• 한국약용작물학회지
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• 제12권1호
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• pp.1-9
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• 2004

200종의 식물추출물을 대상으로 PEP 저해활성을 측정한 결과, 81종의 식물추출물은 $10{\sim}30%$ 범위의 저해율을, 28종의 식물추출물은 10%이하의 저해율을 나타내어 총 200종의 식물추출물 중 약 55% 정도의 식물추출물이 30%이하의 낮은 PEP 저해활성을 나타내었다. 반면에, Prunus mume (매화나무) 잎 (95.3%), 노루발과 (Pyrolaceae)의 Pyrola japonica (노루발) 잎과 줄기 (93.2%), 물레나물과 (Hypericaseae)의 Hypericum ascyron (물레나물) 지상부 (90.2%), 범의귀과(Saxifragaceae)의 Astilbe chinensis var. typica (노루오줌) 지상부 (90.1%), 보리수나무과 (Elaeagnaceae)의 Elaeagnus umbellata (보리수나무) 잎과 줄기 (90.1%)등 5종의 추출물들이 5 ppm에서 PEP에 대하여 90% 이상의 강한 저해활성을 나타내었다.

• 약학회지
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• 제41권2호
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• pp.153-160
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• 1997

One hundred and seventy crude drugs were screened for prolyl endopeptidase (PEP) inhibitors. Among them, 80% methanolic extract of 18 medicinal plants such as Polygonum cuspi data, Sanguisorba officinalis, Eugenia caryophyllata, Rubus coreanus, Cinnamomum cassia (Cassiae Cortex and Cinammomi Ramulus), Rheum palmatum, Ulmus pumila, Sorbus commixta, Areca catechu, Uncaria sinensis, Terminalia chebula, Caesalpinia sappan, Nelumbo nucifera, Machilus thunbergii, Paeonia moutan, Elscholtzia patrini and Cynomorium coccineum inhibited more than 70% of PEP activity at a concentration of 40 ppm. The active principles of P. moutan, M. thunbergii, T chebula, A. catechu, S. commixta, R. palmatum, R. coreanus, E. caryophyllata and P. cuspidata were transferred into organic solvents, which showed more than 75% inhibition at 5 ppm.

• Journal of Applied Biological Chemistry
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• 제49권3호
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• pp.110-113
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• 2006

Prolyl endopeptidase (PEP, EC 3.4.21.26, also referred to as prolyl oligopeptidase) degrades proline containing, biologically active neuropeptides such as vasopressin, substance P and thyrotropin-releasing hormone by cleaving peptide bonds on carboxyl side of prolyl residue within neuropeptides of less than 30 amino acids. Evaluation of PEP levels in postmortem brains of Alzheimer's disease patients revealed significant increases in PEP activity. Therefore, a specific PEP inhibitor can be a good candidate of drug against memory loss. Upon our examination for PEP inhibitory activity from micronutrients, ascorbic acid (vitamin C) showed small but significant PEP inhibition (13% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$). Palmitic acid showed almost no PEP inhibition. However, 6-O-palmitoyl ascorbic acid ($\underline{1}$) showed 70% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$ indicating that hydrophobic portion of the compound $\underline{1}$ may facilitate the inhibitory effect. $IC_{50}$ value of compound $\underline{1}$ was $12.6{\pm}0.2{\mu}M$. The primary and secondary Lineweaver Burk and Dixon plots for compound $\underline{1}$ indicated that it is a non-competitive inhibitor with inhibition constant (Ki) value of $23.7{\mu}M$.

• Applied Biological Chemistry
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• 제42권4호
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• pp.351-355
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• 1999

갈화로부터 항치매 효과를 기대할 수 있는 prolyl endopeptidase(PEP) 저해제를 분리하기 위하여 갈화의 methanol 추출물을 chloroform 및 ethyl acetate로 분배추출 후 chloroform 가용성 분획에 대하여 silica gel, Sephadex LH-20 column chromatography 및 RP-HPLC를 행한 결과, $FeCl_3$,에 양성 반음을 나타내는 4종의 화합물을 얻었다. 이들을 $^1H-$, $^{13}C-$, $^2D-NMR$ 및 MS 등을 이용하여 분석한 결과, 각각 tectorigenin, genistein, 5,7-dihydroxy-4',6-dimethoxyisoflavone, 5-hydroxy-6,7,4'-trimethoxyisoflavone으로 동정하었으며 HMBC, HMQC를 통하여 $^{13}C-NMR$ signal들을 assign함으로써 기 보고된 data들의 오류를 바로잡았다. PEP에 대한 이 화합물들의 $IC_{50}$값은 각각 5.30 ppm$(17.7\;{\mu}M)$, 10.39 ppm$(38.5\;{\mu}M)$, 13.92 ppm$(44.3\;{\mu}M)$, 20.61 ppm$(62.8\;{\mu}M)$이었다.

