• 한국식품영양과학회지
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• 제22권6호
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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.

• 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.

• KSBB Journal
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• 제13권5호
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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.

• 미생물학회지
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• 제48권3호
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• pp.225-227
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• 2012

백색부후균류가 가지는 리그닌 분해효소들은 기질특이성이 넓기 때문에 다양한 난분해성 화합물들을 분해할 수 있다. 본 실험에서는 3가지 다른 방법을 사용하여 laccase와 manganese peroxidase가 각각 도입된 아교버섯 형질전환체의 배양 상등액 효소를 고정화 효소로 만들어 대표적 염료의 하나인 Remazol Brilliant Blue R (RBBR)의 탈색을 실험하였다. 그 결과 알긴산을 효소 용액과 직접 반응하여 만든 고정화 효소에서 48시간 반응 후 약 75% 탈색을 보였다. 비록 한번 사용했던 고정화 효소를 재사용하였을 경우 탈색능이 10-15% 정도 감소되었으나 본 실험에서 제시한 방법이 리그닌 분해효소의 고정화 효소 활용에 기여할 것으로 기대한다.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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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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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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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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• 제44권2호
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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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• 제23권4호
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• pp.350-354
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• 2008

본 연구에서는 나노담체를 이용하여 효소를 고정화 하였으며 고정화 효소의 활성도, 재사용 가능성, pH 영향 및 시간에 따른 안정성을 검토하였다. 고정화 효소의 활성도는 PAMP를 사용한 경우 가장 빠른 것으로 나타났으며 이때 Vm값은 0.169 mM/min였고 Km값은 0.263 mM 이었다. 이는 PS/PSMA를 사용한 경우보다 2배 이상의 효소 반응속도 향상을 보여주었다. 그림을 통하여 효소의 재활용가능성을 제시하였으며 여러 번 재사용한 경우에도 활성도를 잃지 않고 유지하였다. 고정화한 트립신은 공통적으로 pH증가에 따라 활성도가 증가하였으며 PAMP, PANI, DEAE, CMC, PS/PSMA 순으로 활성도가 높음을 확인 할 수 있었다. 트립신이 자체 효소를 분해하는 특성을 고려할 때 고정화 되지 않은 트립신의 안정성은 매우 낮게 된다. 본 연구에서 사용된 담체 중에 크기가 아주 작은 담체인 PAMP와 PANI의 경우에는 활성도가 급격히 감소하는 현상을 보였으나 비교적 크기가 큰 다른 담체의 경우에는 고정화 효소의 안정성이 우수한 것으로 나타났다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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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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• 제18권4호
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• pp.306-311
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• 2003

융합단백질의 절단을 위해 EK를 고정화하여 액상 절단반응과 같은 80％의 절단수율을 얻을 수 있었다. 그리고 니켈 친화칼럼을 이용하여 간단한 정제공정을 구축하였다. 공유결합한 EK의 경우 니켈친화 결합한 EK보다 높은 재접힘 수율을 나타내었고 풀림과 재접힘을 이용하여 효소의 초기 활성을 회복함에 따라서 반복사용을 통한 경제적인 절단공정을 구축할 수 있게 되었다. 그러나 고정화 과정에서 효소의 활성이 감소하는 문제점과 고정화 수율을 높이기 위한 연구가 필요하다.