• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.969-978
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• 2014

The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at $20^{\circ}C$, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at $37^{\circ}C$. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of $1,069.4{\pm}42.4U/mg$ protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and $40^{\circ}C$, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded $A{\alpha}$ and $B{\beta}$ chains of fibrinogen quickly, but not the ${\gamma}$-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The $K_m$ and $k_{cat}/K_m$ of AprE2 was 0.56 mM and $3.10{\times}10^4S^{-1}M^{-1}$, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• 제13권1호
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• pp.31-35
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• 2006

The ubiquitin conjugating enzyme 2 (E2) is core component of ubiquitin proteasome pathway (UPP) which represents a selective mechanism for intracellular proteolysis in eukaryotic cells. The E2 has been implicated in the intracellular transfer of ubiquitin to target protein. We show here the involvement of E2 in antiviral immune of Bombyx mori to Bombyx mori nuclear polyhedrosis virus (BmNPV). In this study, mRNA fluorescent differential display PCR (FDD-PCR) was performed with BmNPV highly resistant silkworm strain NB and susceptible silkworm strain 306. At 24 h post BmNPV infection, FDD-PCR with the arbitrary primer AP34 showed that one cDNA band was down-regulated in the midgut of resistant strain, but highly expressed in susceptible strain. The deduced amino acid sequence of this cDNA clone share 99% identity with the recently published B. mori ubiquitin conjugating enzyme E2 (Genbank NO: DQ311351). Fluorescent quantitative PCR corroborated down regulation of E2 in resistant strain. We there conclude that BmNPV infection evokes strong response of susceptible strain including activation of UPP. BmNPV may evolve escape mechanisms that manipulate the UPP in order to persist in the infected host. In addition, the identification of down-regulation of E2 in resistant strain, as well as structure data, are essential to understanding how UPP operates in silkworm antiviral immune to BmNPV disease.

• Journal of Applied Biological Chemistry
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• 제43권4호
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• pp.240-245
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• 2000

Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.

• Journal of Nutrition and Health
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• 제31권2호
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• pp.153-158
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• 1998

The purpose of this study was to investigate the effect of vitamin E supplementation on the lipid peroxidation and activities of antioxidative enzymes in the pancreas of diabetic KK mice. KK mice were fed high ft diet containing 20% corn oil(wt/wt), and sacrificed at 2 months of diabetes. A hish vitamin E diet consisted of the high fat diet supplemented with an excessive amount of 이-$\alpha$-tocopheryl acetate (2080IU/kg diet). The incidence of diabetes mellitus was 61% when mice were fed the high fat diet, but was 44% when mice were fed the high vitamin E diet, Vitamin E supplementation fhus seems to have the effect of decreasing of decreasing the onset of diaetes. In the diabetic group, we found increases of MDA (malondialdehyde) and antioxidative enzyme activities. Treatment with vitamin E did not modify the level of fasting blood glucose. However, MDA and antiosicative enzyme activities in diabetic mice were decreased by the high vitamin E diet. Increased levels of lipid peroxidation products suggests the occurrence of oxidative damage in the pancreas of diabetic mice. The increased level of antiosicative enzyme activities could be due to an adaptive response to conditions of increased peroxidative stress. Significant normalization on catalase activity was noted in vitamin E supplemented animals.

• 대한화학회지
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• 제12권4호
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• pp.142-145
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• 1968

The relative activity of glucose-6-phosphate dehydrogenase in E. coli was measured at 340 $m\mu$ with a spectrophotometer. The synchronized E. coli cells in exponential phase were treated with Phage($T_2$) ghost, and used as a enzyme solution directly. This assay method supposed to be useful for the continuous determination of enzyme activity in E. coli.

• 한국수산과학회지
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• 제31권6호
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• pp.829-834
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• 1998

옥수수 전분가수분해물의 용해도는 D.E. 값이 높을수록 증가하였고, 10 이상에서는 거의 차이가 없었다. $T_g^{'}$ 값은 D.E. 값이 증가함에 따라 감소하는 직선적인 관계를 나타내었다. Alkaline phosphatase의 동결계에서 거동은 glass cynamic mechanism, 즉 효소의 가수분해 속도는 첨가된 물질의 $T_g^{'}$ 이하의 온도에서 지연되거나 억제되었다. 옥수수전분 가수분해물은 동결계에서 유리전이온도를 높임으로서 동결계가 유리상태로 되고 따라서 점도는 높아지고 단백질은 분자간 접촉의 기회가 줄어들어 단백질이 보호된다는 cryostabilization mechanism으로 설명 가능하였다.

