Construction And Application Of Porous Aerogel Beads Immobilized Enzymes

Although immobilized enzyme has solved the problems of difficult separation,high production cost,and difficulty in repeated recovery and utilization of free enzyme,it is still the focus of research to find the appropriate fixation method,design the carrier with excellent synthesis performance and controllability,and optimize the application technology.Graphene has excellent chemical stability,good biocompatibility,large mechanical properties and large specific surface area,as well as hydrophobicity and lipophilicity,which are conducive to further application of immobilized enzymes in oil-water separation and catalytic hydrolysis of oil.Moreover,the porous network structure of chitin molecule and the abundant hydroxyl and amino groups in the molecule can assist glutaraldehyde crosslinking agent to form covalent bonds with the enzyme,so as to effectively solve the problem of enzyme leaching and improve the stability of the enzyme.Therefore,the preparation of porous beadss from graphene and chitin composites will have important value of application and research in immobilized enzyme.In this paper,an optimized activity of lipase can be achieved and tuned through chemical reduction of graphene oxide.Then,reduced graphene oxide beads（rGOB₄）had been fabricated through a facile self-assembly method.The morphology,specific surface area,and mechanical properties of the rGOB₄were investigated by scanning electron microscopy（SEM）,Brunauer-Emmett-Teller（BET）,and static mechanics properties test,respectively.The results revealed that the rGOB₄ showed a three-dimensional micro-nano porous structure and surface hydrophobicity,which played an important role in improving their adsorption properties and catalytic activity of Candida rugose lipase（CRL）.CRL was directly immobilized onto the beads by physical adsorption with the maximum adsorption capacity of 117.67 mg/g under the optimized condition,the highest activity observed was 1.6 times more than that of free lipases.The immobilized lipase（rGOB₄@CRL）exhibited good temperature,thermal,and reuse stability in comparison to free CRL.In the glycerolysis reaction of olive oil,the conversion rate of triglycerides（TG）in free enzyme reaction system was 67.83%,while the conversion rate of TG in rGOB₄@CRL reaction system could reach 74.87%,which was higher than that of the former.Moreover,rGOB₄@CRL was further used to adsorb emulsified oil due to the excellent oil-absorbing ability of rGOB₄,and then producing monoglyceride（MG）and diglyceride（DG）in situ by catalyzing the glycerolysis reaction.When the oil concentration was 4 mg/m L,the conversion rate of TG could reach 89.70%.Then,to improve the operational stability of immobilized enzyme,chitin/graphene oxide（Ch/GO）composite aerogel beads were fabricated by a simple and environmentally friendly method for lipase immobilization.The morphology and specific surface area of these beads were investigated by scanning electron microscopy（SEM）and Brunauer-Emmett-Teller（BET）,respectively.The results revealed that the prepared beads showed three-dimensional porous structure,and GO was successfully bound to chitin with enhanced surface area and mechanical strength.CRL was immobilized on Ch/GO aerogel beads under optimum conditions by adsorption and subsequent cross-linking with glutaraldehyde which is added in the presence of the unbound proteins by conjugation.The maximum immobilization capacity of Ch,Ch/GO-5,Ch/GO-10 were 129.60,142.03,152.03 mg/g_support.The immobilized lipases exhibited good temperature and thermal stability in comparison to free CRL.After recycling 5 times,the immobilized lipases remained more than90%of their initial activity and the activity of Ch/GO-10 could remain 84.2%of free CRL.With these desired characteristics,the design of Ch/GO composite aerogel beads for lipase immobilization may have potential applications in various lipase-based industrial processes.Finally,in order to further improve the porous structure of the material and the mass transfer rate of the immobilized enzyme,Ch/GO composite beads were obtained through emulsion template method.P ure chitin and Ch/GO composite solution was used to stabilize Pickering emulsion.The results of characterization showed that beads with hierarchical pore structure were obtained,and the specific surface area and pore volume of aerogel beads increased with the oil phase volume ratio in emulsion template,while mechanical strength was the opposite.Laccase was immobilized onto Ch/GO-30 aerogel beads and it was found that the optimum p H and temperature for the activity of immobilized laccase was 3.4 and 55℃.The immobilized laccase exhibited good thermal stability in comparison to free enzyme.After recycling 10times,the immobilized laccase remained more than 90%of their initial activity.It has also proved that laccase immobilized on Ch/GO-30 hierarchical porous beads performed well in dye degradation.The Ch/GO hierarchical porous aerogel spheres prepared by emulsion template method,with controllable pore size and number,significantly increased specific surface area and significantly improved mass tra nsfer rate,will have greater application potential in the field of immobilized enzymes.