• 한국지역사회생활과학회지
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• 제16권4호
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• pp.95-102
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• 2005

현재 알츠하이머 질환의 주요한 치료제는 아세틸콜린 분해 저해제(acetylcholinesterase inhibitors)이나, tacrine의 경우 간독성 때문에 지금은 거의 사용되지 않고 있다. 이외에 알츠하이머 질환의 잠재적 치료로서 항산화제와 항아밀로이드 요법이 대두되어 치매의 예방 및 치료를 위한 무독성의 약물 개발을 위해서 많은 연구가 수행되고 있으나 아직까지 치매의 발병 기전이나 뚜렷한 효능을 가진 치료제에 대한 보고가 없는 실정이다. 따라서 본 연구에서는 새로운 자원에서 알츠하이머 질환의 치료제로서의 가능성을 탐색하고자 전초 추출물 35종과 부위별 추출물 36종의 식물 추출물 71종에서 DPPH 라디칼 소거능과 PEP 활성 저해율을 측정하였다. 항산화 활성을 살펴 본 결과, 식물 전초에서는 땃딸기(Fragaria yezoensis)에서 $90.4\%$의 라디칼 제거 효과를 보였고, 은행나무(Gingko biloba), 치자나무(Gardenia jasminoides for. grandiflora), 산철쭉(Rhododendron yedoensa var. poukhanense)등의 줄기, 털진달래(Rhododendron mucronulatum var. ciliatum), 치자나무(Gardenia jasminoides for. grandiflora)와 물개암나무(Corylus sieboldiana var. mandshurice)의 잎 부위에서 $88\%$ 이상의 높은 라디칼 제거 효과를, 산수유(Cornus officinalis) 열매 부위 추출물에서는 약 $95\%$ DPPH 라디칼 제거 효과를, 은행나무(Gi템 biloba), 우산고로쇠(Acerokamotoaum)등의 뿌리 부위에서 $85\%$ 이상의 DPPH 라디칼 제거 효과를 보였다. PEP활성의 저해율을 측정하여 비교한 결과, 식물 전초의 경우 딸기(Fragaria ananassa), 땃딸기(Fragaria yezoensis), 고추나물(Hyperircum erectum)에서 높은 저해율을 줄기 부위의 경우 다래(Actinidia arguta), 산철쭉 (Rhododendron yedoensa var. poukhanense)의 줄기 부위, 산철쭉(Rhododendron yedoensa var. poukhanense)과 생열귀나무(Rosa davurica), 삼나무(Cryptomeria japonica), 털진달래(Rhododendron mucronulatum var. ciliatum)의 및 부위에서 90$\%$ 이상의 높은 PEP 활성 저해율을, 산수유(Cornus officinalis)의 열매 추출물이 95.3$\%$의 저해율을, 우산고로쇠(Acer okamotoanum) 뿌리 추출물이 $83.3\%$의 저해율을 나타냈다. 식물 추출물 71종에서 땃딸기(Fragaria yezoensis) 전초, 산수유(Cornus officinalis) 열매 부위의 경우 DPPH 라디칼 소거능과 PEP 활성 저해율에서 모두 $90\%$ 이상의 활성을 보이는 등 DPPH 라디칼 소거능과 PEP 활성 저해율 사이에 유의수준 0.000에서 양의 상관관계가 있었다. 따라서 PEP 활성 저해 메커니즘에 대한 항산화 작용의 영향을 더욱 더 조사할 필요가 있을 것으로 판단되며, 산수유와 땃딸기와 같은 식물 자원은 항산화활성과 PEP 활성 저해능이 모두 높으므로 알츠하이머 질환의 치료 및 예방 물질로서의 개발 가능성이 큰 것으로 보인다.

