The Properties And Synergistic Effects Of Polysaccharide Monopolygenase From Chaetomium Thermophilum

Most of the energy exploited and utilized belongs to non-renewable energy,which will eventually be exhausted.Cellulose biomass provides the posibility of energy substitution because of its rich and renewable characteristics,and the utilization of cellulose biomass will play an important role to solving the energy crisis.Lignocellulose can be degraded by a series of enzymatic reactions to produce monosaccharides,which can then be converted into energy materials.Therefore,how to degrade cellulose efficiently and quickly has become an important direction of technological breakthroughs.In this study,the polysaccharide monooxygenase of Chaetomium thermophilum AA9family?Auxiliary Activity family 9?was expressed in Pichia pastoris,and we explored the optimum reaction temperature,pH and other properties of those enzyme.After the enzymatic degradation of Phosphoric Acid-Swollen Cellulose,the products were analyzed by TLC and MALDI-TOF-MS.The regioselectivity of PMO oxidation was determined,and the synergistic effects of PMO on three kinds of cellulases,namely,exogenous cellulase CBH1,endocellulase EG1 and beta glucanase CT2,were determined.The Chaetomium thermophilum were cultured in microcrystalline cellulose to extract RNA,and reverse transcribed into cDNA.Then 12 PMO gene sequences were amplified by PCR with designed primers.pPICZ?A-PMO recombinant expression vector plasmid was constructed using pPICZ?A yeast methanol inducible expression vector.The vector plasmid was transformed by electric shock to construct PMO yeast engineering strain.After 7 days of methanol-inducible fermentation,the expressed protein was purified by protein purifier after precipitation with ammonium sulfate.The purity and molecular weight of the protein were detected.With the high expression,PMO0728,PMO0762,PMO5456,PMO6622 and PMO14091 were continued the follow-up experiment.By setting a series of temperature gradients and pH gradients to react separately,the optimum reaction temperature of polysaccharide monooxygenase was 50°C and the optimum reaction pH was 5.0.Under the optimum reaction conditions,PASC was degraded by five enzymes,and then TLC analysis of the reaction products showed that PMO5456 had more products and best activity.TLC analysis of PASC degradation products by PMO5456 showed that the soluble products contained G2-G6 oligosaccharides,which proved that the enzyme had the activity of degrading PASC.To further determine the oxidation mode of PMO5456,the reaction products were analyzed by MALDI-TOF-MS,and the corresponding molecular weight was analyzed according to the peak size.Among the products,C1 oxidized oligosaccharides?m/z+16?and C4 oxidized oligosaccharides?m/z-2?or C6 oxidized oligosaccharides?m/z-2?peaks were found.It can be determined that there is C1 oxidation,but it is impossible to distinguish the respective peaks of C4 and C6 oxidation.In order to distinguish C4 from C6oxidation,Br₂ oxidation was used to further oxidize the substrates,and the products were analyzed by MALDI-TOF-MS.C1 oxidized oligosaccharides will not be oxidized by Br2and their molecular weight remains unchanged;C4 oxidized oligosaccharides react with Br2,and the molecular weight of the products after oxidation increases by 14 compared with that before oxidation;if C6 oxidized oligosaccharides exist,they will also be oxidized by Br2,and the molecular weight of the products will increase by 30 compared with that before oxidation;therefore,after Br₂ oxidation,the molecular weight of C4 and C6 products will be different.The peak in MALDI-TOF-MS analysis map,it can be inferred that there are both C4 oxidation and C6 oxidation during the degradation of PASC by PMO5456.The oxidation mode of PMO5456 enzyme is C1,C4 and C6.As a member of the auxiliary activity family,PMO has an important role in improving cellulase activity.CBH1,EG1 and CT2 enzymes from Chaetomium thermophilum were selected and added into PASC pretreated with PMO5456.The activity of CBH1,EG1 and CT2 enzymes was determined by DNS method and compared with the original enzyme activity.The results showed that the three cellulase activities increased with the time of PMO5456 treatment.After PMO5456 treatment,the activity of endo-cellulase EG1increased by 3.6 times,that of exo-cellulase CBH1 by 2.2 times and that of?-glucosidase CT2 by 0.6 times.Finally,the oxidation modes of PMO5456 were determined,including C1,C4 and C6.The activity of endo-cellulase EG1 was significantly increased by PMO5456,followed by exo-cellulase,and the activity of?-glucosidase was not significantly increased by PMO5456.