• 생명과학회지
• /
• 제20권11호
• /
• pp.1656-1661
• /
• 2010

난황단백질 가수분해를 위한 알칼리성 단백질분해효소를 5가지 담체 Duolite A568, Celite R640, Dowex-1, Dowex 50W 그리고 Silica gel R60 에 고정화하였다. Duolite A568의 경우에 24.7%의 최대 고정화 효율을 나타내었다. 자유 효소와 고정화 효소에 대한 최적의 pH는 각각 8과 9였고, 최적의 pH는 고정화에 의해 염기성으로 1만큼 증가하였다. 그러나 최적 온도는 자유 효소와 고정화 효소 모두 $50^{\circ}C$로 같았다. 고정화 효소가 자유 효소에 비해 높은 열 안정성을 보였다. 재사용 회분식 공정에서 10 cycle 동안 효소활성은 초기 활성의 86%를 유지하였다. 연속 공정을 위한 충진층 반응기에서 여러 유속에 대한 장기 조업에서 효소 활성의 안정성 평가하였는데 낮은 유속일수록 높은 활성을 유지하였다. 연속 조업에서 casein과 난황 단백질을 사용하여 원료에 대한 고정화 효소의 활성에 대한 영향을 조사하였다. 96시간 연속 조업에서 casein의 경우는 초기 활성의 83%를 유지하였고 난황 단백질의 경우는 초기 활성의 61%를 유지하였다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제8권4호
• /
• pp.257-262
• /
• 2003

A straightforward and effective method is presented for immobilizing enzymes on a microchip platform without chemically modifying a micro-channel or technically microfabricating a column reactor and fluid channel network. The proposed method consists of three steps: the reconstitution of a nitrocellulose (NC) membrane on a plane substrate without a channel network, enzyme immobilization on the NC membrane, and the assembly of another substrate with a fabricated channel network. As a result, enzymes can be stably and efficiently immobilized on a microchip. To evaluate the proposed method, two kinds of enzymatic reaction are applied: a sequential two-step reaction by one enzyme, alkaline phosphatase, and a coupled reaction by two enzymes, glucose oxidase and peroxidase, for a glucose assay.

• Bulletin of the Korean Chemical Society
• /
• 제18권1호
• /
• pp.44-50
• /
• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제27권2호
• /
• pp.329-332
• /
• 2013

In present preliminary report, we showed the possibility of silk sericin (SS) in enzyme immobilization. SS beads were prepared and enzymes were immobilized on it. The specific activity of immobilized a-chymotrypsin retained more than 87% compared to the free enzyme. The immobilized a-chymotrypsin has better stability against ethanol especially those immobilized on SS beads coagulated in methanol. Immobilized trypsin and lipase had also comparable apparent activity compared to free enzyme. Our result indicates that SS could be a good candidate for enzyme immobilization support due to its hydrophilicity.

• Journal of Microbiology and Biotechnology
• /
• 제17권9호
• /
• pp.1430-1436
• /
• 2007

[ ${\alpha}$ ]-Galactosidase was immobilized in a mixture of k-carrageenan and locust bean gum. The properties of the free and immobilized enzyme were then determined. The optimum pH for both the soluble and immobilized enzyme was 4.8. The optimum temperature for the soluble enzymes was $50^{\circ}C$, whereas that for the immobilized enzyme was $55^{\circ}C$. The immobilized enzyme was used in batch, repeated batch, and continuous modes to degrade the raffinose-family sugars present in soymilk. Two hours of incubation with the free and immobilized ${\alpha}$-galactosidases resulted in an 80% and 68% reduction in the raffinose oligo saccharides in the soymilk, respectively. In the repeated batch, a 73% reduction was obtained in the fourth cycle. A fluidized bed reactor was also designed to treat soymilk continuously and the performance of the immobilized ${\alpha}$-galactosidase tested at different flow rates, resulting in a 90% reduction of raffinose-family oligosaccharides in the soymilk at a flow rate 40 ml/h. Therefore, the present study demonstrated that immobilized ${\alpha}$-galactosidase in a continuous mode is efficient for reducing the oligosaccharides present in soymilk, which may be of considerable interest for industrial application.

