Study On Regulation And Mechanism Of Enzymatic Saccharification Process Of Wheat Straw

The preparation of fuel ethanol from lignocellulose is of great significance to alleviate the global energy crisis.However,the natural resistance structure of lignocellulose limits its high value conversion and utilization.Therefore,it is necessary to take effective measures to improve its utilization rate and reduce the transformation cost.In this study,wheat straw was used as raw material,ultrasonic assisted xylanase pretreatment and ionic liquid assisted ball milling pretreatment were respectively used to promote enzymatic saccharification of wheat straw,while overcoming the problems of high energy consumption and heavy pollution existing in the traditional pretreatment process.By means of substrate characterization,component determination and enzyme protein tracking,the changes of structure and properties of wheat straw in two pretreatment processes were studied from the perspectives of substrate,enzymatic hydrolysis and enzyme,and the mechanism of improving enzymatic hydrolysis rate of wheat straw was revealed.At the same time,in view of the disadvantages of cellulase,such as poor stability,high price and difficult recycling,the cellulase was immobilized on polyurea microspheres and three-dimensional polyurea nanofibers,and the cellulose hydrolysis system based on immobilized cellulase was constructed,which strengthened and promoted the catalytic performance of cellulase,The influence mechanism and law of metal ions and glucanase on cellulase activity were explored,which provided a theoretical basis for diversified promotion of cellulose hydrolysis and saccharification.The main research contents of this study are as follows:（1）The effect and mechanism of ultrasonic assisted xylanase pretreatment on enzymatic saccharification of wheat straw were studied.Ultrasonic pretreatment increased the surface roughness of wheat straw through ultrasonic cavitation,destroyed the phenylpropane skeleton structure of wheat straw lignin molecule,exposed the hemicellulose inside,and increased the accessibility of xylanase to hemicellulose.Xylanase can effectively remove hemicellulose by opening theβ-1,4-glycosidic bond in wheat straw hemicellulose.The collapse of the molecular structure of hemicellulose destroyed the uronic ester bond andβ-ester bond between hemicellulose and lignin,and the lignin was stripped from wheat straw.The removal of hemicellulose and lignin increased the component content and crystallinity of wheat straw cellulose,improved the accessibility of cellulase to cellulose,enhanced the hydrophilicity of wheat straw,reduced the unproductive adsorption of lignin to cellulase,and promoted the enzymatic hydrolysis of wheat straw cellulose.Under the optimized conditions,69.1%hemicellulose and 49.7%lignin from wheat straw were removed by ultrasound assisted xylanase pretreatment.After ultrasonic assisted xylanase pretreatment,the glucose yield of wheat straw reached 64.2%,which was 5.2,2.5 and 2.0 times that of untreated,individual ultrasonic pretreatment and individual xylanase pretreatment,respectively.27.8 g glucose can be prepared by enzymatic hydrolysis and saccharification of 100 g wheat straw after ultrasonic assisted xylanase pretreatment.At the same time,xylanase pretreatment realized the regeneration of xylose in hemicellulose,and 18.7 g xylose could be obtained after xylanase pretreatment.（2）The effect and mechanism of ionic liquid[Emim][OAc]assisted ball milling pretreatment on enzymatic hydrolysis and saccharification of wheat straw were explored.[Emim][OAc]has a strong ability to dissolve lignocellulose.It dissolved lignin by opening the C-O and C-C bonds between phenylpropane units of lignin molecules,stripped hemicellulose by destroying the hemicellulose hydrogen bond network,and destroyed the dense structure of cellulose by breaking the intramolecular and intermolecular hydrogen bonds of cellulose.After[Emim][OAc]pretreatment,86.7%lignin and 90.6%hemicellulose in wheat straw were removed,and the molecular size of cellulose was reduced to obtain cellulose nanofibers,realizing the separation and purification of cellulose.The separation and fragmentation of cellulose improve the pretreatment efficiency of ball milling.Ball milling pretreatment further destroyed the crystalline structure of cellulose nanofibers,reduced the crystallinity of cellulose,increased the content of amorphous cellulose and increased the specific surface area of cellulose through mechanical forces such as shear force,friction force and impact force.The physical and chemical fragmentation treatment under the synergistic action of ionic liquid and ball milling greatly improved the accessibility of cellulase to cellulose,thus promoting the enzymatic hydrolysis and saccharification of wheat straw cellulose.Under the optimized conditions,after ionic liquid assisted ball milling pretreatment,the glucose yield of wheat straw reached 81.1%,which was 6.7,3.2 and 1.4 times that of untreated,individual ball milling pretreatment and individual ionic liquid pretreatment,respectively.After ionic liquid assisted ball milling pretreatment,29.8 g glucose can be produced during the enzymatic hydrolysis and saccharification of 100 g wheat straw.The pretreatment of ionic liquid can effectively remove lignin and hemicellulose and realize the fractionation of hemicellulose and lignin.After ionic liquid pretreatment,25.9 g of hemicellulose and 15.5 g of lignin can be obtained.