• 한국식품영양과학회지
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• 제24권4호
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• pp.563-569
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• 1995

Myrosinase from Korean cabbage(Bogdoli) was purified and its enzymatic properties were investigated. Myrosinase from the Korean cabbage was purified by DEAE Bio-Gel Sepharose, Concanavalin-A, and Mono-Q column chromatography and exhibited a 55KD molecular weight with a single band on the gel of SDS-PAGE. The enzyme was purified about 21-fold compared to its crude enzyme and a specific activity of purified enzyme was 15, 120units/mg. Optimum pH of the myrosinase was 7.0 in both phosphate and Tris-HCl buffer solutions, the enzyme was stable at pH 6.5~7.0. Optimum temperature of enzyme was 37~38$^{\circ}C$. The enzyme activity was significantly inhibited by Cu2+ and Hg2+, but enhanced by ascorbic acid, resulting in a maximum activity at 1mM ascorbic acid. Among the ascorbic acid analogues, dehydro-ascorbic acid did not affect, whereas others showed a little effect on the enzyme activity, but less than ascorbic acid itself. Reducing agents such as 2-mercaptoethanol and dithiothreitol had no effect on the enzyme activity, but the enzyme activity was enhanced when 2-mercaptoethanol was mixed with ascorbic acid.

• Journal of Plant Biotechnology
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• 제4권1호
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• pp.39-44
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• 2002

The fast-migrating cationic peroxidase isozyme, named RC3, was purified from rice (Oryza sativa cv. Nak-Dong) callus. Purification of the enzyme was accomplished by ammonium sulfate fractionation, CM-cellulose ionexchange chromatography, and Sephacryl S-100 gel filtration. The molecular mass of the enzyme was about 34 KDa as determined by SDS-PACE and 38 KDa by Sephacryl-100 gel filtration. The pI value of the enzyme was 8.9. Antiserum against RC3 was raised in rabbits, and anti RC3 antiserum reacted with RC3 isozyme by Ouchterlony double immunodiffusion. The optimum pHs and Km values of the enzyme for various substrates were determined. Kinetic studies with various substrates showed that RC3 had very low Km value of 0.01 mM for ferulic acid and ascorbic acid. However, the enzyme did not use esculetin as a substrate.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.307-311
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• 2000

Abstract Extracellular chitinase was purified from the culture liquid of the marine bacterium, Bacillus sp. LJ-25 , and its enzymatic properties were examined. The purified chitinase exhibited a single band on SDS-PAGE and the molecular weight was estimated to be approximately 50 kDa. The optimum pH and temperature for the enzymatic activity were 7.0 and $35^{\circ}C$, respectively. The activity of the chitinase was strongly inhibited by $Zn^{2+}$ and slightly inhibited by $Ba^{2+},{\;}Co^{2+},{\;}Mn^{2+},{\;}and{\;}Cu^{2+}$. The purified chitinase did not hydrolyze $p-nitrophenolN-acetyl-{\bata}-D-glucosaminide{\;}(GlcNAc)_2$ and Micrococcus lysodeikticus cells, which are known to be the substrates for exo-type chitinase. Among the hydrolyzates of colloidal chitin, $(GlcNAc)_2$ was in the highest concentration with small amounts of GlcNAc and $(GlcNAc)_3$..

• BMB Reports
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• 제44권1호
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• pp.64-69
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• 2011

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and $50.0^{\circ}C$, respectively and seasonal variation. The Michaelis-Menten constant ($K_M$) for $H_2O_2$ was 5.27 mM, and the velocity maximum ($V_{max}$) $1.31\;nmol\;min^{-1}$, while the enzyme turnover was $0.148\;s^{-1}$. Finally, the presence of $Ca^{2+}$ and $Mg^{2+}$ enhanced the PD-cP activity, with $Mg^{2+}$ 1.4-fold more effective than $Ca^{2+}$.

• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1381-1387
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• 2012

We applied enzymatic hydrolysis and tangential flow filtration (TFF) to purify a novel anticancer peptide from Mytilus coruscus (M. coruscus) and investigated its anticancer properties. To prepare the peptide, eight proteases were employed for enzymatic hydrolysis. Pepsin hydrolysates, which showed clearly superior cytotoxic activity on prostate cancer cells, were further purified using a flow filtration system using a TFF and consecutive chromatographic methods. Finally, a novel anticancer peptide was obtained, and the sequence was identified as Ala-Phe-Asn-Ile-His-Asn-Arg-Asn-Leu-Leu. The peptide from M. coruscus effectively induced cell death on prostate, breast and lung cancer cells but not on normal liver cells. This is the first report of an anticancer peptide derived from the hydrolysates of M. coruscus.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제40회 국제학술심포지움
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• pp.90-90
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• 2003

• 한국식품영양과학회지
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• 제25권4호
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• pp.687-694
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• 1996

한국산 겨자에서 myrosinase를 분리 및 정제하고, 이들의 효소학적 특성을 조사한 결과는 다음과 같다. 겨자 myrosinase를 DEAE-cellulose, Concanavalin A-Sepharose 및 FPLC Soporose 6 칼럼을 이용하여 분리 및 정제하였을 때 적어도 3개의 이성효소가 존재하는 것으로 나타났으며, Myrosinase II-2의 최종 비활성도는 57.lunits/mg, 정제도는 약 248배였다. SDS-PAGE상에서 myrosinase(II-2)의 단일밴드를 확인한 결과, 그 분자량은 약 67KD로 추정 되었다. 최적 pH는 phosphate 및 Tris-HCI 완충액에서 7.0이 가장 활성이 높았고, 그 효소는 pH 7.0에서 안정하였으며, 최적 활성을 나타내는 온도는 $37^{\circ}C$ 부근이었고, $40^{\circ}C$ 이상에서는 비교적 불안정하게 나타났다. Ascorbic acid의 영향은 1mM에서 가장 안정하였으며, 그 이상의 농도에서는 변화가 없었다. 망간과 마그네슘 및 나트륨은 효소활성을 촉진시키는 것으로 나타났으며 구리, 수은 및 철 이온은 약간 저해하였다. Ascorbic acid analogue 중 dehydroascorbic acid는 효소 활성을 저해하였으며, 나머지 것들은 거의 영향을 미치지 않았다. 2-Mer-captoethanol과 dithiothreitol과 같은 환원제는 효소활성을 억제하였으나 이들과 ascorbic acid를 함께 첨가 하였을 때는 활성이 다소 증가하였다.

• 생명과학회지
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• 제8권3호
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• 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.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.370-378
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• 1989

토양으로부터 역가높은 CGTase를 분비하는 호알칼리성 미생물을 분리하였으며, 동정 결과 Bacillus circulans로 판정되었다. 배양액 중의 CGTase를 ammonium sulfate 침전, DEAE-Sephadex 그리고 Sephadex G-100 column chromatography로 분리, 정제하여 단일 단백질 band를 얻었다. 정제된 CGTase의 분자량은 약 93,000, 최적 pH와 온도는 6.0, $50^{\circ}C$였으며, pH와 온도안정성은 5.5-11, $65^{\circ}C$까지였다. Soluble starch를 기질로 할 때의 $V_{max}$ 와 $K_{m}$ 값은 각각 0.16$\mu$mole $\beta$-CD/min, 14.3mg soluble starch/mi이였고 24시간 반응액의 $\alpha$-：$\beta$-:${\gamma}$-CD 의 생성비율은 1：8.1：1.9로서 $\beta$-CD를 우선적으로 합성하였다. 기질로 glucose와 maltose를 사용하였을 때 CD합성작용이 없었으며, sweet potato 그리고 cornstarch를 사용하였을 때 가장 높은 CD합성작용을 보였다. 어느 수준 이상의 과다한 CGTase 첨가경우에는 $\alpha$-CD생성이 급격히 증가하였다. 또한 정제된 CGTase는 stevioside에로의 당전이성을 갖고 있었다.