Dong-Min Chung

A New Fibrinolytic Enzyme (55 kDa) from Allium tuberosum: Purification, Characterization, and Comparison

Chives have been used both as food and as medicine. Previously, two fibrinolytic enzymes, ATFE-I (90 kDa) and ATFE-II (55 kDa), were identified in chives (Allium tuberosum), a perennial herb. In the present work, ATFE-II was purified by ion-exchange chromatography followed by gel filtration. In addition, the enzyme properties of ATFE-I and ATFE-II were compared. The molecular mass and isoelectric point (pI value) of ATFE-II were 55 kDa and pI 4.0, respectively, as revealed using one- or two-dimensional fibrin zymography. ATFE-II was optimally active at pH 7.0 and 45°C. ATFE-II degraded the Aα-chain of human fibrinogen but did not hydrolyze the Bβ-chain or the γ-chain, indicating that the enzyme is an α-fibrinogenase. The proteolytic activity of ATFE-II was completely inhibited by 1 mM leupeptin, indicating that the enzyme belongs to the cysteine protease class. ATFE-II was also inhibited by 1 mM Fe²(+). ATFE-II exhibited high specificity for MeO-Suc-Arg-Pro-Tyr-p-nitroaniline (S-2586), a synthetic chromogenic substrate of chymotrypsin. Thus proteolytic enzymes from A. tuberosum may be useful as thrombolytic agents.

Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel

A new zymogram method, silver-stained fibrin zymography, for separation of protease bands and activity detection using a single substrate gel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE in a gel containing fibrin, the gel was incubated in enzyme reaction buffer and the zymogram was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined. Furthermore, proteases of high molecular weight were clearly and sharply resolved.

Quercetin Glucoside Profiling of Fresh Onion ( Allium cepa) and Aged Black Onion Using HPLC-ESI/MS/MS

Quercetin is a major flavonoid present in onions, which acts as an antioxidant. Quercetin exists both as a free compound and conjugated with carbohydrates, primarily as glucosides in onion. Aged black onion was made through a 30 day aging process in which the onions were kept in an environment of 60℃ and high humidity (90% RH). Quercetin and quercetin glucosides were assayed in onion bulbs before and after the aging process, using high performance liquid chromatography-electrospray ion trap mass spectrometry (HPLC-ESI/MS/MS). Quercetin mono- and diglucosides were identified in fresh onion bulbs, whereas quercetin aglycone was the only form present in aged black onion bulbs. These findings indicate that the quercetin mono- and di-glucosides present in fresh onions undergo complete deglycosylation during the aging process. Such profiling will provide a rapid method that can be used to assess changes in the two major quercetin glycosides during the aging process of onion bulbs.

Effect of NaCl on Hydrolytic Activity of Leucine Aminopeptidase from Bacillus sp. N2

Salt stability of enzymes is a crucial practical factor in the food industry. Previously, leucine aminopeptidase (LAP) was purified from Bacillus sp. N2. Here, we present the salt effect of LAP using synthetic substrates. LAP had a hydrolytic activity for L-leucine--nitroanilide in high concentrations of NaCl (up to 4 M), but not for other neutral salts (LiBr, LiCl, NaBr, KBr, and KCl). It hydrolyzed various synthetic di-peptide substrates with hydrophobic and hydrophilic amino acids at the C-terminal Xaa region, in the presence of 0-4 M NaCl. The result indicated that the hydrolytic action of LAP is not dependent on the hydrophobicity of the amino acid side chain at the scissile bond of the substrate. Remarkably, the hydrolytic activity of LAP was 1-3 folds higher than those of other LAPs and aminopeptidases in 4.5 M NaCl, suggesting that NaCl-tolerant LAP might be used in the food industry as cheese and anchovy sauce.

Spectrophotometric Assay for Determination of Chlorogenic Acid Using Green Pigment Formation and Quantitative Analysis of Chlorogenic Acid in Blueberry Leaf

본 연구는 클로로겐산의 함량을 측정하기 위한 흡광도 방법을 구축하였으며, 구체적으로는 클로로겐산이

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

50^{\circ}C

Purification and characterization of a novel fibrinolytic enzyme from chive (Allium tuberosum)

와 글라이신 및 알칼리 조건하에서 녹색반응이 일어난다는 현상을 이용했다. 녹색형성은 일련의 클로로겐산 농도에 의존성을 가졌다. 본 연구에 따른 클로로겐산의 측정방법을 이용하여 블루베리에 함유된 클로로겐산의 함량을 분석하였다(12.42 mg/g d.w). 이러한 방법은 저가의 비용과 빠른 시간으로 많은 시료를 대량으로 쉽게 클로로겐산의 함량은 측정할 수 있는 효과를 가진다. 【We developed a spectrophotometric assay for the quantitative determination of chlorogenic acid based on the formation of green pigment at

Purification and Characterization of a Subtilisin D5, a Fibrinolytic Enzyme of Bacillus amyloliquefaciens DJ-5 Isolated from Doenjang

50^{\circ}C

녹색반응을 이용한 클로로겐산의 함량측정을 위한 흡광도 분석법과 블루베리 잎에 함유된 클로로 겐산의 함량분석

under glycine and alkaline conditions in 96-well plates. The formation of green pigment was linear with a series of chlorogenic acid concentration (0-

Rapid concentration of some bacteriocin-like compounds using an organic solvent

300\;{\mu}M