• Applied Science and Convergence Technology
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• v.26 no.6
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• pp.157-163
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• 2017

Immobilized enzymes have become the subject of considerable interest due to their excellent functional properties such as reusability, cost-effectiveness, and optimality during the past decades. Enzyme immobilization technology is not only used in industrial processes, but also a component technology of products for medical diagnostics, therapy, food industry, bio energy, and biomaterial detection. In this review, new methods for enzyme immobilization are introduced, and the advantages and disadvantages of a variety of techniques in enzyme immobilization will be also discussed.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.512-518
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• 2023

To address inherent weaknesses such as low mechanical strength and limited enzyme loading capacity in conventional chitosan or alginate beads, an additional step involving the exchange of anionic surfactants with hydroxide ions was employed to prepare porous chitosan hydrogel capsules for enzyme immobilization. Consequently, excellent thermal stability and long-term storage stability were confirmed. Furthermore, coating the porous chitosan hydrogel capsules with polydopamine not only improved mechanical stability but also exhibited remarkable enzyme immobilization efficiency (97.6% for M1-D0.5). Additionally, it was demonstrated that the scope of application for chitosan hydrogel beads, prepared using conventional methods, could be further expanded by introducing an additional step of polydopamine coating. The enzyme immobilization matrix developed in this study can be selectively applied to suit specific purposes and is expected to be utilized as a support for the adsorption or covalent binding of various substances.

• BMB Reports
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• v.44 no.2
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• pp.87-95
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• 2011

Enzymatic catalysis has been pursued extensively in a wide range of important chemical processes for their unparalleled selectivity and mild reaction conditions. However, enzymes are usually costly and easy to inactivate in their free forms. Immobilization is the key to optimizing the in-service performance of an enzyme in industrial processes, particularly in the field of non-aqueous phase catalysis. Since the immobilization process for enzymes will inevitably result in some loss of activity, improving the activity retention of the immobilized enzyme is critical. To some extent, the performance of an immobilized enzyme is mainly governed by the supports used for immobilization, thus it is important to fully understand the properties of supporting materials and immobilization processes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties. Furthermore, a great many work has been done in order to improve the activity retention and stabilities of immobilized enzymes. Some introduce a spacer arm onto the support surface to improve the enzyme mobility. The support surface is also modified towards biocompatibility to reduce non-biospecific interactions between the enzyme and support. Besides, natural materials can be used directly as supporting materials owning to their inert and biocompatible properties. This review is focused on recent advances in using polymeric materials as hosts for lipase immobilization by two different methods, surface attachment and encapsulation. Polymeric materials of different forms, such as particles, membranes and nanofibers, are discussed in detail. The prospective applications of immobilized enzymes, especially the enzyme-immobilized membrane bioreactors (EMBR) are also discussed.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.100-108
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• 1991

Molecular characteristics and improving methods for thermal stability of enzyme have been considered. Intrinsic and extrinsic stabilizing mechanisms are two governing principles for enhanced thermal stability of enzyme in molecular basis. Factors contributing to the former and the latter mechanisms may be involved in the enhanced thermal stability of enzyme complementarily. Also, the methods for improving thermal stability of enzyme which comprise reaction in organic solvent system, chemical modification, immobilization, sequential unfolding and refolding, gene manipulation techniques and enzyme-antibody complexing are reviewed.

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

• Current Research on Agriculture and Life Sciences
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• v.3
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• pp.166-173
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• 1985

Several enzyme immobilization methods has been compared for immobilization of pepsin. Carboxymethyl cellulose and diethylaminoethyl cellulose were activated with Hcl and with NaOH, and were used for immobilization of pepsin. Sepharose-4B was activated cyanogen bromide, and was used for immobilization of pepsin. Porous glass beads were derivatized with 3-aminopropyitrlethoxysilane and with succinicanhydride, and were used for immobilization of pepsin. The results abtained were summarized as follow, 1. 10 mg/gr. dry bead and 15mg/gr. dry bead of pepsin were absorbed to CM-cellulose and DEAE-cellulose, 20 mg/gr. dry bead and 27 mg/gr. dry bead were coupled to CM-cellulose and DEAE-cellulose with glutaraldehyde respectively. Enzyme yields were 22% and 24% of soluble pepsin. 2. 16 mg/gr. dry bead of pepsin was attached to cyanogen bromide activated sepharose-4B, 19mg/gr. dry bead was cross linked to the activated bead with glutaraldehyde. Immobilized enzyme activity was 23% of soluble pepsin. 3. 40 mg/gr. dry bead of pepsin was conjugated to the derivatized glass beads. Immobilized enzyme activity was 45% of soluble pepsin.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1978.04a
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• pp.96.2-96
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• 1978

