• Korean Journal of Food Science and Technology
• /
• v.11 no.3
• /
• pp.192-199
• /
• 1979

With cells of Streptomyces spp K-45 isolated from soil, the immobilization of glucose isomerase by a series of treatments ; heat, carefully manipulated drying, extrusion with a thickening agent, and glutaraldehyde-induced crosslinking, was presented. This was aimed to obtain a mechanically stable form of whole cell containing glucose isomerase. The resulted pellet form had a good mechanical strength, compared with a commercial product, and showed 26 % of the activity recovery. The specific activity was 48.1 units per g of the dry material. The immobilized glucose isomerase generally showed properties similar to those of the soluble enzyme ; optimal pH at $7.5{\sim}9.0$, optimal temperature at $80{\sim}85^{\circ}C$, activation energy of 10.9 kcal/mole, and $K_m$ for glucose of 10.9M. The immobilized enzyme was very thermostable and pH stable.

• KSBB Journal
• /
• v.15 no.1
• /
• pp.42-48
• /
• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• Microbiology and Biotechnology Letters
• /
• v.43 no.3
• /
• pp.300-305
• /
• 2015

The glutamate decarboxylase gene (gadB) from Lactobacillus plantarum WCFS1 was cloned and expressed as an N-terminal hexa-histidine-tagged fusion protein in Escherichia coli BL21 (DE3) as the host strain. Purified glutamate decarboxylase (GAD) was immobilized onto porous silica beads by covalent coupling. The pH dependence of activity and stability of the immobilized GAD was significantly altered, when compared to those of the free enzyme. Immobilized GAD was stable in the range of pH 3.5 to 6.0. The resulting packed-bed reactor produced 41.7 g of γ-aminobutyric acid/l·h at 45℃.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.666-670
• /
• 2001

Levofloxacin is L-form stereoisomer of ofloxacin. It has better antibacterial activity than D-oflxacin. In this study, levofloxacin was produced enantioselectively by using high density culture of recombinant E. coli containing a foreign esterase gene. Final cell concentration was 89 g/L at the end of fed-batch culture and the cells were used for levofloxacin production after IPTG induction at the optimized condition. For the immobilization of recombinant E. coli. 1.5% sodium alginate showed the best result to maintain enzyme activity and enantioselectivity.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.468-472
• /
• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• Proceedings of the Korean Society of Sericultural Science Conference
• /
• 2003.10a
• /
• pp.55-56
• /
• 2003

Silk fibroin (SF) is one of the typical protein polymer produced by silkworm, Bombyx mori. SF has been used as textile fiber and surgical suture fur thousands of years due to its unique gloss, handle, and mechanical properties. Recently, SF has been intensively studied to diverse usage for biotechlological and biomedical fields because of their reproducibility, environmental compatibility, non-toxicity, and biological compatibility. Based on its biocompatibility, the possible uses of regenerated SF have been proposed including substrate for cell culture[1], enzyme immobilization[2], and matrix for drug release[3]. (omitted)

• BMB Reports
• /
• v.38 no.5
• /
• pp.517-525
• /
• 2005

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

• KSBB Journal
• /
• v.29 no.4
• /
• pp.239-243
• /
• 2014

Recently, there is a growing interest in efficient biomass pretreatment and saccharification processes to produce biofuels and biochemicals from renewable non-food biomass resources. In this study, glucose was produced from cellulose by immobilizing cellulase enzyme on chitosan beads which was reported to have high pH and temperature stability. The immobilized amounts of cellulase on chitosan beads linearly increased with increasing the concentrations of cellulase solution. The glucose production increased to 7.2 g/L from 1% carboxymethyl cellulose (CMC) substrate when immobilized at 20% cellulase solution. The maximum specific activity was 0.37 unit/mg protein when immobilized at 8% cellulase solution. At pH 7 and $37^{\circ}C$, the optimum reaction composition was 0.5 g beads/L from 1% CMC substrate. At this condition, the conversion to glucose completed at ca. 20 min.

• Proceedings of the KIEE Conference
• /
• 1999.11d
• /
• pp.984-986
• /
• 1999

In the case of immobilizing of glucose oxidase in organic polymer using electrosynthesis, the glucose oxidase obstructs charge transfer and mass transport during the film growth. This may lead to short chained polymer and make charge-coupling weak between the glucose oxidase and the backbone of the polymer. That is mainly due to insulating property and net chain of the glucose oxidase. Such being the case, it is useless to increase in amount of glucose oxidase more than reasonable in the synthetic solution. We establish by means of qualitative analysis that amount of immobilized glucose oxidase can be improved by adding a hole ethyl alcohol in the synthetic solution. As ethyl alcohol was added by 0.1mol $dm^{-3}$ in the synthetic solution, the faradic impedance of resultant electrode was increased about five times as much as the case of ethyl alcohol free in the solution, and mass transport was limited more than over. That is due to insulating property and net chain of the glucose oxidase. Moreover, in ultraviolet spectra of the synthetic solution, the adsorption peak at 285nm corresponding to glucose oxidase was decreased. It suggests increase in amount of immobilized glucose oxidase.

• Bulletin of the Korean Chemical Society
• /
• v.23 no.3
• /
• pp.385-390
• /
• 2002

Laccase could be successfully assembled on an amine-derivatized platinum electrode by glutaraldehyde coupling. The enzyme layer formed on the surface does not communicate electron directly with the electrode, but the enzymatic activity of the surf ace could be followed by electrochemical detection of enzymatically oxidized products. The well-known laccase substrates, ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) and PPD (p-phenylenediamine) were used. ABTS can be detected down to 0.5 ${\mu}M$ with linear response up to 15 ${\mu}M$ and current sensitivity of 75 nA/ ${\mu}M.$ PPD showed better response with detection limit of 0.05 ${\mu}M$, linear response up to 20 ${\mu}M$, and current sensitivity of 340 nA/ ${\mu}M$ with the same electrode. The sensor responses fit well to the Michaelis-Menten equation and apparent $K_M$ values are 0.16 mM for ABTS and 0.055 mM for PPD, which show the enzymatic reaction is the rate-determining step. The laccase electrode we developed is very stable and more than 80% of initial activity was still maintained after 2 months of uses.