Study On The Strategy To Enhance Enzymatic Hydrolysis Of Lignocellulose By Cellulase From Penicillium Oxalicum

Lignocellulose is an abundant renewable resource in nature.Converting lignocellulosic biomass into cellulosic ethanol and bulk chemicals through bioconversion is of great significance to alleviate the current energy and resource crisis.During the bioconversion,the lignocellulose is degraded into fermentable sugar with cellulase is a key step,but the low degradation efficiency and high production cost of cellulase are the key bottlenecks hindering the industrial production of cellulosic ethanol now.Penicillium oxalicum is a widely used strain producing cellulase with the advantages of high stability and relatively complete enzyme system,and lots of work on improvement of the cellulase from P.oxalicum have been conducted in our laboratory,but the degradation ability of the enzyme system still needs to be further improved to reduce the cost of the enzyme used in industrial production of cellulosic ethanol.It has been proved by literatures and our previous studies that adding accessory proteins to P.oxalicum cellulase system or modifying cellulase molecules are effective ways for optimizing cellulase system and improving the degradation ability of cellulase system.Based on the above background,this thesis investigated the enhancement of various accessory proteins on degradation ability of P.oxalicum cellulase system and its promotion mechanism,molecular modification of cellobiohydrolase I(CBHI)to improve the enzyme efficiency,and effect of accessory enzymes on degradation of DDGS(Distillers Dried Grains with Solubles),a main by-product from corn ethanol industry,with P.oxalicum cellulase system.Through these studies,it is expected that the degradation efficiency of the enzyme system to be further improved It is expected that the hydrolysis efficiency of the cellulase system will be further improved..The main research contents and results of this study were as follows:1.The promotion and mechanism of the differential protein in cellulase systems from Trichoderma reesei and P.oxalicum on degradation of lignocellulose with P.oxalicum cellulaseThree differential proteins,including cellulose induced proteins Cip1,Cip2 and xyloglucanase Cel74A,which exist in the cellulase system of T.reesei but not in the cellulase system of P.oxalicum,were heterologously expressed,and their effects on the degradation ability of P.oxalicum cellulase systems were studied.By adding these proteins to the enzymatic hydrolysis system of P.oxalicum cellulase,it was found that Cip2 and Cel74A had a certain promotion effect on the degradation efficiency of P.oxalicum cellulase,while Cip1 had significant promoting effect on the degradation of various pretreated lignocelluloses,and its promoting effect was better than that of some auxiliary proteins such as BSA(bovine serum albumin)reported in the literatures,in particular,for the substrates with high lignin content such as liquid hot water pretreated corn stover(LPCS)and corncob residue(CCR)from dilute acid pretreatment of concob,the promoting effect of Cip1 on the enzymatic hydrolysis was better than that of commercial cellulase produced by P.oxalicum when adding the same amount of protein.The mechanism of Cip1 promoting effect was further explored,and it was found that Cip1 protein did not show the activities of polysaccharide lyase and conventional cellulose degrading enzymes.Cip1 protein could destroy the crystal structure of cellulose in lignocellulosic material and reduce the non-productive adsorption of cellulase on lignin,thus promote enzymatic hydrolysis of lignocellulose.2.Enhancement of different sources carbohydrate binding modules(CBMls)on enzymatic degradation of lignocellulose with P.oxalicum cellulase systemFungal cellulases typically contain CBM1 and its role is thought to specifically recognize substrates.By heterologously expressing different CBM1s of cellobiohydrolases(CBHI)derived from T.reesei,P.oxalicum and Penicillium funicillium,respectively,we investigated their effect on the hydrolysis efficiency of the P.oxalicum cellulase system.Adding these different sources CBM1s to the P.oxalicum cellulase system,it was found that all the CBM1s could promote enzymatic saccharification of lignocellulose and increase the glucan conversion during enzymatic hydrolysis,and its enhancement effect was significantly higher than that of accessory proteins reported in some literature,such as BSA.It indicated that these CBM1s could be used