Studies On Properties And Promotion Mechanisms Of Lignocellulose Degradation-related Enzymes In Penicillium Oxalicum

The research into the biodegradation of lignocellulosic biomass is of both biological and industrial significance.To improve the economy of bioconversion of lignocellulose to fuels and chemicals,lignocellulolytic enzymes which are critical for the generation of fermentable sugars need to be engineered for their compositions and properties.Penicillium oxalicum,an industrial cellulase-producing microorganism,has more diverse lignocellulolytic enzymes and higher hemicellulase activities comparing with Trichoderma reesei.Current existing cellulase preparations produced by P.oxalicum still have high-cost problems in large-scale industrial production of bioethanol.Searching for the key components in lignocellulose degradation is critical to reduce the cost of cellulase usage at present.The main results of the thesis are as follows:1.Construction of a lignocellulose degradation-related enzyme library of P.oxalicum and characterization of its membersTwelve kinds of lignocellulolytic enzymes from P.oxalicum were expressed homologously or heterologously to construct a lignocellulose degradation-related enzyme library,which includes two cellulases,eight hemicellulases and two auxiliary enzymes.Enzymatic properties of the members in the library were studied,including specific activities,the optimal temperature and pH of activities,temperature and pH related stabilities of activities.Supplementation of BGL1 led to a boost in glucose yield of the cellulase mixture produced by P.oxalicum high-yield mutant strain JU-A10-T during the hydrolysis of delignified corncob residue and corncob residue.Therefore,equivalent glucose yield was achieved using a lower enzyme dosage.2.Synergistic promotion of the hydrolysis of pretreated substrates by the supplementation of a-L-arabinofuranosidase Abf43D and other hemicellulasesThe supplementation of ?-L-arabinofuranosidase Abf43D and endo-?-1,4-mannase Man5A led to boosts in glucose yield of P.oxalicum cellulase preparation SP during the hydrolysis of hot water pretreated corncob and corncob residue.To further explore the promotion mechanism of Abf43D,we found that Abf43D might remove the arabinose substituents from xylan backbone and work synergistically with endo-?-1,4-xylanase Xyn10A during the degradation of hemicellulose to make cellulose more accessible to cellulases.Moreover,Abf43D supplementation was capable of mitigating the xylo-oligosaccharide inhibition of cellobiohydrolase in the enzyme mixture.3.The effect of aldonolactonase AltA supplementation on P-glucosidase activity and glucose yieldD-Glucono-1,5-lactone was shown to have a very strong inhibitory effect on?-glucosidase,a vital enzyme during cellulose degradation.Supplementation of AltA was able to relieve the decrease of ?-glucosidase activity obviously with a stimulation of glucose yield.HPLC-ESI-Q-TOF MS proved that the concentration of D-glucono-1,5-lactone decreased in the presence of AltA.The boosting effect disappeared when singlet oxygen quencher sodium azide and metal ion chelator EDTA were added to the saccharification system to inhibit the activities of oxidative enzymes.4.The degradation of lignocellulose by lytic polysaccharide monooxygenase-aldonolactonase-p-glucosidase system and the optimization of its proportionIt was first confirmed that LPMO1-AltA-BGL1 system could synergistically hydrolyze phosphoric acid-swollen cellulose.Also,supplementation of this system to commercial cellulase preparation SP could promote the degradation of microcrystalline cellulose and pretreated biomass substrates.The lactone concentration decreased and ?-glucosidase activity increased in the presence of AltA during saccharification.Design Expert software was used to create a mixture design scheme to optimize the proportions of LPMO1,AltA and BGL1 in this system.The optimized proportions of LPMO1,AltA and BGL1 was 3:8:14.