• Korean Journal of Microbiology
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• v.40 no.3
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• pp.183-188
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• 2004

Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractions designated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal physiological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g., EDTA, NaCl, dithiothreitol, $\beta$-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, demonstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of $\beta$-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzyme activities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I from Stenotrophomonas sp. OK-5 was $^1MSDLLNADAVVQLFRTARDS^20$ and exhibited 70% sequence homology with that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI) shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xanthomonas campestris (81 %), X. axonopodis (75%), Streptomyces avermitilis(30%), whereas they had low homology with that from P. putida KT2440 (pnrB) (16%).

• Applied Biological Chemistry
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• v.26 no.1
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• pp.65-72
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• 1983

The composition of four rice protein groups is greatly affected by the extraction conditions. The extraction amounts of albumins and glubulines primarily depended on the temperature rather than the method of extraction. The total amount of glutelins, the major components of rice storage proteins, could be extracted by a successive extraction processes, extraction with 0.5% SDS-0.1M borate buffer(pH 8.3) followed by extraction with 0.5% SDS-0.6% ${\beta}-mercaptoethanol-0.1M$ borate buffer(pH 8.3). The extracted amounts of glutelin with these solvents were 54.1 and 45% respectively. The further purification of SDS soluble glutelins was achieved by Sephadex G-150 gel column chromatography. The molecular weight of the components in four protein groups has been estimated by SDS-polyacrylamide gel electrophoresis with or without ${\beta}-mercaptoethanol.$ The comparison of albumins and globulins by starch gel electrophoresis at pH 3.1 permitted us to identify seven rice varieties. However, at pH 8.95, the specific bands for Japonica type rice varieties were observed.

• Korean Journal of Microbiology
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• v.25 no.4
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• pp.297-297
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• 1987

A $C_{1}$ enriched cellulase fraction was separated from culture filtrate of anaerobic Clostridium thermocellum by hydroxyapatite column chromatography. The separated fraction showed strong synergistic action with $C_{x}$ component (endo-$\beta$-1, 4-glucanase) in digestion of crystalline cellulose, similar to the other aerobic cellulolytic microorganisms. Unlike the $C_{x}$ component the $C_{1}$ enriched fraction was rapidly inactivated by oxidation at the atmospheric condition. The enzyme activity was significantly enhanced by the addition of reducing agents, especially $\beta$-mercaptoethanol, which indicates that a $C_{1}$ component has a lot of sulfhydryl groups essential for the enzyme activity. The effect of metal ions on $C_{1}$ activity was also investigated. The $C_{1}$ fraction was found to be thermally stable compare to endo-$\beta$-1,4-glucanase. Optimal temperature and pH were found to be 60.deg.C and 6.0, respectively.

• Applied Biological Chemistry
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• v.22 no.4
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• pp.191-197
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• 1979

${\beta}-Tyrosinase$ was purified and crystallized from cells of Escherichia intermedia A-21 grown in a medium supplemented with 0.2% L-tyrosine. Molecular weight of its subunit, Km value and absorption spectra were determined. Crystallization methods were also studied to eliminate any unnecessary procedures. The results obtained were as follows: 1. The purification procedure included ammonium sulfate fractionation, dialysis against potassium phosphate buffer, pH 6.0 and pH 7.0, and DEAE-Sephadex column chromatography. In the column chromatography, 11 mg of protein was applied per ml of DEAE-Sephadex for efficiency. 2. Steps of protamine sulfate treatment and Sephadex G-150 gel filtration could be eliminated for this enzyme from the known procedures. 3. The purified enzyme was dissolved in 0.01M potassium phosphate buffer containing 2-mercaptoethanol, with a concentration of 20mg/ml. Crystalline enzyme, which appears as hexagonal rods, was obtained by adding solid fine powdered ammonium sulfate to the enzyme solution. 4. Absorption maxima of the enzyme appeared at 340 and 430nm when associated with pyridoxal phosphate. 5. Km value of the enzyme for L-tyrosine was $2.31{\times}10^{-4}M$ and the molecular weight of its subunit was determined by SDS-polyacrylamide electrophoresis to be approximately 50,000.

• The Korean Journal of Mycology
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• v.17 no.1
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• pp.1-6
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• 1989

The mycelia of Pyricularia oryzae were treated with enzyme solution mixture consisting of Driselase, ${\beta}-Glucuronidase$, Cellulase, and Macerozyme R-10 for the production of protoplasts. More protoplasts were formed from mycelia of race KJ 101 of P. oryzae than that of race KI 315a in the enzyme mixtures. The number of protoplasts was decreased in the untreated control three hrs after the enzyme treatment, whereas the number was increased in the treatments with 10, 50 and 100 mM 2-mercaptoethanol, respectively. The number of protoplasts increased to reach maximum at five hrs after treatment of 10 mM 2-mercaptoethanol, but the least was found in 200 mM. The protoplasts of P. oryzae in a liquid medium containing 2.5% yeast extract, and 2% dextrose reverted to the mycelia after five hrs shaking incubation at $27^{\circ}C$. Some protoplasts produced yeast like buds and the buds were developed to irregularly shaped chains of cell protruded a germ tube like hypha from the distal cell. Once in a while a germ tube like hypha protruded directly from the protoplasts. Except in the first type of reversion, other protoplasts reverted to the normal mycelia. The reversion frequency was highest on PDA with stabilizer of 0.6 M KCl. No reversion of protoplasts occurred on water agar regaardless oftreaatments.

