• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.39-45
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• 2002

Activity staining on the native polyacrylamide gel electrophoresis (PAGE) of a cell-free extract of Rhodococcus sp. TK6, grown in media containing alcohols as the carbon source, revealed at least seven isozyme bands, which were identified as alcohol dehydrogenases that oxidize cyclohexanol to cyclohexanone. Among the alcohol dehydrogenases, cyclohexanol dehydrogenase II (CDH II), which is the major enzyme involved in the oxidation of cyclohexanol, was purified to homogeneity. The molecular mass of the CDH II was determined to be 60 kDa by gel filtration, while the molecular mass of each subunit was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The CDH II was unstable in acidic and basic pHs, and rapidly inactivated at temperatures above $40^{\circ}C$ . The CDH II activity was enhanced by the addition of divalent metal ions, like $Ba^2+\;and\;Mg^{2+}$. The purified enzyme catalyzed the oxidation of a broad range of alcohols, including cyclohexanol, trans-cyclohexane-1,2-diol, trans-cyclopentane-l,2-diol, cyclopentanol, and hexane-1,2-diol. The $K_m$ values of the CDH II for cyclohexanol, trans-cyclohexane-l,2-diol, cyclopentanol, trans-cyclopentane-l,2-diol, and hexane-l,2-diol were 1.7, 2.8, 14.2, 13.7, and 13.5 mM, respectively. The CDH II would appear to be a major alcohol dehydrogenase for the oxidation of cyclohexanol. The N-terminal sequence of the CDH II was determined to be TVAHVTGAARGIGRA. Furthermore, based on a comparison of the determined sequence with other short chain alcohol dehydrogenases, the purified CDH II was suggested to be a new enzyme.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.251-259
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• 1990

생전분 분해력이 강력한 glucoamylase를 생산하는 균주로서 Asp.usamii IAM 2185를 선정하였다. 밀기울배지에서의 효소생산의 최적 initial pH는 6.0-8.0, 최적 배양온도는 25-$30^{\circ}C$, 최적 배양시간은 72시간이고, 밀기울배지에 ammonuim nitrate와 albumin의 첨가는 효소의 생산을 약간 증가시켰다. 황산암모늄분획, CM-cellulose와 DEAE-cellulose column chromatography에 의하여 효소를 정제하였고, 정제효소의 specific activity는 34.3U/mg.protein, 수율은 10.3 이었다.

• Applied Biological Chemistry
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• 제46권3호
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• pp.176-182
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• 2003

한국재래간장으로부터 혈전용해효소를 강하게 생산하는 균주를 선발하고 이를 Bacillus subtilis K7로 동정하였다. B. subtilis K7이 생산하는 혈전용해성 protease를 정제하여 분자량을 확인한 결과 21,500 Da이었다. 정제된 효소의 최적반응조건은 $40^{\circ}C$와 pH 9.0이었으며 pH 5.0라서 12.0까지 안정하고 $50^{\circ}C$에서 20분간 열처리한 후에도 50%이상의 효소활성을 가지며 EDTA, CDTA 및 iodoacetat에 실활하는 효소이었다. 이 효소의 fibrin에 대한 Km 값은 $1.8{\times}10^{-2}$ M이었다.

• Nutritional Sciences
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• 제9권4호
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• pp.317-322
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• 2006

Soybeans are well-known as allergenic foods. Koreans consume large amounts of soybean foods, such as kochujang, which have gone through the fermentation process. To lower the allergenicity of these foods, we prepared hypo allergenic kochujang with aloe extract (AK). A sensory evaluation was conducted along with a clinical evaluation that used a double-blind, placebo-controlled food challenge (DBPCFC) test These tests were designed to evaluate the acceptability of the fermented foods. In comparison to normal kochujang (NK), AK elicited a higher sensory test score, and the rate of positive reactions in atopic dermatitis patients during the DBPCFC test was reduced. Methods of protein extraction, protein quantitation with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and protein identification using two-dimensional (2D) gel electrophoresis were performed for both NK and AK to compare the functional factors. We found a reduction in the levels of high molecular proteins even though the bands of the proteins had not entirely disappeared, indicating that the boiling and fermentation process changed the soybean protein patterns. The rate of the reduction of high molecular proteins was more effective in the AK. In conclusion, AK can be recognized as a food with hypoallergenic effect.

