• Journal of Pharmacopuncture
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• v.17 no.1
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• Food Science and Biotechnology
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• v.18 no.2
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• pp.488-493
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• 2009

A protein isolate was prepared from black soybean (Glycine max L. Merrill) that possessed higher antioxidant activity than ordinary white soy protein isolates. The isolate was partially hydrolyzed by alcalase to reduce the allergenicity of black soybean. Alcalase remarkably reduced the molecular mass of the major soybean allergens that have molecular weights of 53, 38, and 24 kDa. Hydrolytic breakdown occurred more effectively in Gly m Bd 30K than in Gly m Bd 60K or Gly m Bd 28K. Alcalase hydrolysis increased the solubility and hydrophobicity of the black soybean protein isolate. The foaming activity and stability of black soybean proteins were highly increased by the partial hydrolysis.

• Korean Journal of Food Science and Technology
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• v.29 no.6
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• pp.1125-1130
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• 1997

The effects of enzyme combination, pH, acid washing and enzyme treatment sequence were investigated in the hydrolysis of soy protein. Comparing Alcalase vs. Neutrase/Alcalase, it appeared that Neutrase/Alcalase was more efficient than Alcalase alone, as the highest degree of hydrolysis (DH) was seen in Neutrase/Alcalase. A surprisingly high DH (more than 60%) was observed with Flavourzyme in the second hydrolysis. The separation of insolubles from the first hydrolysis had little effect on the second hydrolysis. When the washing water from the first hydrolysis was reused in the next hydrolysis, the DH and protein recovery were increased. The addition of calcium ion showed not so much positive effects by the stabilization of Neutrase on the Protein hydrolysis. The use of carbohydrase and repeated acid washing gave positive effects on DH. The simultaneous treatment using endoprotease and exoprotease with pH adjustment improved DH significantly.

• Toxicological Research
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• v.14 no.4
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• pp.525-533
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• 1998

The covalent interactions of toluenediisocyanate (TDI) with macromolecules were investigated both in vitro and in vivo. In vitro incubations of 2,4- and 2,6-TDI with DNA or proteins resulted in dose-dependent formation of TDI-protein and TDI-DNA adducts. TDI-treated DNA was highly resistant to enzymatic digestion and thermal hydrolysis, but was readily hydrolyzed under acidic conditions by releasing its corresponding toluenediamine (TDA), suggesting that TDI caused the crosslinking of DNA. Reaction of TDI with albumin and globin resulted in the formation of several adducts, and some adducts were formed in blood of TDI-treated rats in a dose-dependent fashion. Administration of TDI to rats resulted also in a dose-dependent binding of TDI to hepatic tissue. Levels of TDI-albumin adducts were 10 times higher than those of TDI-globin adducts; the biological half lives of TDI-albumin and TDI-globin adducts were 1.2 and 12.5 days, respectively. Globin adducts were detected up to 28 days after the treatment. Hepatic TDI protein adducts were persistent for a substantial period whereas the levels of hepatic TDI-DNA adduct were decreased rapidly. These results indicate that the isocyanato group of TDI is not readily hydrolyzed under physiological conditions, is transported to other organs, and is bound to DNA and/or proteins without further metabolic activation. As the adducted products degrade in the body, TDA is released and introduced to the liver. TDA may additionally bind to hepatic tissue after metabolic activation. Thus, the toxic effect of TDI exposure is considered to persist during the lifetime of the adducted biological macromolecules.

• Journal of Animal Science and Technology
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• v.63 no.1
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• pp.114-124
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• 2021

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

• Food Science of Animal Resources
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• v.42 no.4
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• pp.625-638
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• 2022

This study aimed to carefully investigate the effect of hydrolysis using Flavourzyme on meat quality, antioxidative status, and taste-related compounds in breast of Samgyetang that was supplemented with black garlic (BG). Four different treatment groups were compared: (1) conventional Samgyetang (control), (2) Samgyetang hydrolyzed with Flavourzyme (1%, v/w) (FS), (3) Samgyetang made with the BG extract without hydrolysis (NBG), and (4) BG samgyetang pre-treated with Flavourzyme (1%, v/w) in a water bath at 55℃ for 2.5 h and hydrolyzed before being processed (HBG). All the treatment groups were cooked by retorting at conditions 121℃ and 1.5 kg/cm² for 1 h. Improved umami profiles through the increase of umami-related nucleotides (5c-GMP, 5'-IMP) and free amino acids-aspartic acid and glumtamic acid, in Samgyetang breast was recorded following hydrolysis. The HBG group tended to impart stronger scavenging activity toward free radicals compared with the other two groups, while not differing with NBG group regarding suppressing malondialdehyde. Textural properties were improved through hydrolysis, wherein the shear force value decreased from 2.29 kgf in the control to 1.19 and 1.25 kgf in the FS and HBG group. Moisture percentages were highly retained, with the redness score increasing and the lightness color decreasing following hydrolysis. In conclusion, the results of this study can be a preliminary information of the effect of hydrolysis pre-treatment for BG samgyetang. Further experiments are required to compare various enzymes along with its organoleptic acceptances.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.2
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• pp.246-255
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• 1996

