• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.181-187
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• 1993

Steroid $\Delta^1$-dehydrogenase which introduces a double bond into the 1, 2 positions of steroid ring A was purified from Arthrobacter simplex, an excellent biotransformer of hydrocortisone into prednisolone. Hydrocortisone-induced cells were disrupted by vigorous agitation with glass beads, and a solubilized enzyme was obtained after centrifugation at 100, 000$\times$g for 90 minutes. The enzyme was purified 123-fold in three steps of chromatographic procedures with 13% yield. The last step of testosterone-agarose affinity column decisively contributed to the successful purification. The molecular weight of the enzyme was estimated to be 98, 000 by SDS-PAGE and 100, 000 by gel filtration, indicating that this enzyme behaves as a monomer. The enzyme showed demands for artificial electron acceptor, and among the several reagents tested, phenazine methosulfate acted as the most effective electron acceptor. Subcellular distribution of this enzyme was studied by centrifugation experiment. Comparison of the enzyme activities in pelleted membrane and cytosol fractions suggests that the enzyme may be a weakly attached peripheral membrane protein in vivo. But considerable amounts of enzyme was solubilized without any additional treatments for membrane protein.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.375-379
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• 2002

Methyl mercaptan oxidase was successfully induced in Thiobacillus thioparus TK-m using methyl mercaptan gas, and was purified for the detection of mercaptans. The purification procedure Involved a DEAE (diethylaminoethyl) -Sephacel, or Superose 12, column chromatography with recovery yields of 47.5 and 48.5%, and specific activities of 374 and 1240.8 units/mg-protein, respectively, The molecular weight of the purified methyl mercaptan oxidase was 66.1kDa, as determined by SDS-PAGE. The extract, from gel filtration chromatography oxidizes methyl mercaptan, producing formaldehyde, which can be easily detected by the purpald-coloring method. The optimized temperature for activity was found to be at 55$\^{C}$. This enzyme was inhibited by both NH$_4$Cl and (NH$_4$)$_2$SO$_4$, but was unaffected by either KCl or NaCl at less than 200 mM. With K$_2$SO$_4$, the activity decreased at 20 mM, but recovered at 150 mM. In the presence of methanol, full activity was maintained, but decreased in the presence of glycerin, ethanol and acetone 43, 78 and 75%, respectively.

• 생명과학회지
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• 제8권5호
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• pp.598-603
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• 1998

1. 어류에서 분리한 V. anguillarum KS410의 용혈소를 분리하여 배양 상등액, 황산암모늄 침전, DEAE-cellulose column chromatography, Sephadex G-200 gel 여과 등의 과정을 통하여 36배 정제하였고 최종 회수율은 2.3%였다. 2. 정제된 용혈소의 SDS-PAGE상의 전기영동 결과는 38 Kda의 분자량을 보유하였다. 3. 온도에 대한 정제 용혈소는 45$^{\circ}C$까지는 안정하였으나 그 이상의 온도에서는 급격히 실활되어 이열성인 것으로 나타났다. 4. NaCl 농도에 따른 V. anguillarum 용혈소의 안정성은 1%가 가장 안정하였고 4%이상에서는 안정성이 감소하였다. 5. 정제된 용혈소의 pH 안정성은 6-9 사이었다. 6. 금속이온에 대한 정제 용혈소는 $Ca^{2+}, Cu^{2+}, Zn^{2+}, Fe^{2+}$ 첨가시에는 실활되었으나 $Mg^{2+}$첨가에는 영향을 받지 않았다. 7. 정제용혈소는 EDTA에 대하여 전혀 활성에 저해를 받지 않았으나 $FeCl_3$에서는 저해를 받았다.

