Study On Low-dimensional Polymer Substrate For Immobilization Of Cellulase

Bioethanol produced from cellulose has the advantages of abundance and renewability of the raw materials,which is of great significance to alleviate the energy shortage of the world and achieve sustainable development of the energy.The conversion of cellulose into fermentable sugar catalyzed by cellulase is a key step in the production of cellulosic ethanol.However,the high cost of enzyme has become a bottleneck problem for the large-scale production of cellulosic ethanol.Immobilization of cellulase with appropriate carrier provides the ideal strategy to solve the obstacle in industrial application,which can enhance the structural stability of cellulase,improve its environmental tolerance,and realize its recycling.Considering massive insoluble residues produced in the cellulose hydrolysis system,polymer carrier materials have the advantages of low cost,easy separation and recovery over the commonly used nano/micro particle carriers,which make them has great potential in industrial application.However,polymeric materials,especially polyolefin,always suffered low surface energy and reaction inertness,which need to be modified for immobilization of cellulase.Additionally,owing to cellulose is insoluble in water,the interface of polymer materials should be rationally designed to enhance the catalytic efficiency of immobilized cellulase.Based on the above background,in this dissertation,functional polymer brushes with various structures were introduced on different low-dimensional polymer supports by the photo-induced surface graft polymerization to achieve covalent immobilization of cellulase.The main results are followed:1.Styrene-maleic anhydride copolymer(PMS)and polyacrylic acid(PAA)were introduced on the surface of polypropylene(PP)non-woven fabrics by UV-induced surface graft polymerization and used for immobilization of cellulase.Effects of different immobilization methods on the catalytic performance of cellulase were explored.Firstly,the influences of light intensity,irradiation time,monomer concentration and photoinitiator concentration on graft efficiency were studied.Then the aldehyde modified PP non-woven fabric carrier(PP-CHO)was prepared by the sequential reaction of PMS with ethylenediamine and glutaraldehyde.Cellulase was immobilized on three substrates containing acid anhydride(PMS modified PP,PP-PMS),aldehyde group(PP-CHO)and carboxyl group(PAA modified PP,PP-PAA)respectively.After 10 cycles usage for hydrolysizing sodium carboxymethylcellulose(CMC),the activity of cellulase immobilized on PP-PMS,PP-CHO,and PP-PAA could be retained for 70%,80%and 95%respectively.The pH stability of cellulase immobilized by PP-PMS was higher than that of free enzyme,and relative activity of cellulase could still remain 65%at pH 8.0,while the activity of free enzyme dropped to 10%.The activities of endo-glucanase(EG),cellobiohydrolase(CBH)and ?-glucosidase(BG)of cellulase immobilized by PP-PAA were 78.55%,59.66%and 55.06%of the free enzyme,respectively.The results from hydrolysis of microcrystalline cellulose(MCC)showed that cellulase immobilized by PP-PAA had good reusability and retained 60%of the initial enzyme activity after 7 cycles,while the activity of cellulase immobilized by PP-CHO decreased to 30%after 3 cycles.2.A novel strategy for immobilization of cellulase on block polymer modified PP fiber was developed.Firstly,a cross-linked copolymer of glycidyl methacrylate(GMA)and polyethylene glycol diacrylate(PEGDA)was introduced on the surface of PP fiber by UV-induced graft polymerization.Subsequently,the surface amination was achieved by the reaction of ethylenediamine with the surface epoxy group,which further reacted with 2bromoisobutyryl bromide(BIBB)to introduce the initiator of atom transfer radical polymerization(ATRP).Finally,PEGMA-b-PGMA block copolymers containing polyethylene glycol(PEG)hydrophilic spacer and epoxy groups were prepared by sequential surface-initiated ATRP of PEGMA and GMA on the surface of PP fibers,and then cellulase was immobilized by reaction with epoxy groups.After immobilization,the temperature stability and pH stability of cellulase was significantly improved,and 62%of the initial enzyme activity was retained at 80?,which was higher than the 47%of free enzyme.At pH 8.0,20%of the initial enzyme activity was retained,which was twice of the remaining activity of the free enzyme.The storage stability was also significantly improved.The relative activity of immobilized cellulase decreased to 90%after storage for 25 days,while that of free cellulase was 80%.And immobilized cellulase retained 60%of initial activity after catalyzing CMC for 6 cycles.3.Based on the visible-light-induced controlled/living graft polymerization,a dual-enzyme layered immobilization strategy was developed for the co-immobilization of ?-glucosidase(BG)and cellulase on the surface of LDPE films.Firstly,the PEG hydrogel layer was attached on the surface of LDPE by visible-light-induced graft polymerization,and BG in precursor solution was in situ encapsulated into the hydrogel layer.Due to the mild reaction conditions(visible light irradiation and room temperature),the immobilized BG could still maintain high activity.When the loading amount of BG was 0.025 mg/cm2,the immobilization efficiency of BG was about 94%.Additionally,the immobilized BG could retain about 93%of original activity compared with free BG.The reusability of immobilized BG was investigated using p-nitrophenol-?-D-glucopyranoside(p-NPG)and cellobiose as substrates,and more than 95%of the original activity was maintained after 10 cycles.On this basis,by utilizing the residual dormant group on the surface of PEG hydrogel layer,a secondary graft polymerization of AANa was performed under visible light,and a bilayer graft structure with the inner layer of PEG hydrogel and the outer layer of PAANa brush was formed.Cellulase was covalently bonded to PAANa brush by coupling reaction between amino group and carboxyl group.The activity of dual-enzyme system determined by CMC was 7.63 U/mg,which was 87.2%of free cellulase activity.Synergistic effect of the immobilized BG and cellulase was demonstrated as well.Compared with the single cellulase immobilization system and the isolated BG/cellulase immobilization system,the dual-enzyme system exhibited 82%and 20%increase in catalytic activity,respectively,using filter paper as the substrate.The reusability of the two enzymes was significantly improved after immobilization.The dual-enzyme system could maintain 93%of initial activity after repeating 10 cycles with CMC as substrate,and 89%of initial activity could be retained after 6 cycles when filter paper was used as the substrate.