• KSBB Journal
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• 제28권4호
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• pp.225-229
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• 2013

Bovine Carbonic anhydrase (BCA) was immobilized on a submicro-porous membrane through covalent immobilization. The immobilization was conducted on the porous membrane surface with the treatment of polyethyleneimine, glutaraldehyde, and the anhydrase, in sequence. The immobilization was confirmed using X-ray photon spectrometer. The pH values of carbon-dioxide saturated solution with buffer were monitored with respect to time to calculate the catalytic activities of hydration of carbon-dioxide for free and immobilized CA. The catalytic rate constant values for free CA, immobilized CA on polystyrene nanoparticles, and immobilized CA on a porous cellulose acetate membrane were 0.79, 0.67, and 0.56 $s^{-1}$, respectively. Reusability was studied up to 10 cycles of $CO_2$ sequestration. The activity for the CA immobilized on the membrane was kept to 95% after 10 cycles, and comparable to the CA on the nanoparticles. The stabilities for heat and storage were also investigated for the three cases. The results suggested that the CA immobilized the membrane had the least loss rate of the activity compared to the others. From this study, the porous membrane was feasible as a carrier for the CA immobilization in hydration and sequestration of carbon-dioxide.

• KSBB Journal
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• 제13권1호
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• pp.52-57
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• 1998

The present work proposes a simple electrochemical method applicable to any immobilization processes of oxidase using a Clark type oxygen electrode as a base transducer. The present work suggests an optimal immobilization technique among three different methods of glucose oxidase(GOD) onto one side of $37[\mu}$mthick blend membranes, composed o 80% of cellulose triacetate and 20% of polycaprolactone, on the basis of the maximum Michaelis-Menten parameter(Vm) determined by either steady state or transient analyses. The electrode system was made of disk type gold cathode(4mm diameter) and Ag/AgCl anode. One side of the blend membrane was in contact with the cathode surface while the other side was immobilized with GOD either in covalent-bond or cross-linked forms, the latter being covered by $25{\mu}$m thick dialysis membrane of cellulose acetate. The resultant current density was on-line monitored by a potentiostat while glucose level was varied from 1 to 20 mM. The present study shows that direct cross-linking of GOD with glutaraldehyde was mostly preferred for fabrication of glucose sensor, on the basis of resultant kinetic parameters from either steady state or transient analyses.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.297-301
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• 2004

The dTDP-D-glucose 4,6-dehydratase from Salmonella enterica was immobilized using covalent binding to cyanogen bromide activated sepharose. The immobilized enzyme was used to produce dTDP-4-keto-6-deoxy-D-glucose, a key sugar intermediate that can be used economically to produce diverse classes of unusual sugars appended in various antibiotics. The enzyme was immobilized on the sepharose after activation with cyanogen bromide. The maximum immobilization (80.03％) was achieved after 14 h of coupling. The covalently immobilized enzyme was stable, and an average of 78.4 ％ conversion was achieved until 120 h of immobilization when it was repeatedly used. Similar conversion was noticed for the first batch using the enzyme entrapped-hydrogel but activity was gradually decreased in the following batches. The production of dTDP-4-keto-6-deoxy-D-glucose by using an immobilized enzyme has high potential for commercial application.

• Bulletin of the Korean Chemical Society
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• 제30권2호
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• pp.389-396
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• 2009

MFI structural zeolite (ZSM-5 or Sililcalite) was treated with HF solution to introduce mesoporous channels in the microporous crystals. Inner mesopore size could be controlled from 2.5 to 3.5 nm by changing the concentration of HF solution. The pore structure of HF-treated MFI zeolite was studied by instrumental analysis. The active Co (III) salen complex monomers were successfully anchored non-covalently on the surfaces of mesoporous MFI-type zeolite. These heterogeneous catalysts could be applied in asymmetric ring opening of terminal epoxides by phenol derivatives. It showed very high enantioselectivity and yield up to 95% in the catalytic synthesis of optically active $\alpha$-aryloxy alcohol compounds.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.205-210
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• 2013