To develop a natural antioxidative peptide, the gelatin was extracted from fish (Yellowfin sole) skin by hot $water(50^{\circ}C)$ extraction method and hydrolyzed with Alcalase, pronase and collagenase through a continuous 3-step membrane reactor. Each step enzymatic hydrolysates were determined the antioxidative activity and their synergistic effects, compared with $\alpha-tocopherol$ and butylated hydroxytoluene (BHT). Also, we tried to investigate the antioxidative disposition of peptide which was successfully separated by gel filtration, ion-exchange chromatography, and HPIC in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide (TBHP). Second step enzymatic hydrolysate (SSEH) among all hydrolysates and $\alpha-tocoperol$ was showed the strongest antioxidative activity. The optimum concentration of antioxidative activity for SSEH was $1\%(w/w)$ in linoleic acid. The synergistic effects were increased in using the hydrolysate with tocopherol and BHT. In the presence of the peptide isolated from SSEH, supplemented hepatocytes exposed to TBHP showed that delayed cell killing and decreased significantly the lipid peroxidation, compared with hepatocytes not cultured with isolated peptide.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.4
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• pp.494-499
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• 2010

In this study, Sargassum coreanum was enzymatically hydrolyzed to prepare water-soluble extracts by using five carbohydrates (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavozyme and Alcalase) and their potential antioxidant activity were evaluated. The Celluclast and Neutrase extracts of Sargassum coreanum exhibited better DPPH radical scavenging activities (92.42% and 92.78%, respectively) and hydrogen peroxide ($H_2O_2$) scavenging activities (58.28% and 57.97%, respectively) compared to those of other enzymatic extracts. These results suggest that Sargassum coreanum would be a good raw materials for antioxidant and enzymatic hydrolysis would be a good strategy to prepare antioxidant extracts from seaweeds.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.4
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• pp.427-431
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• 1999

This study was conducted to investigate the inhibitory activity of water extracts and its enzymatic hydrolysates from algae against angiotensin-I converting enzyme (ACE). The 7 kinds of algae were extracted with water at $50^{\circ}C,\;70^{\circ}C$ and $98^{\circ}C$. ACE inhibitory activities of water extracts were the highest at $70^{\circ}C$, and those of ceylon moss, layer, green layer, sea mustard, seaweed fusiforme sea tangle and sea staghorn were $10.9\%,\;9.3\%,\;8.9\%,\;8.2\%,\;7.5\%,\;7.1\%$ and $7.0\%$, respectively. Layer, green laver sea mustard and ceylon moss of high ACE inhibitory activities among the 7 kinds of water extracts were hydrolyzed by maxazyme and papain during 24hrs. ACE inhibitory activity of enzymatic hydrolysates was higher than that of water extracts, and was the highest in enzymatic hydrolysates of laver among the tested samples. In laver hydrolysates by proteases, the highest ACE inhibitory activity and peptide-nitrogen contents were observed at 8 hours hydrolysis and the hydrolysates by maxazyme showed relatively higher activity than those by papain(31.3 and $27.9\%$, respectively). But peptide-nitrogen contents were greater in papain hydrolysates than in maxazyme.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.503-511
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• 2008

An edible marine red alga, Grateloupia filicina, collected from Jeju Island of Korea was hydrolyzed by cheap food-grade carbohydrases (Viscozyme, Celuclast, AMC, Termamyl, and Ultraflo) to investigate its anticoagulant activity. Among the tested enzymatic extracts of G. filicina, a Termamyl extract showed the highest anticoagulant activity. Anion-exchange chromatography on DEAE-cellulose and gel-permeation chromatography on Sepharose-4B were used to purify the active polysaccharide from the crude polysaccharide fraction of G. filicina. The purified sulfated polysaccharide (0.42 sulfate/total sugar) showed ${\sim}1,357kDa$ molecular mass and was comprised mainly of galactose(98%) and 1-2% of glucose. The sample showed potential anticoagulant activity on activated partial thromboplastin time (APTT) thrombin time (TT) assays. The purified G. filicina anticoagulant (GFA) inhibited the coagulation factor X (92%), factor II (82%), and factor VII (68%) of the coagulation cascade, and the molecular interaction (protein-polysaccharide) was highly enhanced in the presence of ATIII (antithrombin III). The dissociation constant of polysaccharide towards serine proteins decreased in the order of FXa (58.9 nM) >FIIa (74.6 nM) >FVII (109.3 nM). The low/less cytotoxicity of the polysaccharide benefits its use in the pharmaceutical industry; however, further studies that would help us to elucidate the mechanism of its activity are needed.