• Journal of Applied Biological Chemistry
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• 제55권3호
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• pp.195-198
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• 2012

Four chromone derivatives (1-4) were isolated from the clove buds (Syzygium aromaticum). Of these, two chromone C-glucosides (1 and 2) showed significant PEP inhibition with $IC_{50}$ values of $1.48{\pm}0.02$ and $1.74{\pm}0.03{\mu}M$ and $K_i$ values of $0.27{\pm}0.02$ and $0.50{\pm}0.05{\mu}M$, respectively. They also exhibited strong antioxidant capacities against the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt radical system with $EC_{50}$ values of $4.13{\pm}0.04$ and $4.79{\pm}0.03{\mu}M$, respectively.

• 한국미생물·생명공학회지
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• 제24권2호
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• pp.191-196
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• 1996

The bacteriolytic enzyme produced from Bacillus subtilis SH-1 was purified and characterized, and its molecular weight was determined. The bacteriolytic enzyme activity was increased about 66.5 times via purification with recovery yield of 18.5%. The optimum pH and temperature of this enzyme were 9.0 and 50$\circ$C. The enzyme was stable within a pH range of 6.0-10.0 and unstable above 60 . The molecular weight of the enzyme was estimated to be 23,000 dalton in a form of monomer with no other subunits. Effect of the enzyme on the lysis of bacteria engaged in food posion was tested. The lysis degree was below 31% against Gram negative bacteria and above 48% in Gram positive bacteria. The values higher than 73% were obtained against Vibrio sp. and Listeria sp. As the turbidity of dissolved peptidoglycan clecreases, the free amino group levels were increased. And, based on hydrolysis of casein, this enzyme was thought to be an endopeptidase.

• Journal of Microbiology
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• 제41권1호
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.798-803
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• 1999

Prolyl endopeptidase [PEP; EC 3.4.21.26], a serine protease which is known to cleave peptide bonds on the carboxy side of a proline residue, plays an important role in the degradation of proline-containing neuropeptides that have been suggested to participate in learning and memory processes. An abnormal increase in the level of PEP, which can lead to generation of $A{\beta}$, is also suggested to be involved in Alzheimer's type senile dementia. In the course of screening PEP inhibitors from Basidiomycetes, the mushroom Polyozellus multiplex exhibited a high inhibitory activity against PEP. Two active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification, using silica gel, Sephadex LH-20, and Lobar RP-18 chromatography. The chemical structures of these compounds were identified as thelephoric acid and 12-acety1-2,3,7,8-tetrahydroxy-[12H]-12-hydroxymethylbenzobis[I.2b,3.4b'] benzofuran-11-one (kynapcin-9) by spectral data including UV, IR, MS, HR-MS, $^1H-,{\;}^{13}C-$, and 2D-NMR. The $IC_{50}$ values of the thelephoric acid and kynapcin-9 were 0.157 ppm (446nM) and 0.087 ppm (212nM) and their inhibitor constants ($K_i$) were 0.73ppm ($2.09{\;}\mu\textrm{m}$) and 0.060 ppm (146 nM), respectively. Furthermore, they were non-competitive with a substrate in Dixon plots.

• Molecules and Cells
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• 제42권1호
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• pp.79-86
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• 2019

Endolysins are bacteriophage-derived enzymes that hydrolyze the peptidoglycan of host bacteria. Endolysins are considered to be promising tools for the control of pathogenic bacteria. LysB4 is an endolysin produced by Bacillus cereus-infecting bacteriophage B4, and consists of an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall binding domain (CBD). LysB4 was discovered for the first time as an L-alanoyl-D-glutamate endopeptidase with the ability to breakdown the peptidoglycan among B. cereus-infecting phages. To understand the activity of LysB4 at the molecular level, this study determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin. The LysB4 EAD has an active site that is typical of LAS-type enzymes, where $Zn^{2+}$ is tetrahedrally coordinated by three amino acid residues and one water molecule. Mutational studies identified essential residues that are involved in lytic activity. Based on the structural and biochemical information about LysB4, we suggest a ligand-docking model and a putative endopeptidase mechanism for the LysB4 EAD. These suggestions add insight into the molecular mechanism of the endolysin LysB4 in B. cereus-infecting phages.