Thiol-ene polymerization is a versatile tool for several applications. Here we report the preparation of epoxide groups containing thiol-ene photocurable polymeric support and the covalent immobilization of ${\alpha}$-amylase onto these polymeric materials. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) coupled with SEM was used to explore the chemical composition. The polymeric support and the immobilization of the enzyme were characterized by FTIR analysis. SEM-EDS and FTIR results showed that the enzyme was successfully covalently attached to the polymeric support. The immobilization efficiency and enzyme activity of ${\alpha}$-amylase were examined at various pH (5.0-8.0) and temperature ($30-80^{\circ}C$) values. The storage stability and reusability of immobilized ${\alpha}$-amylase were investigated. The immobilization yield was $276{\pm}1.6$ mg per gram of polymeric support. Enzyme assays demonstrated that the immobilized enzyme exhibited better thermostability than the free one. The storage stability and reusability were improved by the immobilization on this enzyme support. Free enzyme lost its activity completely within 15 days. On the other hand, the immobilized enzyme retained 86.7% of its activity after 30 days. These results confirm that ${\alpha}$-amylase was successfully immobilized and gained a more stable character compared with the free one.

• Journal of Microbiology and Biotechnology
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• 제33권1호
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• pp.127-134
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• 2023

Laccase activity is influenced by copper (Cu) as an inducer. In this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe₂O₄) nanoparticles (NPs) to improve enzyme stability and potential applications. The Cu/Fe₂O₄ NPs functionally activated by 3-aminopropyltriethoxysilane and glutaraldehyde exhibited an immobilization yield and relative activity (RA) of 93.1 and 140%, respectively. Under optimized conditions, Cu/Fe₂O₄ NPs showed high loading of laccase up to 285 mg/g of support and maximum RA of 140% at a pH 5.0 after 24 h of incubation (4℃). Immobilized laccase, as Cu/Fe₂O₄-laccase, had a higher optimum pH (4.0) and temperature (45℃) than those of a free enzyme. The pH and temperature profiles were significantly improved through immobilization. Cu/Fe₂O₄-laccase exhibited 25-fold higher thermal stability at 65℃ and retained residual activity of 91.8% after 10 cycles of reuse. The degradation of bisphenols was 3.9-fold higher with Cu/Fe₂O₄-laccase than that with the free enzyme. To the best of our knowledge, Rhus vernicifera laccase immobilization on Cu or Cu/Fe₂O₄ NPs has not yet been reported. This investigation revealed that laccase immobilization on Cu/Fe₂O₄ NPs is desirable for efficient enzyme loading and high relative activity, with remarkable bisphenol A degradation potential.

• 한국미생물·생명공학회지
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• 제20권1호
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• pp.20-24
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• 1992

Inulinase의 효율적인 재사용을 위하여 vinylsulfone activated agarose에 endo- 및 exoinulinase를 고정화시켰다. Gram gel당 exoinulinase는 400U, endoinulinase는 80U까지 고정화시킬 수가 있었고 열안정성은 exoinulinase 에서 증가되었다. 두 고정화 효소의 혼합비율에 따른 synergistic effect는 endo/exo가 0.5-0.1일 대 가장 컸으며, synergistic effect는 혼합되지 않은 상태의 고정화 효소에 비해 그 활성이 약 1.7배 증가하였다. 두 고정화 효소의 최적 pH는 4.4-5.0 범위이었으며 operational stability는 batch reactor에서 20번 반복된 실험결과 어떠한 효소활성의 감소도 보이지 않았다.

