Preparation And Application Of Immobilized Enzyme Onto Magnetic Nanocrystalline Cellulose

Enzymes are important and highly efficient natural catalysts.In recent years,the use of enzyme in industrial biotechnology has been exploded.However,the drawbacks of free enzymes,including high cost,poor operational stability,and challenges in recovery and reuse,have limited industrial applications of enzymes.Enzyme immobilization by using novel enzyme carriers and novel immobilization process were both efficient ways of solving such problems.In recent years,nanocrystalline cellulose(NCC)has gained increasing attention in the enzyme immobilization because of their excellent physical chemistry properties.Nevertheless,the stable dispersion of the NCC in aqueous solution makes it difficult to recycle from the reaction system,this limiting their applications.Thus,it is particularly meaningful to develop a new technique for preparation of the magnetic NCC(MNCC).Also,Immobilization of enzyme onto the MNCC carrier via novel enzyme immobilization methods is significative.Dipeptides,used as functional food ingredients,play an important role in the food and pharmaceutical industry.Enzymatic synthesis of dipeptides has become more favorable due to its mild catalytic conditions but suffer from low yield.Recently,deep eutectic solvents(DESs)have emerged as promising green alternatives to conventional and unconventional solvents(such as ionic liquids).As inexpensive,sustainable and biocompatible solvents,DESs have been successfully employed for biocatalytic process with improved activity and stability of enzyme as well as minimized side effects.Thus,investigation of the enzymatic dipeptide synthesis in DES-containing media is interesting and significant.This dissertation carried out the following aspects of research and exploration:Novel MNCC-A and MNCC-B were prepared via coprecipitation-electrostatic-self-assembly technique and coprecipitation-crosslinking technique,respectively.Results showed that MNCCs exhibited rod-like morphologies and the Fe3O4 MNPs were well-dispersed onto the surface of the NCC.MNCCs exhibited the characteristic diffraction peaks of both NCC and Fe3O4 MNP,but the relative intensity of the Fe3O4 MNP decreased.In order to improve the saturated magnetizations of MNCC,epichlorohydrin was used to replace sodium tripolyphosphate as the cross-linker and formed MNCC-B.The highest saturated magnetization of MNCC-B was significantly higher than that of MNCC-A(10.1 emu/g v.s 6.9 emu/g).Increase of the ferric salts and decreased of the chitosan content could increase the saturated magnetizations of the MNCC.The mechanism study showed that(1)the electrostatic interaction between NCC and chitosan and that between chitosan and Fe3O4,were the key driving forces for the MNCC-A formation;(2)the electrostatic interaction between NCC and chitosan,and the crosslinking of chitosan and NCC by epichlorohydrin,were the key driving forces for the MNCC-B formation.These results demonstrated that the coprecipitation-electrostatic-self-assembly technique and coprecipitation-crosslinking technique were both effective methods to prepared MNCC.Papain was immobilized onto MNCC-B-3 enzyme carrier by a traditional activation-crosslinking process.The results showed that under the optimal conditional the papain was immobilized onto MNCC with an activity recovery 74.5% and a protein loading amount 8.9 mg/g.The immobilized papain exhibited higher stability and catalytic performance than free enzyme.Also,the pH and temperature adaptability was superior to its free counterpart.A cheap and efficient domestic papain was successfully immobilized onto the MNCC-B-3 by a precipitation-cross-linking method.The optimal preparation conditions was as followed: 1.5 mg PA was incubated with 4.5 mg MNCC in 0.5 m L pH 7.0 PBS at 0oC and then precipitated with 7.5 m L anhydrous alcohol and cross-linked with 1.92% GA for 2 h.The resulting nano-biocatalyst PA@MNCC showed high PA loading(333 mg/g)and enzyme activity recovery(more than 80%).The stabilities,catalytic performance and enzyme-substrate affinity of PA@MNCC were greatly superior to that of its free counterpart.Also,the pH and temperature adaptability were superior to its free counterpart.The FTIR spectroscopy assay revealed that the ?-helix content of PA@MNCC was significantly higher than that of free PA.This might explain the enhancement of the structure rigidity and stabilities of the PA@MNCC after immobilization.Finally,five enzymes were successfully immobilized onto MNCC by using the precipitation-cross-linking method.These results demonstrated that this is a universal applicable enzyme immobilization technology.In order to investigate the interaction between papain and MNCC carrier,molecular simulation was performed by using Gromacs software.Interaction could be observed between MNCC and papain according to the radial distribution function(rdf)and portential energy analysis,and the average distance of papain and MNCC decrease with the increase of interaction intensity.Also,the increase of the structural stability of the papain-MNCC composite might be attributed to(1)the presence of MNCC reduced the number of H-bonds between papain and water and formed new hydrogen bond between MNCC and papain;(2)in the catalytic triad Cys25-Hisl59-Asnl75,the distance between Hisl59-Asnl75 decreased significantly.Finally,the secondary structure content assay showed an increase of ?-Helix and a decrease of loop.Furthermore,the as-prepared PA@MNCC was successfully used as an efficient biocatalyst for the synthesis of N-(benzyloxycarbonyl)-alanyl-glutamine(Z-Ala-Gln)dipeptide in the DES-containing media,with a high yield(about 71.5%).The optimal reaction condition were as followed: the water contents of the media,optimal temperature,acyl donor/nucleophile ratio,triethylamine concentration were found to be 16.6%,50 oC,2.5-3,560 mM,and the obtained yield of Z-Ala-Gln was about 71.5%.Based on these meaningful results,Z-Ala-His was enzymatic synthesized by PA@MNCC in DES solution.Under the optimal reaction condition,the obtained yield of Z-Ala-His was about 68.4%.Moreover,the yield of Z-Ala-His via PA@MNCC-catalytic reaction in different DES solutions was compared investigated.Results illustrated that the enzymatic dipeptide synthesis method based on PA@MNCC catalyst and DES-containing media is effectual.This study not only enriches our knowledge of the enzyme immobilization,but also provides an efficient and novel method for the enzymatic-catalytic synthesis of dipeptide.