• 한국임상수의학회지
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• 제12권1호
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• pp.905-910
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• 1995

GC 함유량(72%)이 높은 Mycobacterium paratuberculosis의 DNA의 PCR 증폭시 특이성과 생산성을 높이기 위하여 PCR 반응액에 denaturant인 DMSO, glycerol, formamide, Tween 20과 NP 40를 첨가하였다. Denaturant를 첨가하지 않은 상태의 PCR에서는 다수의 비특이적인 DNA가 관찰되었으며 표적 DNA 생산량이 낮았다. 모든 denaturant는 PCR의 특이성과 생산물의 생산량을 증가시했으며, 이들 중 DMSO, glycerol, farmamide와 NP 40는 높은 농도에서 생산량을 증가시켰다. Tween 20은 낮은 농도에서 생산량을 증가시켰다. Denaturant를 첨가하였음에도 불구하고 대부분의 반응에서 1 또는 2개의 비특이적인 DNA가 관찰되었다.

• 대한의생명과학회지
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• 제2권2호
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• pp.241-247
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• 1996

G+C 함량이 높은 primer를 이용한 중합효소 연쇄반응을 실시하는 경우 높은 annealing 온도로 인하여 특이 염기서열의 합성정도가 매우 미약하게 나타나는 경우가 많이 있다. 이와 같은 문제점을 보완하기 위하여 glycerol, formamide 및 dimethyl sulfloxide (DMSO) 등의 변성제를 반응용완충액에 첨가하고 중합효소 연쇄반응을 실시하여 그 결과를 비교 검토하였다. G+C 함량이 낮은 Borrelia burgdorferi의 Lyl 유전자의 primer set인 Bb 679와 Bb 680를 이용한 중합효소 연쇄반응에서는 변성체 첨가에 따른 합성 DNA의 양의 변화가 뚜렷하지 않았다. 그러나 G+C 함량이 높은 primer set인 Mycobacterium paratuberculosis의 IS900 유전자의 IS900/150C와 IS900/921를 이용한 중합효소 연쇄반응을 유도한 경우에는 변성제를 첨가함에 따라 합성된 DNA의 양의 증가가 뚜렷하였으며, 2.5％ glycerol과 1.25％ formamide를 혼합 첨가한 경우와 또는 2.5％ DMSO를 반응용 완충액에 첨가하였을 때 비특이적인 증폭비율이 낮아 특이 염기서열의 합성 결과가 양호한 것으로 나타났다.

• 생명과학회지
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• 제22권6호
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• pp.778-787
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• 2012

카드뮴에 의해 유도되는 담배의 생장, 엽록소 함량, rubisco와 rubisco activase에 미치는 SA의 영향과, 이에 대한 변성제의 효과를 연구하였다. 담배 기내생장에 대한 SA의 최적농도를 찾기 위해, $10^{-6}$ mM - $10^2$ mM SA를 처리하여 9주간 생장시킨 결과, $10^{-4}$ mM SA에서 가장 높은 생장을 보였다. SA와 카드뮴을 4개의 실험구(대조구, SA, 카드뮴, 카드뮴 + SA)로 하여 생장, 엽록소 함량 및 rubisco와 rubisco activase의 함량과 활성을 측정한 결과, 카드뮴 > 카드뮴 + SA < 대조구 카드뮴 + SA < 대조구 ${\beta}$-mercaptoethanol은 활성을 촉진시켰으며, urea, thiourea, guanidium-HCl은 억제시켰다. 이는 L-cysteine과 ${\beta}$-mercaptoethanol은 변성에 관여하지 않으며, urea, thiourea, guanidium-HCl은 변성에 관여하였음을 의미한다. Rubisco activase의 활성에 대한 변성제들의 영향을 조사한 결과, 5종 모두는 비처리구와 비교하여 현저한 영향은 나타내지 않았다.