• 한국미생물·생명공학회지
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• 제31권4호
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• pp.368-376
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• 2003

AA를 AA-2G로 전환하는 CGTase를 고정화하여 AA-2G 대량 생산의 가능성을 고찰한 결과, CNBr-sepharose 4B가 가장 효과적인 담체로 판명되었고, CGTase의 고정화 최적 조건과 고정화 CGTase에 의한 AA-2G 생산의 최적조건 및 재사용성을 검토하였다. 최적 고정화 조건은 효소량 24,000 units/g resin으로 9시간 반응하여 약 18,000 units/g resin의 최고 고정화율을 얻을 수 있으며, pH 5.0(50 mM sodium citrate buffer)용액에 12% AA-Na와 8% soluble starch를 기질로 하여 800 units/ml의 고정화 CGTase를 첨가한 후 $37^{\circ}C$에서 100 rpm으로 교반하면서 25시간 반응하여 약 18 mM의 AA-2G 최고량을 얻을 수 있었다. 또한 0.015 mM의 $CaCl_2$를 첨가하여 고정화 CGTase의 재사용성을 관찰한 결과, 5회까지 50% 이상의 AA-2G 생산율로서 그 가능성을 입증할 수 있었다. 그리고 효소의 재사용성이란 측면에서, 본 고정화의 다른 한 방법인 ultrafiltration(한외 여과)에 의해서는 Millipore사의 YM 10 membrane을 이용하여 먼저 단백질량과 효소 활성 변화를 측정하여 그 가능성을 확보할 수 있었으며, 기질 20 ml을 사용하여 AA-2G를 생성시킨 후, 한외여과에 의해 효소만을 회수하여 연속 반응해 본 결과 8회까지 50%의 생성률을 유지하였다. 따라서 한외 여과는 CNBr-sepharose 4B와 함께 효율적인 고정화의 한 방법으로 판명되었으며, 앞으로 이들 고정화 효소를 이용한 연속 반응 시스템의 구축이 뒤따라야 할 것이다.

• KSBB Journal
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• 제5권2호
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• pp.141-149
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• 1990

Aspergillus niger유래의 $\beta$-glucosidase를 (1)glutarald-ehyde를 가교제로 한 chitin과 chitosan담체에 covalent linkage (2)Amberite IRA93과 DEAE-cellulose담체에 succinylation시킨 후 흡착, 그리고 (3)alginarte, polyacry-lamide를 각종 가교제를 이용 entrapment등의 방법으로 고정화 하였다. Glutaraldehyde를 가교제로써 chitosan을 활성화시킨후 $\beta$-glucosidase를 고정화 시켰을때 효소활성 회수율이 31.5%로 가장 높았고, 또한 column형 반응기에서의 15일 경과 후 효소활성 유지도도 69%로서 가장 우수하였다. 또한 succinylation시킨 효소를 Amberite IRA93 담체에 흡착시켰을 때 효소활성 회수율은 24.7%였고 효소 활성유지도는 62%였다. 반면에 entra-pment 방법에 의한 $\beta$-glucosidase의 고정화는 효소의 계속적인 용출로 $\beta$-glucosidase의 고정화에는 적합하지 않았다. Chitosan담체에서의 고정화 최적조건을 조사한 결과 가교제인 glutaraldehyde의 최적농도는 0.4%였고, glutaraldehyde의 최적 반응 pH는 4.8이었다. 또한 column형 반응기를 이용하여 cellobiose로부터 glucose로의 전환율을 조사하여 고정화 효소의 효용성을 검토하였다.

• 한국미생물·생명공학회지
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• 제43권3호
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• pp.300-305
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• 2015

효율적인 γ-aminobutyric acid (GABA)의 생산을 위해 Lactobacillus plantarum WCFS1로부터 글루탐산 탈탄산효소(glutamate decarboxylase, GAD)를 대장균에 발현, 정제 후 silica beads에 covalent coupling 방법을 이용하여 고정화하였다. 고정화된 효소의 특성을 고정화하지 않은 효소와 비교한 결과, 모든 pH의 범위(pH 3.5–6.0)에서 80% 이상의 활성을 나타내었으며 pH 안정성과 열 안정성 모두 증대되었다. 이 고정화 효소를 packed-bed reactor에 충진하여 GABA의 생산성을 확인한 결과 1리터당 1시간에 최대 41.7 g의 GABA 생산이 가능한 것으로 확인되었다.