as synergistic proteins of P.oxalicum cellulase to improve the degradation ability of the cellulase system.Further research found that the enhanced effect of CBM 1 on the enzymatic hydrolysis efficiency was not only related to the source of CBM 1,but also to the characteristics of lignocellulose,while was affected by the reaction conditions of enzymatic hydrolysis such as the protein dosage,the substrate concentration.By analyzing the adsorption kinetics of CBM1 and the main cellulase components such as cellobiohydrolase(CBH),endoglucanase(EG)and β-glucosidase(BG)in the P.oxalicum extracellular enzyme system on cellulose and lignin extracted from LPCS,it was found that CBM1 protein had a higher affinity for cellulose and lignin than these cellulases.The changes of different enzyme activities of free cellulase in the hydrolysis reaction system before and after adding CBM1 was also investigated,and the binding conformation and various binding energies of CBM 1 with cellulose and lignin were predicted by molecular docking.It was speculated that,the CBM1 with high affinity to the substrate can displace part of the cellulase adsorbed on lignin during the enzymatic hydrolysis,resulting in more cellulase being free and released into the hydrolysis system,thus enhancing the degradation efficiency of the cellulase system.3.Rational modification of cellobiohydrolase CBHI of P.oxalicum based on the properties of CBM1Fourteen CBM1s derived from cellulase from T.reesei,P.oxalicum,P.funicillium,Aspergillus niger and Chaetomium thermophilus were respectively heterologously expressed in Escherichia coli.By studying the adsorption properties of these CBM1s on cellulose and lignin,it was found that,compared to the CBMPoCBHI derived from P.oxalicum CBHI,the CBMPfCBHI derived from P.funicillium had stronger adsorption on cellulose and weaker adsorption on lignin,the adsorption ability on both cellulose and lignin were stronger for the CBMTrCip2 derived from T.reesei Cip2,but were weaker for the CBMTrSwo1 derived from T.reesei Swo1(swollenin).By replacing the corresponding domains of P.oxalicum CBHI with above three CBM1s,the enzyme CBHI was modified and found that the mutant enzymes had the specific enzymatic activity similar to that of the CBHI from P.oxalicum on the soluble substrate pNPC,but the mutant enzymes PRP(PCBHI-CBMPfCBHI)and PRTC(PCBHI-CBMTrCip2)had higher degradation ability on filter paper(FP),NaOH pretreated corn stover(NPCS)and LPCS than the CBHI from P.oxalicum.4.Accessory enzymes enhanced enzymatic hydrolysis of DDGS with cellulase system from P.oxalicum cellulaseDDGS is the main by-product from the dry process of corn ethanol production.By hydrolyzing cellulose in DDGS into fermentable sugar with cellulase,then fermentation to produce ethanol,the total yield of ethanol could be increased and the quality of DDGS was also improved.The cellulase systems produced from several engineering strains of P.oxalicum and T.reesei were used to degrade DDGS,and found that the enzyme system secreted by P.oxalicum RBB was more suitable for the hydrolysis of DDGS.The conditions of enzymatic hydrolysis with the cellulase RBB,such as the cellulase dosage,the substrate concentration,and the particle size of DDGS,were preliminarily optimized.It was found that when the amount of RBB cellulase was higher than 4 mg protein/g dry substrate,increasing the amount of enzyme had little effect on the conversion rate of glucan in DDGS.Appropriately reducing the particle size of DDGS was beneficial to increase the glucan conversion rate,but excessive reduction in size was not conducive to the enzymatic hydrolysis of DDGS.Within the range of experimental conditions,the solid concentration had little effect on the enzymatic hydrolysis efficiency of DDGS.Further,the effects of accessory enzymes on degradation of DDGS with P.oxalicum RBB cellulase were studied.The accessory enzymes include different single enzyme components of cellulase and hemicellulase,and amylase,pectinase,etc.It was found that,by adding arabinofuranosidase,pectinase and amylase,the degradation efficiency of DDGS with cellulase RBB was effectively improved.Besides,by using DDGS instead of carbon source in the fermentation medium to induce the strain to produce cellulase,the enzymes activities of xylanase,arabinofuranosidase and pectinase in the cellulase system were significantly higher than that of cellulase produced by fermentation using the original medium,and degradation of DDGS using the cellulase induced by DDGS was also improved.