（3）The cellulase immobilized on polyurea microspheres was prepared and the effect of immobilized on the catalytic performance of cellulase was investigated.Using isophorone diisocyanate as raw material,polyurea microspheres containing abundant covalent immobilizing sites of enzyme proteins were prepared by precipitation polymerization.Using glutaraldehyde as a molecular scaffold,a large number of highly reactive primary amino groups on the surface of polyurea microspheres can covalently bind to the amino groups of the side chains of cellulase protein through schiff base reaction,and achieve multi-point covalently immobilization of cellulase.The unique chemical structure of polyurea microspheres reduces the preparation cost of immobilized carrier.The covalent immobilization method enhanced the catalytic stability of immobilized cellulase.The catalytic activity of cellulase was retained under mild immobilization conditions.Under the optimized conditions,the activity recovery of immobilized cellulase and the amount of immobilized cellulase were 77.9%and 39.2 mg/g,respectively.SEM,TEM and AFM characterization showed that polyurea microspheres were monodispersed with uniform morphology and had smooth and clean surface,with a diameter of about 10μm.When the cellulase was immobilized on the surface of polyurea microspheres,the particles or peaks on the surface of the microspheres,the absorption peak of C=N bond in FT-IR of the immobilized cellulase,the appearance of S element in EDS energy spectrum,and the reduction of thermal weight loss of the immobilized cellulase all proved that the cellulase was successfully immobilized on the surface of polyurea microspheres by covalent binding.The immobilized cellulase has strong acid and base resistance,thermal stability,storage stability and process experiment stability.After recycling for 8 times,the immobilized cellulase still showed high catalytic activity（75%）,indicating that it had good recycling performance.（4）Three dimensional polyurea nanofibers immobilized cellulase were prepared and the effect of immobilization on the catalytic performance of cellulase was investigated.In order to solve the problems of small specific surface area of polyurea microspheres and high preparation cost of nanofibers,ultrafine three-dimensional polyurea nanofibers（PUNF）were prepared by homogeneous continuous feeding assisted precipitation polymerization with toluene diisocyanate and 4,4-diaminodiphenyl ether as raw materials.In the homogeneous continuous feeding process,the gradual increase of monomer concentration of the reaction system reduces the random collisions between short chains of polymers,and the ordered collisions and combinations occur under the directional induction force of polymerization reaction,forming three-dimensional polyurea nanofibers.The three-dimensional structure increases the specific surface area of polyurea nanofibers and reduces the steric hindrance of enzyme immobilization.The chemical structure of polyurea makes PUNF contain abundant primary amino groups with high reactivity.These advantages increase the feasibility of PUNF as an enzyme immobilization carrier.The specific surface area and surface amino group of PUNF were 119.46 m²/g and 57μmol/g,respectively.After immobilization,the appearance of granular ridges on PUNF surface,the formation of C=N bond absorption peak in FT-IR of immobilized cellulase,the reduction of specific surface area and pore volume of PUNF,and the decrease of thermal weight loss of immobilized cellulase all proved that cellulase was successfully immobilized on the surface of PUNF through covalent binding.Under the optimized conditions,the enzyme immobilization amount and enzyme activity recovery reached 87.9 mg/g and 79.1%,respectively.According to the specific surface area of PUNF,the number of amino groups on PUNF surface and the amount of enzyme immobilization,each cellulase protein molecule was immobilized on the surface of PUNF by 43 amino groups.PUNF immobilized cellulase showed strong acid and base tolerance,thermal stability,storage stability and process experiment stability.After recycling for 10 times,the catalytic activity of PUNF immobilized cellulase was still high（79%）,indicating that PUNF had excellent recycling performance.（5）The effects and mechanism of metal ions(K⁺、Na⁺、Ca²⁺、Mg²⁺、Zn²⁺、Cu²⁺、Ni²⁺、Co²⁺、Mn²⁺、Fe³⁺)and glucanase on the catalytic performance of Aspergillus niger cellulase（An C）were investigated.K⁺promotes the activity of An C by reducing the transition/ground state transition energy barrier before and after the binding of enzyme protein and substrate molecules;The smaller ion radius of Na⁺,the shorter M⁺-O pair distance and the lower surrounding electron density lead to the weaker interaction between Na⁺and enzyme protein,which has less effect on An C activity;The coordination of Ca²⁺and Mg²⁺with amino acid carboxyl group of enzyme protein peptide chain enhances the rigidity of protein molecular structure and improves the thermal stability of An C;Cu²⁺,Ni²⁺,Co²⁺,Mn²⁺,Zn²⁺and Fe³⁺coordinate with tryptophan indolyl,cysteine sulfhydryl and histidine imidazolyl in the peptide chain of cellulase protein,destroy the secondary and tertiary structure of cellulase protein and inhibit the exertion of enzyme activity.Because the coordination kinetics of zinc ligand does not constitute a typical stereochemical constraint of transition metals,Zn²⁺has the weakest inhibitory effect on An C among the six transition metal ions.Glucanase randomly cuts off the cellulose molecular chain and converts cellulose into fiber oligosaccharides with different lengths,so as to reduce the size of cellulose and promote the microfibrilization of cellulose,which provides sufficient substrate for cellobiohydrolase in cellulase system.When glucanase and cellulase act on cellulose together,they showed a significant synergistic effect on the hydrolysis and saccharification of cellulose.The synergistic effect of glucanase and cellulase in the initial stage of enzymatic hydrolysis was more robust than that in the later stage.The combination of glucanase and cellulase can improve the rate of cellulose hydrolysis and glucose yield.