genes could be normally expressed in the gut of horses by RT-PCR.These results indicate that thermophilic fungi can colonize in the horse gut and can grow normally at the horse body,the PMOs gene of the AA9 family can also be expressed normally.We also extracted the PMOs proteins of AA9 family in horse stomach,and used DEAE-Sepharose anion exchange chromatography to separate and purify the protein.The isolated and purified proteins were identified by LC-MS/MS,and the identification results showed that 6 kinds of AA9 family PMOs proteins were successfully matched,of which three were from T.terrestris,and one from S.thermophilum,one from C.thermophilum and one from T.aurantiacus.In addition,using HiPMO1 and Ct PMO1 and Ta AA9 A enzymes to treat PASC at 37?,and then the products were analyzed by MALDI-TOF-MS and HPLC-RID,we found that there were C1 oxidized oligosaccharides in the products.The above results directly demonstrated that there are PMOs enzymes of the AA9 family expressed by thermophilic fungi in the horse gut,and that PMOs enzymes can oxidatively cleave cellulose at the temperature of horses.In spite of these,C1 and C4 oxidized cellulose were also found in the insoluble cellulose of termites' feces,which proved that oxidative decomposition of cellulose also exists in the gut of termites.In conclusion,firstly,this study confirmed there are two oxidation types,C1 and C4,in the process of oxidative cleavage of cellulose by HiPMO1 enzyme.And HiPMO1 can synergize with cellulases in cellulose degradation.Secondly,we proved that the oxidative cleavage reaction of cellulose is mainly composed of C1 and C4 oxidation modes in horse gut.Finally,it is clear that there are AA9 family PMO proteins expressed by thermophilic fungi in horse gut.And they can oxidatively cleave cellulose in horse gut.Therefore,we found new biological functions of PMOs and elucidate the biological significance of thermophilic fungi in animal guts. |
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