• 한국식품영양학회지
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• 제5권2호
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• pp.144-149
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• 1992

Invertase was extracted from Korean ginseng(Panax ginseng C. A. Meyer) leaf with deionized water, and then prepared by ammonium sulfate(0.4~0.6 Sat.) fractionation, the enzymological properties of the invertase were investigated, and the results obtained were as follows. The optimum pH and temperature of the enzyme were pH 6.0 and 4$0^{\circ}C$ respectively. The enzyme was stable in the pH range of pH 6.0 to 8.0, and at the temperature below 4$0^{\circ}C$. The enzyme was inactivated completely by the treatment with some proteases(pepsin, trypsin, papain and ficin) and protein denaturants(8M urea and 6M guanidine-HCI), but not with glycosidases (a-amylase, $\beta$-amylase and glucoamylase). The enzyme catalyzed specifically the hydrolization of the $\beta$-fructofuranosides such as sucrose and inulin.

• KSBB Journal
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• 제13권2호
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• pp.133-140
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• 1998

Using rlFN-$\alpha$ and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.567-573
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• 1996

Chitinase (EC 3.2.1.14) from culture fluid of Bacillus licheniformis KFB-C14 was purified 66-folds to homogenity in overall yield of 21% by ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl and TSK-Gel HW-55F column chromatography. The enzyme protein had a molecular weight of about 86,000 and was composed of one subunit. The enzyme was significantly stable not only at high temperature but also on treatment with organic solvents and protein denaturants such as SDS, urea and guanidine-HC1. The optimum temperature and pH for reaction was 60$\circ $C and 6.0, respectively. The enzyme activity was inhibited by only Mn$^{2+}$ ion, but not inhibited by EDTA, N- ethylmaleimide and pCMB. The enzyme had high activity with colloidal chitin (V$_{max}$: 421) and commercial chitin (V$_{max}$: 480), but not with typical substrates of exo type chitinase. The thermostable chitinase had an useful reactivity for producing functional chitooligosaccharide, showing the production of (GlcNAc)$_{1}, (GlcNAc)$_{3}$, and (GlcNAc)$_{2}$ as major product.

• BMB Reports
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• 제31권2호
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• pp.130-134
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• 1998

An aldolase gene has been cloned from Methanococcus jannaschii. The coding region of the gene has been expressed in E. coli using a pET system to a level of 30% of total cellular proteins. The protein was purified to more than 95 % homogeneity by heat treatment and ion exchange chromatography. The protein performed an aldol condensation reaction with glyceraldehyde as substrate and dihydroxyacetone phosphate as a carboxyl donor. The protein was determined to be a type II aldolase which requires the $Zn^{2+}$ ion as a metal cofactor. This enzyme has a broad range of optimum pH (7-9) and temperature ($50-80^{\circ}C$). It shows strong stability against heat, chemical denaturants, as well as a high percentage' of organic solvents. The half-life of this enzyme at $85^{\circ}C$ is more than 24 h and it maintains more than 90% of aldolase activity in the presence of 6 M urea, 50% acetonitrile, or 15% isopropyl alcohol.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.308-312
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• 1991

B.thuringiensis subsp. darmstadiensis 73E10-2의 내독소에 대한 화학적 처리에서 고농도의 중성염(4M NaBr), 유기용매(50% acetone), 변성제(4M urea) 및 중성 계면활성제(10% triton X-100)로 내독소를 처리하였을 때 모기유충에 대한 독력의 소실이 거의 나타나지 않았으나, guanidine HCL이나 $CCl_4$ 또는 양이온 및 음이온 계면활성제로 처리함으로써 그 독력이 크게 소실되었다. 또한, 내독소의 sulfhydryl기의 변형은 모기유충에 대한 독력에 영향을 나타내지 못하였으나, lysine기 변형으로 내독소의 독력이 거의 완전히 소실되었다.

• BMB Reports
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• 제38권2호
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• pp.177-181
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• 2005

Effects of common electrophoretic reagents, reducing agents ($\beta$-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.235-243
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• 2019

A special eosinophilic agarase exo-type ${\beta}$-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a ${\beta}$-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were $50^{\circ}C$ and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at $50^{\circ}C$, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type ${\beta}$-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.