• Applied Biological Chemistry
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• v.32 no.2
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• pp.85-90
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• 1989

High resolution two-dimensional (2-D) electrophoresis with isoelectrofocusing in the first dimension and electrophoresis in sodium dodecyl sulfate in acrylamide gradient gels in the second dimension has been used to produce maps of proteins, extracted from rice seeds with 2% sodium dodecyl sulfate/5% 2-mercaptoethanol. Six rice cultivars-three Japonica types and three Tongil(high-yielding) types-at six maturities were studied. Composite map was constructed and more than 300 polypeptide spots were counted in the pH range of $5.2{\sim}8.3$ and molecular range of $20,000{\sim}100,000$. Vast differences were observed between varieties and between maturities in the maps.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.285-290
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• 2008

Rahnella aquatilis strain AY2000 produces an anti-yeast substance (AYS), however activity of the AYS has a declining tendency during storage. To investigate what has been decreased activity of the AYS, the AYS was treated with various physicochemical agents in this paper. The activity of AYS was decreased by heat treatment. Thiol reagent such as $\beta$-mercaptoethanol or dithiothreitol was also another factor decreasing the activity of AYS. However, pH, EDTA, and NaCl were not factors decreasing the activity of AYS. Use of methanol to precipitate the AYS was also decreased the activity of AYS. The activity of AYS was not lost after Sepharose S-400 gel filtration. However, the AYS activity was completely lost, when a polysaccharide and a unknown substance (230 nm absorption) among components of the AYS was separated by DEAE-cellulose chromatography. MIC of the AYS against S. cerevisiae was usually determined at $7.8-15.6{\mu}g/ml$.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.73-73
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• 2003

본 연구는 돼지 체세포의 clonal cell lines을 효율적으로 확립할 수 있는 배양방법을 제시하기 위하여 실시하였다. 50일령의 돼지태아로부터 섬유아세포를 회수하여 2회 계대배양 후 단일 세포를 96-well plate와 4-well dish에서 배양하여 배양액내의 FBS농도(10, 30, 50%), 첨가제(catalase, $\beta$-mercaptoethanol; BME), 세포의 크기(<16, 16~20, 20<) 및 형태(smooth, rough)에 따른 cell line 확립 효율을 검토하였다. FBS 농도에 따른 clonal line 확립 효율과 PD를 검토한 결과, 효율에 있어서 30% FBS 처리구(5.1%)가 10%(0.3%) 및 50%처리구(2.1%)보다 비교적 높게 나타났으나 유의적 차이는 없었으며, PD의 경우는 10, 30, 50%처리구에서 각각 36.7, 29.4, 26.3 시간으로 FBS 농도가 증가할수록 PD 시간이 유의적으로 짧아졌다(p<0.05). 배양액 첨가제에 있어서 BME 가시 무처리구나 catalase 첨가시보다 유의적으로 높은 효율을 보였으며(11.4%, 3.2%, 3.2%; p<0.05), PD 시간은 짧게 나타났다(23.6, 25.5, 28.1; p<0.05). 그러나 catalase는 cell line 확립 효율에 영향을 미치지 않는 것으로 나타났다. 세포의 크기에 따른 cell line 확립효율은 5.2~8.2%로 크기에 따른 차이는 보이지 않았으며, PD 또한 23.7~27.9 시간으로 세포 크기에 따른 차이는 없었다. 세포의 형태에 따른 세포의 부착율은 smooth(55.8%)구가 rough(73.0%)구보다 유의적으로 낮게 나타났으나(p<0.05), clonal line 확립 효율(7.7%n vs. 6.6%) 및 PD(23.5h vs. 24.0h)에서는 차이가 없었다. 본 연구의 결과, 세포의 형태에 따른 cell line 확립 효율은 큰 차이가 없었으나, 세포 배양액내에 30% FBS와 BME의 첨가로 clonal cell line 확립 효율을 향상시킬 수 있음을 확인할 수 있었다. 또한 cell line 확립 효율이 높아질수록 PD 시간이 짧아지는 경향을 볼 수 있었다.

• Biomedical Science Letters
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• v.6 no.4
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• pp.261-268
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• 2000

Fibrinolytic enzyme (FE-2) was purified from the fruiting bodies of Tricholoma saponaceum using DEAE-Cellulose chromatography and Mono-S column chromatography, The enzyme has a molecular weight of 18.23 kDa and include Zn$^{2+}$ ion as found by ICP/MS. The N-terminal amino acid sequence of the enzyme was A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V It has a pH optimum at pH 7.5, suggested that FE-2 was a neutral pretense. The activity of FE-2 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-2 was increased by $Mg^{2+}$, Zn$^{2+}$, Fe$^{2+}$, and Co$^{2+}$, but the enzyme activity was totally inhibited by Hg$^{2+}$. No inhibition was found with PMSF, E-64, pepstatin and 2-mercaptoethanol. The enzyme hydrolyzed both $A\alpha$ and B$\beta$ chains of human fibrinogen. The $\gamma$ chain was resistant to hydrolysis by FE-2.

• Analytical Science and Technology
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• v.12 no.3
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• pp.256-259
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• 1999

Tyrosinases are related to the enzymatic browning of plants and attract the major scientific interest for the prevention of it. Three tyrosinase isozymes ($P_1$, $P_2$ and $P_3$) from pine needles were purified to homogeneity and characterized the factors that affect their activities. The L-ascorbic acid and ${\beta}$-mercaptoethanol notably inhibited the enzymatic activities of the three isozymes. The sodium diethyldithiocarbamate was a competitive inhibitor of isozymes with the $K_i$ values of $P_1$(0.030 mM), $P_2$(0.015 mM) and $P_3$(0.019 mM), respectively. Their enzyme activities were however, increased by the addition of most metal ions. The optimum pH for the three isozymes was 9.0~9.5 and the optimum temperatures ranged from 55 to $60^{\circ}C$ using L-DOPA as substrate.