Commerial naringinase from Aspergillus niger was partially purified by various methods, and was immobilized to porous alkylamine silica of 30~40 mesh and 400 $\AA\pm10％$ pore diameter that had been activated with 2.5％ glutaraldehyde. About 50~70％ of initial naringinase activity was recovered after the immobilization process. Some enzymatic properties of the immobilized naringinase was investigated and compared with those of the native enzyme. The optimal temper-ature had moved from $40^{\circ}C$ to $55^{\circ}C$ and the heat stability of the immobilized enzyme was better than that of the native naringinase. But no signi-ficant diference in the pH effect on activity was detected. The activation energy of reaction, Ea, was markedly decreased from 14.9 to 8.64 (Kcal/mole) by immobilization.

• Journal of the Korean Wood Science and Technology
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• v.29 no.3
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• pp.104-111
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• 2001

There are some evidence that active enzymatic proteins, e.g. fungal laccase, exist in the naturally occured soil humus. This study was performed to investigate the covalent binding of fungal laccase to the humic acid-iron complex, and to measure laccase activity of immobilized ones. Seven methods were adopted to form the covalent binding of fungal laccase with soil humic acids complexed with iron. Using these seven methods it was possible to change the dimension of spacer arm between laccase and support, and also to regulate the mode of covalent binding of this enzyme. The spacer arm was regulated from 2C to 11C. There was not observed any straight relationship between the spacer arm longitude and the laccase activity after immobilization, but the binding mode more effective than the former. Three out of the seven methods gave the high activity of immobilized laccase, and which active products of laccase immobilization was stable up to 10 days after the process. It is indicated that natural soil condition might be prevented the laccase activation by the toxic influence of some phenolic humic compounds. It was shown, for the first time, the possibilities to obtain the high activity of fungal laccase by binding to humic acids, and especially in complex with iron.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.56-60
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• 1990

A bacterial strain of Brevibacterium sp. CHI was isolated from soil and used to produce an enzyme (nitrile hydratase) necessary for carrying out the bioconversion of acrylonitrile to acrylamide. Various immobilization methods and enzyme stability were investigated. The nitrile hydratase showed the maximum stability at pH 7 for the free cells. EDTA and phenyl methyl sulfonyl fluoride were selected as the protease inhibitor and the enzyme stability was evaluated by changing inhibitor concentration. Acrylamide beads were the best carriers among four carriers we tested in terms of stability and physicoehemical strength. The storage stability of the immobilized cells decreased with increasing acrylamide concentration of the gel phase at 4$^{\circ}C$, and was very low at acrylarnide concentration above 25%.

• Journal of Electrochemical Science and Technology
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• v.8 no.2
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• pp.87-95
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• 2017

Enzymatic fuel cells are promising low cost, compact and flexible energy resources. The basis of enzymatic fuel cells is transfer of electron from enzyme to the electrode surface and vice versa. Electron transfer is done either by direct or mediated electron transfer (DET/MET), each one having its own advantages and disadvantages. In this study, the DET and MET of laccase-based biocathodes are compared with each other. The DET of laccase enzyme has been studied using two methods; assemble of needle-like carbon nanotubes (CNTs) on the electrode, and CNTs/Nafion polymer. MET of laccase enzyme also is done by use of ceramic electrode containing, ABTS (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid]) /sol-gel. Cyclic voltammetric results of DET showed a pair of well-defined redox peaks at $200{\mu}A$ and $170{\mu}A$ in a solution containing 5and $10{\mu}M$ o-dianisidine as a substrate for needle-like assembled CNTs and CNTs-Nafion composite respectively. In MET method using sol-gel/ABTS, the maximum redox peak was $14{\mu}A$ in the presence of 15 M solution o-dianisidine as substrate. The cyclic voltammetric results showed that laccase immobilization on needle-like assembled CNTs or CNTs-Nafion is more efficient than the sol-gel/ABTS electrode. Therefore, the expressed methods can be used to fabricate biocathode of biofuel cells or laccase based biosensors.