• Journal of Microbiology and Biotechnology
• /
• 제8권6호
• /
• pp.696-699
• /
• 1998

Biological oxidation of D-sorbitol to L-sorbose using permeated and immobilized cells of Gluconobacter suboxydans was carried out to investigate the optimum reaction condition. The stabilization effect of cross-linking agents such as glutaraldehyde, tannic acid, and polyethylene imine to prevent the leakage of enzymes from beads containing permeated and immobilized cells of G. suboxydans was examined by the production of L-sorbose from the mixture of D-sorbitol and gluconic acid. The protein concentration effused from immobilized beads treated with only glutaraldehyde was $5.2\mug/m\ell$ after 20 h. The beads of G. suboxydans immobilized with alginate and cross-linked with 0.3% glutaraldehyde was the most useful for the oxidation of D-sorbitol to L-sorbose.

• Membrane and Water Treatment
• /
• 제11권3호
• /
• pp.195-200
• /
• 2020

In this study, a biocatalyst composite was prepared by immobilizing oxidoreductases onto Cu-activated zeolite to facilitate biochemical decomposition of 4-chlorophenol (4-CP). 4-CP monooxygenase (CphC-I) was cloned from a 4-CP degrading bacterium, Pseudarthrobacter chlorophenolicus A6, and then overexpressed and purified. Type X zeolite was synthesized from non-magnetic coal fly ash using acetic acid treatment, and its surfaces were coated with copper ions via impregnation (Cu-zeolite). Then, the recombinant oxidative and reductive enzymes were immobilized onto Cu-zeolite. The enzymes were effectively immobilized onto the Cu-zeolite (79% of immobilization yield). The retained catalytic activity of CphC-I after immobilization was 0.3423 U/g-Cu-zeolite, which was 63.3% of the value of free enzymes. The results of this study suggest that copper can be used as an effective enzyme immobilization binder because it provides favorable metalhistidine binding between the enzyme and Cu-zeolite.

• Bulletin of the Korean Chemical Society
• /
• 제30권1호
• /
• pp.57-60
• /
• 2009

Alcohol oxidase catalyzes the oxidation of short lines alcohol to aldehyde. In this study, alcohol oxidase from Hansenula polymorpha (HpAOD) was induced by addition of 0.5% methanol as the carbon source and purified to electrophoretic homogeneity by column chromatographies. The purified HpAOD was immobilized with DEAE-cellulose particles and its biochemical properties were compared with those of free enzyme. The substrate specificity and the optimum pH of immobilized enzyme were similar to those of free enzyme. On the other hand, the Km values of free and immobilized enzymes for ethanol were 6.66 and 14.65 mM, respectively. The optimum temperature for free enzyme was ${50^{\circ}C}$, whereas that for immobilized enzyme was ${65^{\circ}C}$. Immobilized enzyme showed high stability against long storage. Immobilized enzyme was also tested for the enzymatic determination of ethanol by the colorimetric method. We detected 1 mg/liter ethanol ($1{\times}10^{-4}$% ethanol) by 2,6- dichloroindophenol system. Therefore, the present study demonstrated that immobilized HpAOD has high substrate specificity toward ethanol and storage stability, which may be of considerable interest for alcohol biosensor and industrial application.

• Journal of Pharmaceutical Investigation
• /
• 제15권3호
• /
• pp.93-99
• /
• 1985

For the effective immobilization of urease on chitosan matrix with glutaraldehyde, optimal activation methods were studied, and its enzymatic properties was investigated. In the stability of enzyme. the retained activity of the native urease was 55% after it was soaked af pH 7 for 10 hrs., while the retained activity of immobilized one was about 62% after soaked at pH 6.5-8.5 for the same time. After heat treatment at $60^{\circ}C$ for 10 hrs., the native urease lost the most of its activity, while immobilized urease retained 54% of its activity by the same treatment. The retained activity of immobilized urease did not decrease nearly when it was stored at room temperature for 25 days. From Linweaver-Burk plots, the $V_{max}$ value of native urease was $66{\mu}M/l$ and that of immobilized urease was $41{\mu}M/l$, while Km value 40mM/l for both enzymes was unaltered.

• 한국식품영양과학회지
• /
• 제20권5호
• /
• pp.494-502
• /
• 1991

Diazotized chitin(CHITN) as supports of immobilized enzyme, which was obtained by alkaline hydrolysed chitin with NaN3 and HCI was employed to produce CHITN-Gase with glutaraldehyde as bifunctional reagent. Activities of CHITN-Gase were determined with reaction of p-nitro-pheol-$\beta$-D-glucopyranoside(PNG) in plug flow reactor as a reference of CHITA-Gase. Their optimum temperature, pH, Km and Vmax, mass transfer coefficient (h), effctiveness factor(η)were plotted with variation of flow rate and H/D. Mass transfer coefficient(h) of those enzymes increased because of their flux, as flow rates were increased and controlled by reaction rate. Effectiveness factor(η) of both enzymes were nearly 1.0.