• 대한미생물학회지
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• 제35권2호
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• pp.141-147
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• 2000

We purified enolase from Candida albicans KNIH10 strain which was isolated from a clinical specimen in Korea. The purified enolase was used to detect anti-Candida antibodies in sera of patients with invasive candidiasis. For purification of enolase from the crude extract prepared by French pressure at 20,000 PSI, the fast performance liquid chromatography (FPLC) using DEAE-sepharose column was used. The elutes at $0.3{\sim}0.4\;M$ NaCl in FPLC was purified with homogenity in SDS-PAGE and its enzymatic activity was confirmed in sera of invasive candidiasis with candidemia patient by immunoblotting. The purified enolase indicated no signal (100% specificity) in 40 normal human sera and 75% (6/8) sensitivity in sera of candidemic patients with suspicious invasive candidiasis by immunoblotting.

• 대한방사성의약품학회지
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• 제6권2호
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• pp.69-74
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• 2020

PIB is the first amyloid plaque PET image tracer reported for the first time in 2003, and is considered to be the best and is still being utilized due to its very high uptake and kinetic properties. Initially, it was synthesized by radioisotope labeling using a precursor containing a methoxy methyl protection group, but now it is synthesized using a 6-OH precursor that can be easily synthesized in one step using [¹¹C]methyl triflate. Carbon-11 has several limitations in clinical studies using PET because its half-life is as short as 20 minutes. In this study, in order to overcome the difficulty of this half-life, a rapid method using Sep-Pak was adopted instead of HPLC purification to significantly reduce the burden of the purification process and attempted synthesis. As a result, the synthesis time was shortened by more than 50%, and the yield of the final compound was higher than the previous result and showed relatively high specific radioactivity, confirming that it is a strategic method with high applicability for various precursors having primary amines.

• KSBB Journal
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• 제13권3호
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• pp.320-324
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• 1998

Agar degrading enzyme-agarase-was purified from the culture fluid of Cytophaga so/ ACLJ-18, by acetone precipitation, DEAE-Cellulose, Sephadex G-100 and CM-Sephadex C25 column chromatographies. The molecular weight of purified agarase was estimated to be 24,700 dalton by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for agarase activity were 7.0 and 40$^{\circ}C$, respectively. this agarase was stable in the pH range of 6.5 - 8.0 and 40$^{\circ}C$, and required 0.35M NaCl for optimum activity. And this agarase was inhibited by metal ions such as Ba2+, Cu2+, Co2+, Mn2+, Hg2+, Zn2+, and showed specificity on agar.

• 한국식품과학회지
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• 제23권4호
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• pp.394-399
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• 1991

Katsuobushi에서 곰팡이, 세균, 효모 등 총 70여 균주를 분리하였으며 이중 곰팡이는 가다랑이 추출물에 밀기울을 가한 배지에서 생육이 양호하였다. protease활성이 높고 고미생성도가 적은 균주는 Aspergillus niger로 동정된 OK-63 strain이었으며 배양 6일만에 균체의 최대증식, protease의 최대 효소활성을 나타내었다. 효소정제는 150배 정제, 활성수율은 45%였으며 polyacryamide gel 전기영동에 의해 단일 band로 확인되었다.

• BMB Reports
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• 제38권2호
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• pp.232-237
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• 2005

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by S-hexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.

• 미생물학회지
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• 제49권1호
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• pp.95-98
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• 2013

본 연구에는 대장균에서의 과발현 벡터 개발과 FPLC를 사용한 2단계 컬럼 정제과정을 통해 Pseudomonas alkylphenolia의 alkylphenol hydroxylase의 oxygenase 단백질을 다량으로 정제하는 방법을 개발하였다. 재조합 Escherichia coli BL21(DE3)(pJJPMO2)의 50 g의 wet cake로부터 110 mg의 heterodimer이며 화학량론적 철을 갖는 순수한 단백질을 정제하였으며 147nmole/min/mg의 비활성을 보였다.

• BMB Reports
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• 제41권3호
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• pp.254-258
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• 2008

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of $35-40^{\circ}C$ and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of $Hg^{2+}$ or $Cu^{2+}$ ion. Partial amino acid sequence of the enzyme was also determined.