• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.6
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• pp.797-802
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• 1993

The properties of the alcohol-oxidase from yeasts assimilating of methanol(Hansenula polymorpha CBS 4732, Pichia pastoris CBS 2612 and Candida boidinii CBS 8106) as free(cellules, purified enzymes) and immobilized forms(immobilized cellules, immobilized enzymes) were investigated. Immobilization enhanced the activity and stability of alcohol-oxidase to a certain degree. The optimum temperature of the immobilized alcohol-oxidase was lower than those of the free forms. The pH / activi쇼 profiles of alcohol-oxidase did not change by immobilization, but changed by the microorganisms. When the immobilized cellules were stocked at 4$^{\circ}C$ in 10mM potassium phosphate buffer(pH 7.5 or 8.0), alcohol-oxidase was more stable than those were stocked in potassium phosphate buffer containing 0.65M sucrose. The immobilization modifies the conditions of oxidation on the various substrates. alcohol-oxidase in immobilized forms showed some with higher Km value for methanol than that in free ones.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.20-24
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• 1992

In order to reuse inulinase effectively, a method for immobilizing both endo- and exoinulinase to vinylsulfone activated agarose via covalent bond was investigated. The immobilized enzyme preparation had, respectively, 400 U for exoinulinase activity and 80 U for endoinu- Iinase activity per gram gel. A thermal stability by immobilization had increased in the case of exoinulinase. Optimum pHs for two immobilized enzymes were 4.4 to 5.0. Synergistic effect which depends on mixed ratio of two immobilized enzymes was the best when the mixed ratio of endo/exo lay between 0.1 and 0.5, and its activity of the mixed enzyme increased 1.7 times as compared to that of each immobilized enzyme. Inulinase activities of both of the immobilized enzymes did not change during 20 times experimental runs in a batch reactor.

• KSBB Journal
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• v.13 no.5
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• pp.577-584
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• 1998

A sensitive membrane strip assay for plasma lipoprotein cholesterol that can be performed without handling reagents has been investigated. We previously developed an assay system with immobilized enzymes (cholesterol esterase and cholesterol oxidase) on the surfaces of nitrocellulose membrane(1). In such a case, the amount of enzymes present on the membrane was limited by its surface area and, thus, the detection capability was relatively poor (> 50 mg/dL cholesterol). To overcome this problem, we devised a new system with non-immobilized enzymes by placing them within interstitial spaces of a celullose membrane pad in a dry state. Upon contact with sample medium, the enzymes were immediately dissolved and participated in the reactions with cholesterol in a liquid phase. We constructed a user-friendly system consisting of four membrane pads fro sample application, cholesterol decomposition, color development as signal, and medium absorption to invoke a continuous flow (sequential location from the bottom). A sample containing lipoproteins was added into the application pad by capillary action and transferred to the next pad for decomposition. The decomposition pad (namely, enzyme pad) contained a detergent (sodium cholate) for the destruction of lipoprotein particles, the two enzymes for cholesterol decomposition, and a chromogen (3,3'-diaminobenzidine). As a consequence of the enzyme reactions, hydrogen peroxide was produced, and then reacted in the presence of the chromogen with horseradish peroxidase immobilized on the signal generation pad. Finally, a colorimetric signal directly proportional to the cholesterol concentration was produced. The detection limit determined from this system under optimal conditions was at least 2 times lower than of the enzyme-immobilized system.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.225-227
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• 2012

Lignin degrading enzymes from white rot fungi show broad substrate specificities, and therefore they can degrade variety of recalcitrant compounds. We have used three different protocols for the generation of immobilized laccase and manganese peroxidase crude enzymes from the genetically transformed strains of Merulius tremellosus Fr. These immobilized enzymes were used in the decolorization of Remazol Brilliant Blue R (RBBR), and they showed about 75% decolorization rates during the 48 h reactions. Although the decolorization efficiency decreased by 10-15% after a repeated use of the immobilized enzymes, these can be reused in various degrading reactions.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.718-722
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• 2015

Techniques for immobilizing effective enzymes on nanoparticles for stabilization of the activity of free enzymes have been developing as a pharmaceutical field. In this study, we examined the effect of three different pH conditions of phosphate buffer, as a dissolving solvent for lysosomal enzymes, on the direct immobilization of lysosomal enzymes extracted from Hen's egg white and Saccharomyces cerevisiae. Titanium(IV) oxide (TiO₂) nanoparticles, which are extensively used in many research fields, were used in this study. The lysosomal enzymes immobilized on TiO₂ under each pH condition were evaluated to maintain the specific activity of lysosomal enzymes, so that we can determine the degree of melanin treatment in lysosomal enzymes immobilized on TiO₂. We found that the immobilization efficiency and melanin treatment activity in both lysosomal enzymes extracted from Hen's egg white and S. cerevisiae were the highest in an acidic condition of phosphate buffer (pH 4). However, the immobilization efficiency and melanin treatment activity were inversely proportional to the increase in pH under alkaline conditions. In addition, enhanced immobilization efficiency was shown in TiO₂ pretreated with a divalent, positively charged ion, Ca²⁺, and the melanin treatment activity of immobilized lysosomal enzymes on TiO₂ pretreated with Ca²⁺ was also increased. Therefore, this result suggests that the immobilization efficiency and melanin treatment activity of lysosomal enzymes can be enhanced according to the pH conditions of the dissolving solvent.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.692-695
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• 2003

In this work immobilization technique of enzymes onto the magnetic nanoparticles has been developed for biochip applications. Glucose oxidase and lactate dehydrogenase were immobilized on magnetic nanoparticles via cyanamide and glutaraldehyde. Immobilized enzymes had good operational and storage stability The immobilized glucose oxidase and lactate dehydrogenase were characterized by some factors(pH, temperature, and components of buffer solution etc) which affect the activity, In order to characterize the magnetic nanoparticles, we have used transmission electron microscopy(TEM), X-ray diffraction(XRD) and Fourier transform infrared(FTIR).

• 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.

• KSBB Journal
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• v.23 no.4
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• pp.350-354
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• 2008

Magnetically separable immobilized trypsin was developed and their biocatalytic activity was evaluated for the different immobilization media. The activity, recyclability, pH effect, and stability of immobilized enzymes were evaluated for the different supporting media. The biocatalytic activity of immobilized trypsin was highest with magnetically separable polyaniline (PAMP), and Vm and Km of PAMP were 0.169 mM/min and 0.263 mM respectively. With increasedpH, the biocatalytic activity increased for all supporting materials used. Immobilized enzymes were recycled and recycle activities were over 90% of their original activity after ten times reuse. The operational stabilities of enzymes were greatly improved with enzyme immobilization.

• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.18-21
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• 1996

A continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized on a high porous resin, Diaion HPA25. The optimum pH(5.5) and temperature(55$^{\circ}C$) of the enzyme for activity was unaltered by immobilization, and the immobilized enzyme became less sensitive to the pH change. The optimal operation conditions of the immobilized enzyme column for maximizing the productivity were as follows: 600g/L of sucrose feed concentration, flow rate of superficial space velocity 2.7h-1. When the enzyme column was run at 50$^{\circ}C$, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days of continuous operation, during which high productivity of 1174g/L$.$h was achieved. The kinds of products obtained using the immobilized enzyme were almost the same as those using soluble enzymes or free cells.

• 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.