• Journal of Microbiology and Biotechnology
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• 제2권3호
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• pp.161-165
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• 1992

The gene encoding the bacteriolytic enzyme cell wall peptidoglycan hydrolase from alkalophilic Bacillus sp. was cloned in E. coli using pBR322 as a vector. A recombinant plasmid, designated pYTR451, was isolated and the size of the cloned HindIII fragment was found to be 4.8 Kb. The cell wall hydrolysis activity of an extract of the E. coli harboring the recombinant plasmid pYTR 451 was detected by SDS- polyacrylamide gel containing 0.2% (w/v) purified cell wall of Bacillus sp. The molecular weight of the enzyme was estimated to be about 27, 000 corresponding to the molecular weight of the Bacillus sp. bacteriolytic enzyme. The recombinant plasmid was found to contain the fragment originated from Bacillus sp. YJ-451 chromosomal DNA by Southern hybridization.

• 한국미생물·생명공학회지
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• 제1권2호
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• pp.93-98
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• 1973

The acid protease was isolated from the culture broth of the Penicillum sp. grown in the wheat bran media. 'quot;The crude purification of this enzyme was carried out by extraction with distilled water and precipitated with saturated ammonium sulfate. " The activity of this enzyme was found to be very strong by Folin′s colorimetric method. The results were as follows. 1. The optimum pH of the enzyme activity was at 3.0 and its optimum temperature was 5$0^{\circ}C$. 2. Although the enzyme activity to hydrolyze casein was maximal at 5$0^{\circ}C$, its activity decreased rapidly by about 50％, treated at 5$0^{\circ}C$ for 30 min. When treated at 4$0^{\circ}C$ for 60 min, the enzyme activity decreased to 75％ of original value and did not decrease any more. 3. The enzyme was stable at pH 2.0 to 6.0. 4. This enzyme activity was not effected by metal ions; C $d^{＋＋}$, Z $n^{＋＋}$, $Co^{＋＋}$, H $g^{＋＋}$, M $n^{＋＋}$, P $b^{＋＋}$, $Mg^{＋＋}$, L $i^{＋}$, C $u^{＋＋}$, $Ba^{＋＋}$, A $g^{＋}$, $Al^{＋＋＋}$, $Ca^{＋＋}$, F $e^{＋＋}$, and F $e^{＋＋}$ 5. Also, it was not effected by treatemnt of EDTA.

• Applied Biological Chemistry
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• 제38권2호
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• pp.118-122
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• 1995

Escherichia coli의 오르니틴 트란스카바밀라제는 오르니틴과 카바밀인산으로부터 시트룰린의 합성을 촉진시키는 효소이다. 이 효소의 기능과 구조와의 상관관계, 반응메카니즘 등 생화학적 연구를 하기 위하여 대량의 효소를 추출할 필요가 있다. 본 연구는 오르니틴 트란스카바밀라제의 대량생산 시스템을 확립하기 위하여 E. coli argI 유전자를 E. coli $DH5{\alpha}$ 세포의 염색체 DNA를 추출한 후에 PCR 방법으로 증폭시켜 얻었다. 증폭된 argI 유전자를 단핵생물 단백질 발현벡터인 pKK223-3에 접합시킨 후, 오르니틴 트란스카바밀라제가 존재하지 않은 E. coli TB2 세포에 클로닝 시켰다. 이 세포로부터 생산된 오르니틴 트란스카바밀라제는 암모늄염에 의한 분할, 열변성, 크로마토그래피 등을 사용하여 순수하게 분리하였다. SDS 단백질 전기영동 결과 약 38 kDa 크기의 효소가 순수하게 얻어졌다. 반응속도론적 실험결과 $K_{cat}$은 $1{\times}10^5m^{-1}$, $K_M$은 오르니틴에 대하여는 0.35 mM, 카바밀인산에 관하여는 0.06 mM이 각각 얻어졌다. 이 결과는 야생형 오르니틴 트란스카바밀라제의 반응속도 인자들과 비슷한 값이다. 본 연구는 이들 결과로부터 오르니틴 트란스카바밀라제의 기능을 하는 E. coli argI 유전자가 클로닝 되었음을 확인하였다.

• 대한의생명과학회지
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• 제6권4호
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• pp.261-268
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• 2000

할미송이버섯으로부터 혈전용해효소 (FE-2)를 DEAE-cellulose, Mono-S column으로 분리 정제하였다. 이 효소는 분자량이 18.2 kDa 이었으며, ICP-MS (inductively coupled plasma mass spectrometer) 분석 결과 $Zn^{2+}$을 포함하고 있었다. 15번째까지의 N-terminal amino acid 서열은 A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V이고, pH 7.5에서 활성이 가장 큰 염기성 단백질 분해효소로, EDTA와 1,10-phenanthroline에 의해 활성이 감소되는 metalloprotease였다. $Mg^{2+}$, $Zn^{2+}$, Fe$^{2+}$, Co$^{2+}$의 첨가 시 활성이 증가하였으나 Hg$^{2+}$의 경우 활성이 완전히 소멸되었다. 이 효소 (FE-2)는 단백질 분해효소 저해제인 E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), PMSF (phenylmethylsulfonyl fluoride), pepstatin 과 2-mercaptoethanol의 영향을 받지 않으며, 섬유소원 (fibrinogen)과 반응 시 $A\alpha$ chain과 B$\beta$ chain은 분해시키지만 $\gamma$ chain과는 반응하지 않았다.