• BMB Reports
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• 제31권4호
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• pp.384-390
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• 1998

DNA fragments (51-587 bp) were separated by capillary electrophoresis using entangled polymer, hydroxyethylcellulose, in uncoated fused silica capillary columns. The factors affecting the separation of DNA fragments with hydroxyethylcellulose media were evaluated, i.e., the concentration of buffer and entangled polymer, effects of additives (methanol, ethidium bromide, EDTA), temperature, and injection methods. Maximum performance was obtained by adding 5% methanol in 0.5% hydroxyethylcellulose solution at $30^{\circ}C$. Addition of methanol in polymer media increased the resolution of small size DNA fragments (< 100 bp). On the other hand, addition of ethidium bromide and EDTA, which are commonly used in conventional DNA separation, reduced the resolution of DNA fragments in the polymer solution. It turns out that the separation behavior of DNA in entangled polymer is more sensitive to the running condition compared to that in polyacrylamide gel-filled capillary, but the reproducibility of DNA separation in entangled polymer is reliable.

• 한국미생물·생명공학회지
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• 제15권6호
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• pp.420-424
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• 1987

Saccharomycopsis lipolytica ATCC 44601 과 MX9-11RX8 온도감수성 변이균주의 isocitrate lyase는 조추출액을 ammonium sulfate 분획, Toyo Peal HW-55F gel filtration, DEAE-Cellulose ion exchange chromatography 등의 방법에 의하여 각각 54배, 87배 분리 정제되었다. 정제효소의 subunit 분자량은 59,000이고 Sephadex G-200 gel filtration에 의한 native enzyme 은 230,000이므로 이 효모의 isocitrate lyase는 같거나 비슷한 subunit 4개로 구성된 tetramer이며, 최적 pH는 6.9이었다.

• KSBB Journal
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• 제8권3호
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• pp.287-294
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• 1993

사람의 뇨로부터 유용한 단백질을 동시에 분리하 기 위한 목적으로 효과적인 통합 분리정제 공정이 고안되었다. Ultrafiltration 방법을 이용한 농축과정 과 pH침전법을 전처리 단계로 사용하였고, 이어서 g gel filtration과 흡착, 이온교환, 친화, 그리고 역상 C column을 선택적으로 혼합한 chromatography를 수행하였다. 이 공정으로 정제한 urokinase, epider­m mal growth factor, albumin은 각각 SDS-poly­a acrylamide gel 전기이동상에서 단일의 단백질 띠로 이동하였고, 자신의 단백질 활성을 유지하고 있었다. 이들 세 가지 목적 단백질의 전체 수율은 각각 48 % %, 17%, 46%로 나타났다.

• BMB Reports
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• 제39권2호
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• pp.216-222
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• 2006

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT(dithiothreitol) and 0.2% carrier ampholytes; (b) 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 40mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

• 한국미생물·생명공학회지
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• 제24권2호
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• pp.197-205
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• 1996

The $\beta$-xylosidase was purified 99- fold from the culture supernatant of Pseudo onas sp. CB-33 by ammonium sulfate precipitation, PEI precipita- tion, DEAE-Sephadex column chromatography, Sephadex G-75 gel filtration chromatography and preparative disc gel electrophoresis. Molecular weight of the enzyme was estimated to be 44,000 by SDS polyacrylamide gel electrophoresis. The enzyme has a pH optimum for activity at 7.0 and is stable over pH 6.5-9.0. The optimal temperature of the enzyme was 45$\circ$C, and its enzymatic activity was completely inactivated at 55$\circ$C for 30 min. Km value of the enzyme for p-nitrophenyl-$\beta$-D-xylopyranoside was calculated to be 4.6 mM. The effect of various reagents on the $\beta$-xylosidase activity was investigated. The enzyme activity was completely inhibited by Hg$^{2+}$, Cu$^{2+}$ and Zn$^{2+}$. The $\beta$-xylosidase was inactivated by tryptophan-specific reagent, N-bromosuccinimide and tyrosine-specific reagent, iodine. The enzyme could degrade xylo-oligosaccharides to xylose and the enzyme was competitively inhibited by xylose. The $\beta$-xylosidase and endoxylanase from Psedomonas sp. CB-33 hydrolized xylan synergically. The purified enzyme also showed $\alpha$-L-arabinofuranosidase activity.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.525.1-525
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• 1986

To clone ${\beta}$-glucosidase gene from Cellulomonas sp. a gene library was constructed using E. coli JM83 pUC9. Among 2,500 pseudotransformants obtained, 20 clones developed yellow color on the p-nitrophenyl- -D-glucopyranoside filter paper These 20 clones were classified into three groups based on the results of activity staining using nondenaturating polyacrylamide gel electrophoresis and restriction enzyme digestions. Among the three groups, only one group containing pCEl plasmid has specificity for cellobiose.