The Adsorption Characteristics Of Xylanase Xyn10A From Penicillium Oxalicum Onto Lignin And Construction Of "Weak-lignin-adsorbed" Xylanase

Bio-conversion of renewable lignocellulosic biomass into liquid fuels and bulk chemicals is one of the effective ways to solve the problems such as shortage of fossil resources and energy,environmental challenges,etc.The bioconversion process includes three main steps:pretreatment,enzymatic hydrolysis and fermentation,in which,a key step is that the pretreated lignocellulosic biomass is hydrolyzed with cellulase into fermentable monosaccharides.It was proved by literatures that ammonium sulfite(AS)pretreatment and steam explosion(SE)pretreatment are two pretreatment methods with industrial application prospects.After AS-and SEpretreatment,however,part of lignin was retained in solid substrate,and the residue lignin in the pretreated lignocellulose could hinder the enzymatic hydrolysis of lignocellulose through nonproductive adsorbing cellulase,thus increases the cost of enzymes for the bioconversion of lignocellulose.In addition,the structure and properties of lignin were also changed during pretreatment,which affected the adsorption characteristics of enzyme onto lignin.In recent years,the research about the adsorption of cellulase onto lignin and how to reduce the adsorption is a hot topic.Penicillium oxalicum is one of the main strains for industrial production of cellulase,and xylanase Xyn1OAis one of the main enzyme components of the cellulase produced by Penicillium oxalicum,which plays an important role in the degradation of lignocellulose.The adsorption of xylanase onto lignin have been reported in literature,but,up to now,the interaction and binding mechanism between lignin and xylanase are not clearly understood.The solution of this problem is helpful to theoretically guide people to look for the methods that can effectively reduce the unproductive adsorption of xylanase onto lignin and improve the catalytic efficiency of xylanase.Based on the above background,this thesis systematically studied and compared the adsorption characteristics of xylanase Xyn1OA from Penicillium oxalicum onto lignin extracted from AS-and SE-pretreated corn stover,and investigated the mechanism of differential adsorption of the two different lignin.The effects of environmental factors such as temperature and pH value on the adsorption of lignin were also studied,and then the engineering modification of xylanase molecules were conducted by different strategies to construct the "weak-lignin-adsorbed" xylanase for reducing the adsorption of xylanase onto lignin and improving the efficiency of enzymatic hydrolysis of lignocellulosic biomass.The main research contents and results of this study were as follows:1.Adsorption characteristics and mechanism of xylanase Xyn10A on lignin from AS-and SE-pretreated corn stoverUsing AS-and SE-pretreated corn stover,and untreated corn stover as substrate,the milled wood lignin(MWL)was extracted,respectively,and the adsorption characteristics of xylanase Xyn10A from Penicillium oxalicum onto the lignin samples,including adsorption ability,adsorption kinetics and thermodynamic properties,etc.were studied,and their differences on the absorption characteristics were analyzed.The results showed that,the adsorption capacities of the lignin isolated from AS-and SEpretreated corn stover increased when compared with the lignin isolated from unpretreated corn stover.There was different adsorption characteristics for the lignin from SE pretreated corn stover(SE-MWL)and the lignin from AS pretreated corn stover(AS-MWL)to the xylanase Xyn1OA.Compared to the AS-MWL,the SE-MWL showed faster Xyn10A initial adsorption velocity(13.56 mg g-1 min-1 vs.10.89 mg g-1 min-1),higher adsorption capacity(49.46 mg g-1 vs.27.42 mg g-1)and binding strength(310.6 L g-1 vs.215.9 L g-1),and lower steric repulsion between Xyn10A protein molecules during adsorption.It was found that the binding stability of lignin-protein complex formed by the adsorption of xylanase Xyn10A onto the SEMWL was higher than that of the complex of Xyn10A and AS-MWL,and the Xyn1OA could not be dissociated by washing and centrifugation.The physicochemical properties of the two lignin were analyzed,and it was found that AS-MWL had a low zeta potential,high hydrophilicity and a small number of phenolic hydroxyl groups,which weakened the electrostatic and hydrophobic interaction force between AS-MWL and Xyn10A,resulting in a low adsorption capacity of the lignin to Xyn10A;while SEMWL had high zeta potential and hydrophobicity,and more phenolic hydroxyl groups,which increased the interaction between SE-MWL and Xyn1OA,so the adsorption capacity of SE-MWL to Xyn10A was high.2.Effect and mechanism of environmental factors on the adsorption of Xyn10A onto ligninThe effect of environmental factors such as temperature and pH on the adsorption characteristics of lignin isolated from AS and SE pretreated corn stover to Xyn1OA,and binding stability between lignin and enzyme were studied.The kinetics and thermodynamics analysis of adsorption of the Xyn10A onto the SE-MWL showed that increasing the temperature within a certain range could promote the adsorption of Xyn10A onto lignin,increase the adsorption capacity of lignin,but the increase of pH value could significantly reduce the adsorption rate of Xyn10A onto