Preparation Of Nanocellulose-based Immobilized Materials And Study On The Performance Of Its Immobilized Laccase

As bio-enzymatic treatment of water pollutants is a green and efficient method for pollution control,it has gradually become one of the development directions to resolve the problem of environmental water pollution in the future.Laccase can be used to catalyze the oxidative degradation of phenols,aromatic amines and aromatic carboxylic acids,etc.Because the enzymic catalytic reaction do not produce any toxic by-products,laccase has great application potential in the fields of lignin degradation,decolorization of dyeing wastewater,environmental remediation and so on.However,the disadvantages of free enzyme such as high cost,low stability and difficulty in recycling,limit its application in wastewater treatment.Immobilized enzyme technology is one of the most effective methods to solve the problems.The selection of carrier material,the structure and properties of carrier and the suitable immobilized methods are the important factors affecting the performance of immobilized enzyme.Nanocellulose has many excellent properties including high specific surface,large aspect ratio,ease of chemical modification by surface hydroxyl groups,and good biocompatibility.Its application as immobilized enzyme carrier has become one of the hot spots in nanocellulose research.In this thesis,four kinds of nanocellulose-based carrier materials for enzyme immobilization,including magnetic nanocellulose,nanocellulose/alginate composite hydrogel,nanocellulose/polyacrylamide cryogel,and ordered mesoporous carbon,were developed by functional design and material structure characterization.Laccase was immobilized onto the as-prepared carriers by physical adsorption or covalent binding method.The enzymatic properties of the immobilized laccase and free enzyme were comparatively studied.The applications of immobilized laccase in papermaking wastewater treatment,bisphenol A degradation,dye decolorization and pyrocatechol detection were also explored.The main contents of this doctoral thesis are summarized into four aspects:Firstly,novel magnetic nanocellulose were prepared via electrostatic self-assembly approach.The formation mechanism,surface morphology,structure and magnetic properties of magnetic nanocellulose were characterized by Zeta potential determination,FTIR,SEM,TG and PPMS.Nanocellulose was successfully combined with cationic polyethyleneimine modified Fe₃O₄ nanoparticles,and the electrostatic interaction between them was a key driving force.The magnetic nanoparticles were dispersed on the surface of nanocellulose,and the close interaction between them significantly improved the thermal stability of magnetic nanocellulose.The saturation magnetization of magnetic nanocellulose was 30.9 emu·g^-1,and it could be easily separated from the reaction mixture with an external magnet.Laccase was immobilized on the as-prepared magnetic nanocellulose by physical adsorption.Under the optimal conditions(i.e.,the p H is 6.0,initial concentration of the enzyme is 0.8 mg·m L^-1,and reaction time is 2 h),the protein loading of the carrier was 21.5 mg·g^-1,and the activity of the immobilized laccase was 332.8 U·g^-1.Compared with free enzyme,magnetic nanocellulose immobilized laccase showed wider p H suitability,higher thermal stability and storage performance.When the immobilized laccase was used to treat papermaking wastewater,the COD removal rate of the wastewater can reach more than 20%,the decolorization rate is more than 65%,and the cationic demand is reduced by 38%.Moreover,after magnetic separation and repeated use for 3 times,the immobilized laccase can still maintain a good treatment effect for papermaking wastewater.In order to further solve the problem of weak physical adsorption between enzyme and nanocellulose,the nanocellulose was modified by dopamine.A"bridge"was introduced that could be firmly covalently bound with enzyme molecules,and the immobilized laccase with high reusability was obtained.Dopamine was grafted onto the surface of oxidized nanocellulose by amidation of carboxyl group using EDC and NHS as activators.The modified nanocellulose was compounded with sodium alginate to form nanocellulose/alginate composite hydrogel.Laccase was immobilized onto the as-prepared hydrogel by covalent binding of dopamine and