• 한국미생물·생명공학회지
• /
• 제20권4호
• /
• pp.401-408
• /
• 1992

Cholesterol oxidase를 생산하는 균주를 토양으로 분리하고 이를 배양하여 효소를 생산한 후 정제하여 그 특성을 조사하였다. 본 효소는 CH2 concentrator에 의한 농축, DEAE-cellulose chromatography, Superose 12 column FPLC로 정제하였고, 비활성이 약 31배나 증가하였다. 이 효소는 $50^{\circ}C$, pH 6.0에서 최대 활성을 나타냈고 30-$45^{\circ}C$ 범위에서는 안정하였다. pH에 대한 안정성은 pH 6.0-11.0까지 광범위한 안정성을 보였으며, Km 값은 cholesterol에 대해서 $3.65{\times}10^{-3}$M이며 분자량은 59,500 dalton으로 추정 된다. 또한 $Ca^{2+}$, $Ba^{2+}$, $Fe^{2+}$, Brij 35에 의해서는 효소의 활성이 저해되지 않았지만, $Hg^{2+}$, $Ag^{2+}$ 및 SDS에 의해서는 저해되는 것으로 나타났다.

• 약학회지
• /
• 제41권5호
• /
• pp.638-646
• /
• 1997

The steroid 9${alpha}$-hydroxylase activity has been detected in cytosol fraction, $100,00{\times}g$ supernatant of cell free extract of Mycobacterium fortuitum. The activity was not linear with protein concentration in the assay suggesting 9${alpha}$-hydroxylase is a multicomponent enzyme. The 9${alpha}$-hydroxylase system was partially purified through fractional saturation of ammonium sulfate, strong anion exchange (Mono Q) column chromatography, gel filtration (Superose 12) column chromatography, and testosterone affinity gel chromatography. Ammonium sulfate 50~60% saturated fraction of the cytosol gave 9${alpha}$-hydroxylase activity. For further purification, the half-saturated ammonium sulfate fraction was applied to Mono Q, Superose 12, or affinity gel column. The purification factors of 9${alpha}$-hydroxylase containing fraction after Mono Q, Superose 12, and affinity gel chromatography was 13, 11, and 17 respectively.

• 한국식품영양과학회지
• /
• 제23권6호
• /
• pp.964-972
• /
• 1994

The 4.3-kb gene coding for L-lactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment , DEAE-Sephadex, and NAD++ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200 . The molecular weight of the purfied enzyme was estimated to be about 35, 000 and 140, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. indicating that the enzyme is composed of four identical subunits. THe enzyme for pyruvate reduction and lactate oxdiation was stable at 60 and 75$^{\circ}C$ for 30 min, and the optimal temperatures for both reactions were 60 and 7$0^{\circ}C$, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme of pyruvate reduction was table between pH 5 and 7. more than 90% of enzyme activity was lost at 1mM FeSO4 and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1, 6-bisphosphate.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권6호
• /
• pp.593-598
• /
• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

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

• Mycobiology
• /
• 제35권4호
• /
• pp.219-225
• /
• 2007

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and $28^{\circ}C$. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH ($7{\sim}10$) and temperature ($30^{\circ}C{\sim}70^{\circ}C$) profiles with the optimal for keratinase activity at pH 8 and $45^{\circ}C$. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

• Preventive Nutrition and Food Science
• /
• 제7권1호
• /
• pp.22-27
• /
• 2002

The cellulase from internal organs of edible snails was purified by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel filtration on Sephacryl S-200 and Superose 12 HR 10/30. The specific activity of the purified cellulase was 85.1 units/mg protein with 24.3 purification fold from crude extract. Molecular weight of the enzyme was estimated to be approximately 74,000 dalton by gel filtration chromatography and SDS-PAGE eletrophoresis. T7e isoelectric point of the enzyme was determined to be pH 4.6. The optimum temperature and pH of the enzyme were 5$0^{\circ}C$ and pH 6.0, respectively. The enzyme was stable at 30~5$0^{\circ}C$ and pH 6.0~10.0. It was activates by Mn$^{2+}$, but inhibited by Li$^{2+}$, Zn$^{2+}$, Ag$^{2+}$ and Hg$^{2+}$./TEX> 2+/.

• 한국미생물·생명공학회지
• /
• 제23권1호
• /
• pp.60-67
• /
• 1995

Methanol assimilating yeast, Hansenula sp. MS-364 that has high productivity with methanol as carbon and energy source has been preserved at dept. of Microbiological engineering. Purification and properties of alcohol oxidase (E.C.1.1.3.13: oxygen oxidoreductase) were investigated in the methanol assimilating yeast, Hansenula sp. MS-364. Alcohol oxidase is related to the catalytic reaction that degrades alcohol to aldehyde and peroxide. The methanol oxidizing enzyme was purified by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and gel filtration on Sepharose 6B from cell-free extract. The purified enzyme preparation gave a single band in the sodium dodesyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was calculated to be about 576,000 and molecular weight of subunit was also calculated to be 72,000. The optimal pH and temperature of the enzyme reaction were pH 7.5 and 37$\circ$C, respectively. The enzyme was unstable in acidic pH and higher temperature. The enzyme was not specific for methanol and also oxidized lower primary alcohols. The Km value for methanol was 2.5 mM and that for ethanol was 1.66 mM. The enzyme was heavily inhibited by metal ions such as Hg$^{2+}$, Ag$^{2+}$, Cu$^{2+}$. The high concentration of EDTA and sulfhydryl reagents strongly inhibited the enzyme activity. The component of coenzyme was determined to flavin adenine dinucleotide.

• 한국축산식품학회지
• /
• 제27권1호
• /
• pp.102-109
• /
• 2007

Lactobacillus salivarius subsp. salivarius CNU27 possessed a high level of ${\alpha}$-galactosidase activity. Purified ${\alpha}$-galactosidase was obtained after sonication of harvested cell pellet followed by DEAE-Sephadex A-50 and Mono Q anion exchange chromatography. The specific activity of the purified enzyme was 8,994 units/mg protein which is 17.09 times higher than that in crude extract. The native enzyme was a monomer with a molecular mass of 56,397.1 dalton. The optimum temperature and pH for the enzyme were $40^{\circ}C$ and 6.0, respectively. The enzyme was stable between 25 and $50^{\circ}C$. However, ${\alpha}$-galactosidase activity was lost rapidly below pH 4.5 and above pH 8.5. The enzyme activity decreased to 6.73% and 4.30% of the original activity by addition of $Cu^{2+}$ and $Hg^{2+}$, respectively. Other metal compounds did not affect the enzyme activity significantly. The enzyme liberated galactose from melibiose, raffinose, and stachyose. The rate of substrates hydrolysis was measured by HPLC. Raffinose, stachyose and melibiose were completely decomposed after 24 hr at $40^{\circ}C$.

• Fisheries and Aquatic Sciences
• /
• 제1권2호
• /
• pp.209-215
• /
• 1998

Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.