The Absorption Of Cellulase Onto Lignin And Its Mechanism, And Rational Engineering Of Cellulase

Bioconversion of lignocellulose to fuels and chemicals was carried out to alleviate the energy crisis and pollution problem.Lignocellulose was composed of cellulose,hemicellulose and lignin,in which cellulose could be hydrolyzed by cellulase to fermentable sugar.However,the existence of lignin and hemicellulose hindered the hydrolysis process of cellulose.Pretreatment of substrate was a way to break the "biomass recalcitrance" of lignocellulose and improve the hydrolysis efficiency.However,residual lignin was remained in most pretreated substrate and the residual lignin inevitably led to the decrease of the hydrolysis yield by adsorbing cellulase,blocking the enzyme access to cellulose and reducing the cellulases activity.For now,although the adsorption behavior of lignin with cellulase have been more documented in cocktails and mono-components of cellulase,the mechanism of adsorption between cellulase and lignin was not yet fully understood,which hindered the development of technology for decreasing adsorption of cellulase to lignin.In this study,we explored the adsorption properties and mechanism of lignin,isolated from corn stover after liquid hot water(LHW)pretreatment,to cellulase in cocktail and monocomponent.By comprehensively analyzing adsorption mechanisms of main cellulase components,it disclosed various effect of cellulases-lignin interaction on hydrolysis.This dissertation was helpful for decreasing non-productive adsorption of the cellulase to lignin and negative influence of the adsorption on enzymatic hydrolysis,through engineering enzyme,improving cellulase system by engineering strain and/or adding relative enzyme components,etc.The main contents of this dissertation were described in the following.(1)The adsorption properties of celluases onto ligninsLHW pretreatment removed most hemicellulose and some lignin in corn stover,as well as it improved enzymatic digestibility of corn stover.After LHW pretreatment,the molecular weight of lignin obviously increased,whereas its polydispersity decreased and became more negative.The hydrophobicity and functional groupsin lignin also changed.Adsorption of cellulase from Penicil(P.oxalicum(P.oxalicum)onto lignin isolated from corn stover was enhanced after LHW pretreatment.Different adsorption behaviors were observed in different lignin samples and components of cellulase mixtures,even in different cellobiohydrolases(CBHs),endo-beta-1,4-glucanases(EGs).The greatest reduction in enzyme activity caused by lignin was observed in CBH,followed by that in xylanase and then in EG and?-Glucosidase(Bgl).The adsorption behavior exerted different effects on subsequent enzymatic hydrolysis of various biomass substrates.Hydrophobic and electrostatic interactions may be important factors affecting different adsorption behaviors between lignin and cellulase.For cellulase solution from P.oxalicum,electrostatic action was a main factor influencing the adsorption of EG and xylanase onto lignin in corn stover,while hydrophobicity affected the adsorption of CBH and Bgl onto lignin.(2)The mechanisms of cellulases-lignin interactionUnder the understanding of the adsorption properties of lignin to cellulase cocktail,we further analyzed the mechanisms of the interaction between main cellulase components-lignin and discussed how cellulase-lignin interaction influenced hydrolysis yield.It suggested that CBHI displayed high adsorption affinity to lignin,especially for the lignin isolated from LHW pretreated substrate,and a stable combination of lignin-CBHI was formed.The CBHI adsorbed to lignin still had the catalytic ability on Avicel,although the ability was decreased.The possible reason,by molecular docking,was that the catalytic domain(CD)of CBHI was irreversibly adsorbed onto lignin mainly through a hydrophobic cleft located oppositely to catalytic tunnel,not influencing the catalytic process of the enzyme except for the protein movement.Electrostatic interaction played a significant role in the adsorption of carbohydrate binding domain(CBM)of CBHI to lignin.It also suggested that altering the charges from positive to negative of amino acid on the CBM of CBHI by engineering enzyme may be a feasible way to reduce the adsorption affinity of CBHI onto lignin.Comparing to CBHI,EGII showed lower adsorption ability to lignin,but the adsorption behavior of EGII to lignin influenced the catalytic ability of enzyme.During the adsorption of EG to lignin,CBM and CD both played an important role in the process.Two-step adsorption model was built in the adsorption process of EGII to lignin,in which it assumed that CBM as the domain adsorbed to lignin quickly in the first adsorption step leading the activity of lignin-EGII increased,and CD adsorbed to lignin in the following slow-step leading the decrease of lignin-EGII activity.The optimal binding position of lignin on CD of EGII was consistent with the binding sites of polysaccharide,caused the decrease of catalytic potential of enzyme adsorbed by lignin.From that,it assumed that the adsorption of EGII to lignin maybe the main reason for the decrease of hydrolysis yield.The adsorption properties of two Bgls,Bgll from P.oxalicum and Bgl3 from Aspergillus fmigatus,to lignin were evaluated in this study.Bgll showed lower adsorption ability to lignin and higher binding ability to cellulose,while Bgl3 showed greater adsorption ability to lignin and slight binding ability to cellulose.The binding sites of Bgl3 to lignin were concentrated on the domain Fn?.The positive charges distributed on the Fn? of Bgl3 promoted protein binding to lignin,so elevating the pH value,with decrease in the position charges carried by Fn?,obviously decreased the amount of Bgl3 adsorbed on lignin.It suggested that the different distribution of electrical potential and animo acids on Fn?s may casue the various adsorption properties of Bgll and Bgl3.(3)The rational engineering of cellulasesUnder the study on mechanisms of cellulase-lignin interaction,rational engineering of CBHI,EGII and Bgll were carried out and we further analyzed the binding and hydrolysis properties of engineered enzymes.The modified cellulases,CBHs and EGs,were constructed by mutation and fusion CBMs to cellulase,in which two engineered cellulases CBH-TrCBMV27E,P30D,Link1 and EG-TrCBMV27E,EP30D,Link1 performed well during hydrolysis.Compared to wild-type enzymes,EG-TrCBMV27E,P30D,Lnkl obtained relatively less adsorption ability to lignin and greater affinity to cellulose,especially Avicel.However,for CBH-TrCBMV27E,P30D,Link1,the hydrolysis manner was changed and in favor to hydrolysis process,although the adsorption properties was unexpected.It suggested that the various binding conformations of polysaccharide on CBMs hypothetically resulted in different function of CBMs,including binding,processive and digestive abilities on the fiber surface.Fusion of T.r-CBMV27E,P30D,Link1 to cellulase,both CBHI and EG?,gave the destruction ability of enzyme and increased the accessible surface of substrate to cellulase,enhanced the adsorption and hydrolysis efficiency of cellulase.Because of the important role of Fn? in binding of Bgls to substrate,we constructed the heterogeneous fusion protein Bgll-3 by replacing the Fn? of Bgll by Fn? from Bgl3.Bgl1-3 showed relatively higher adsorption affinity to lignin and lower binding affinity to cellulose versus Bgl1.It confirmed the important role of Fn? on binding ability of Bgls to substrate.In the adsorption of CBHI to cellulose,addition of Bgl1 reduced the amount of CBHI adsorbed on cellulose.Bgls could hydrolyze the cellubiose not cellulose.It implied the binding of Bgls to cellulose may be unfavorable to hydrolysis.