amino group of laccase protein.FTIR,SEM,EDX and enzyme activity analysis confirmed the covalent immobilization of laccase.When the initial enzyme concentration was2 mg·m L^-1 and the p H is 6.0,the optimal enzyme loading was 7.6 mg·g^-1.Meanwhile,the maximum enzyme activity was 462 U·g^-1.Compared with free enzyme,the p H stability,operating temperature range and storage performance of immobilized laccase were improved greatly.The optimum p H of immobilized laccase shifted towards acidic direction.The relative activity of it was more than 90%in p H 4.0～5.5.Although the optimum temperature dropped of5 ^oC,the enzyme activity of the immobilized laccase can still be more than 80%of the maximum activity at 40～65 ^oC.After 15 days of storage,91.8%of their initial enzyme activity remained.After 14 cycles of filtering separation and reuse,the immobilized laccase remained79.6%of its initial activity.The removal of BPA from polluted water by immobilized laccase was also studied.Operating at optimal conditions(i.e.,p H 6.0,50 ^oC,the initial concentration of BPA is 40 mg·L^-1,1.0 g of immobilized laccase),a BPA removal of 82.4%in 12 h was achieved.Degradation kinetics analysis showed that the catalytic degradation rate of BPA was about 1/5 of that of free laccase.However,after adding a small amount of ABTS into the reaction system,the removal rate of BPA by immobilized laccase were significantly increased,and the removal rate reached 98.7%within 12 h.In order to increase the enzyme loading of nanocellulose based carrier and obtain more active sites for immobilized enzyme,the nanocellulose/polyacrylamide with macroporous network structure was prepared.The cryogel was prepared via freezing polymerization method initiated by tetramethylenediamine and ammonium persulfate.In polymerization reaction,nanocellulose was used as skeleton material,acrylamide was monomer,N,N-methylene bisacrylamide was the crosslinking agent,and glycyl methacrylate was the material modifier.The FTIR and SEM characteristic analysis showed that nanocellulose had crosslinked with polyacrylamide to form a kind of hybrid cryogel.The cryogel is a three-dimensional network porous structure with an aperture of about 220μm.It can absorb water and the swelling ratio is up to 520%.After extrusion,it can be quickly restored.It also has good dimensional stability.Laccase was immobilized onto the as-prepared cryogel,where the enzyme’s amino groups react with the epoxy groups by ring-opening reaction.When the initial concentration of the enzyme was 5.0 mg·m L^-1and the p H was 5.0,the enzyme loading of the carrier was up to 51.7mg·g^-1.The immobilized laccase has outstanding thermal stability.When stored at 60 ^oC and70 ^oC for 7 h,it can even retain 36.2%and 24.3%of its initial activity.The immobilized laccase has good decolorization effect on the solution of Congo red,Trypan blue,sunset yellow,solid green FCF and chlorazol black,but it is p H dependent.The decolorization rates of Congo red and chlorozole black were 85.5%and 92.4%under p H 4.0,and 87.4%,93.6%and 92.5%for sunset yellow,solid green FCF and Trypan blue under p H 7.0.Moreover,after repeated use for 5 times,more than 60%of its decolorization effect can still be maintained.Finally,the ordered mesoporous carbon was prepared by evaporation induced self-assembly and high temperature carbonization of nanocellulose template with the pore-forming agent of tetramethoxysilane.The mesoporous carbon not only retained the chiral nematic structure of nanocellulose template,but also endowed the immobilized enzyme with conductivity.A novel enzyme electrode was fabricated by immobilization of laccase onto the as-prepared mesoporous carbon,and the electron transfer between the active center of laccase and the electrode was realized.The electrochemical behavior and performance of electro-catalytic oxidation-reduction for pyrocatechol were investigated.And the immobilized laccase electrode biosensor was successfully used for determination of pyrocatechol.The results suggest that the direct electrochemical reaction of pyrocatechol take place at the surface of enzyme electrode.The enzyme electrode biosensor had good and stable current response on pyrocatechol,and the linear detection for pyrocatechol ranged from 1.1 to 22.8μmol·L^-1 with a selective sensitivity of 0.016 A/（mol/L）and a detect limit of 0.217μmol·L^-1.