lignin.The binding stability of between lignin and Xyn10A was reduced by increasing temperature and pH within a certain range,which was helpful for the dissociation of proteins bound to lignin,but the degree of dissociation was related to the type of lignin.Further studies found that the change of temperature and pH affect the surface charge and hydrophobicity of Xyn10A protein.The increase of temperature and pH led to the decrease in the protein surface charge(from positive to negative)and hydrophobicity of Xyn10A,thus increasing the electrostatic repulsion between lignin and protein,and decreasing the hydrophobic interaction forces,which decreased the absorption ability of Xyn10A onto lignin and the binding stability between lignin with Xyn10A.3.Molecular modification of xylanase to alleviate the non-productive adsorption ability of xylanase onto ligninOn the basis of the above research,xylanase molecules with different enzymatic characteristics and domain compositions from Penicillium oxalicum,Aspergillus fumigatus and Aspergillus niger were screened for applying in molecular modification.The removal,replacement and addition of CBM and Linker domains were performed on the selected xylanase molecules,respectively,and the changes in the adsorption performance of the enzyme molecules onto lignin before and after the modification and their effect on the enzymatic hydrolysis of different pretreatment substrates were compared.It was expected to construct xylanase molecules with "weak-ligninadsorbed" and high enzymatic performance.By single removing the CBM domain of the xylanases derived from Penicillium oxalicum and Aspergillus fumigatus,respectively,and simultaneously removing the CBM and the Linker domains,four deletion mutants,PoXyn10AdC,AfXyn10AdC,PoXyn10AdLC and AfXyn10AdLC,were constructed respectively.It was found that the optimal pH,optimal temperature,pH stability and thermostability of the deletion mutants by removal of CBM domain were hardly changed.Compared with the control protein(PoXyn10A and AfXyn10A),the specific activities of the PoXyn10AdC and AfXyn10AdC were increased by 110%and 20%,respectively,and the adsorption capacity on lignin were decreased by 64%and 67%,respectively.The enzymatic hydrolysis experiments proved that the removal of CBM improved the degradation ability of PoXyn10AdC and AfXyn10AdC on pure xylan substrate,but decreased the degradation rates of xylan in pretreated substrate under low-concentration hydrolysis conditions,and thus also affects the hydrolysis of cellulose,especially for AfXyn10AdC,it was speculated that,due to the lack of CBM in the deletion mutant,the binding ability to xylan in lignocellulose was weakened in the reaction system with low solid concentration compared with the full-domain enzyme molecule.The thermostability and specific activity of the deletion mutants(PoXyn10AdLC and AfXyn10AdLC)by simultaneous removal of CBM and Linker domains were significantly reduced,and showing poor enzymatic hydrolysis performance to substrates,proving that Linker is necessary to ensure the catalytic degradation ability of xylanase.On the basis of the above work,the CBM and Linker of PoXyn10A(PPP)and AfXyn10A(AAA)were further replaced,and the CBM and Linker were also transferred to GH11 family xylanase from the Aspergillus niger with only one catalytic domain(AnXyn11B,11B),and six recombinant xylanases,PAA(PoXyn10ACDLinkerAfXyn10A-CBMAfXyn10A),PAP(PoXyn10ACD-LinkerAfXyn10A-CBMPoXyn10A),APP(AfXyn10ACD-LinkerPoXyn10A-CBMPoXyn10A),AAP(AfXyn10ACD-LinkerAfXyn10ACBMPoXyn10A),BAP(AnXyn11B-LinkerAfXyn10A-CBMPoXyn10A),and BAA(AnXyn11BLinkerAfXyn10A-CBMAfXyn10A)were successfully constructed.Compared with the unmodified protein(PPP),the specific activities of the recombinant proteins PAA and PAP were increased by 64%and 78%,respectively.The enzymatic properties analysis showed that the thermostability of PAA and PAP were improved when compared with PPP.The experiments of lignin adsorption and enzymatic hydrolysis showed that the adsorption performance of PAA and PAP onto lignin did not greatly improve,but the addition of these two recombinant enzymes in the enzymatic hydrolysis system could significantly improve the enzymatic hydrolysis of pretreated lignocellulosic substrates.For example,compared with addition of PPP,the addition of PAA increased the conversion of xylan and glucan at 72 h in ammonium sulfite pretreated corn stover by 27%and 13%,respectively,which should be partly attributed to the high specific activity and high thermostability of the PAA recombinant enzyme.Compared with the AAA(control protein),the enzymatic hydrolysis properties of recombinant proteins APP and AAP were not improved,but the binding ability of the APP to lignin was reduced.Compared with 11B,the specific activities of recombinant proteins BAP and BAA were increased by 132%and 41%,respectively,and the pH stability and thermostability were also improved.Further research found that,compared with the above-mentioned recombinant xylanase PAP,PAA and AAP,the binding abilities of between lignin with the BAP and the BAA were weakened,and the adsorption capacities of lignin to the BAP and the BAA were significantly reduced.Adding the BAP and the BAA into the enzymatic hydrolysis system of pretreated corn stover could improve the enzymatic hydrolysis efficiency of xylan and cellulose at the same time.