• Fisheries and Aquatic Sciences
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• 제19권5호
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• pp.25.1-25.8
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• 2016

The fractionation of serine protease inhibitor (SPI) from fish roe extracts was carried out using polyethylene glycol-4000 (PEG4000) precipitation. The protease inhibitory activity of extracts and PEG fractions from Alaska pollock (AP), bastard halibut (BH), skipjack tuna (ST), and yellowfin tuna (YT) roes were determined against target proteases. All of the roe extracts showed inhibitory activity toward bromelain (BR), chymotrypsin (CH), trypsin (TR), papain-EDTA (PED), and alcalase (AL) as target proteases. PEG fractions, which have positive inhibitory activity and high recovery (%), were the PEG1 fraction (0-5 %, w/v) against cysteine proteases (BR and PA) and the PEG4 fraction (20-40 %, w/v) against serine proteases (CH and TR). The strongest specific inhibitory activity toward CH and TR of PEG4 fractions was AP (9278 and 1170 U/mg) followed by ST (6687 and 2064 U/mg), YT (3951 and 1536 U/mg), and BH (538 and 98 U/mg). The inhibitory activity of serine protease in extracts and PEG fractions from fish roe was stronger than that of cysteine protease toward common casein substrate. Therefore, SPI is mainly distributed in fish roe and PEG fractionation effectively isolated the SPI from fish roes.

• 한국어병학회지
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• 제10권1호
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• pp.31-38
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• 1997

잉어로부터 분리한 Aeromonas hydrophila는 세포 밖으로 여러 종류의 proteases를 생산한다. A. hydrophila의 배양 상층액을 이용한 inhibitor assay를 통하여, 주된 활성을 나타내는 metallopretease와 약한 활성을 나타내는 serine protease가 있음을 알게 되었다. Gelatin SDS-PAGE를 통하여 두 개의 활성 band가 관찰되었으며, 이 들 중 넓게 퍼진 band는 metalloprotease에 특이하게 작용하는 inhibitor인 EDTA에 의해 활성이 상실되었고 따라서 metalloprotease임을 알 수 있었다. 다른 하나는 serine protease에 특이하게 반응하는 inhibitor인 PMSF에 의해 저해되어 serine protease임을 알 수 있었다. 이러한 두 extracellular protease의 활성은 $75^{\circ}C$에서 30 분간 열을 가한 후에도 Gelatin SDS-PAGE상에 남아있었다. 그런데, 주된 metalloprotease는 Sephadex G-75를 이용한 column chromatography를 거친 후 Gelatin Gel상에서 두 개의 band로 분리되었다.

• 한국수산과학회지
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• 제29권3호
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• pp.296-308
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• 1996

혈합육어 (멸치와 전어)와 백색육어 (농어와 도다리)의 육과 내장에서 추출한 조효소에 대하여 천연기질 및 합성 기질에 대한 효소 활성과 disc-PAG 전기영동에서 분리된 효소 단백질의 분포를 선택적 합성기질을 써서 비교 검토하였다. 육에는 SH-proteinase인 cathepsin L, B, 및 H 유사 효소가 분포하는 것을 확인 할 수 있었으며, 이 밖에도 cathepsin G 및 D도 다소 분포하는 것으로 추정되었다. 그리고 내장에는 강한 활성의 chymotrypsin 및 trypsin 유사 효소가 주로 분포하고 있음이 확인되었다. 육과 내장에서 추출한 조효소들은 공통적으로 멸치, 전어, 도다리 그리고 농어의 순으로 활성이 높은 것을 알 수 있었으며, 전체적으로 혈합육어에 분포되어 있는 효소가 백색육어류에 분포되어 있는 효소에 비하여 약 100배정도 높았다. 육과 내장의 조효소를 방어의 근원섬유단백질에 작용시킨 결과, 육 중의 조효소는 myosin heavy chain의 분해가 두드러졌으며, 특히 멸치의 효소가 근원섬유 단백질의 구성 subunit 모두에 대하여 강한 분해능을 보였다. 내장 조효소의 경우는 반응 초기에 myosin heavy chain과 actin을 현저하게 분해시켰으며, 혈합육어인 멸치와 전어 조효소의 분해능이 백색육어인 농어와 도다리의 조효소보다 강한 것을 확인 할 수 있었다. 육의 조효소는 어종에 따라 서로 다른 분해 양상을 보였으며, 내장 조효소는 혈합육어와 백색육어의 효소 활성뿐만 아니라 효소의 분포상으로도 많은 차이가 있었다.

• Journal of Microbiology and Biotechnology
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• 제34권7호
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• pp.1385-1394
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• 2024

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the β-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60℃. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.

• 한국수산과학회지
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• 제44권1호
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• pp.8-17
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• 2011

To identify and examine the distribution of proteolytic inhibitory activity in crude extracts from fish eggs, and to determine the applicability of these protease inhibitors as anti-degradation agents in surimi-based products and fish meat, we compared the inhibitory activities of various extracts from fish eggs to those of commercial proteases, such as trypsin and papain. We used the optimal conditions for the screening of trypsin activity: 30 ug/uL of 0.1% trypsin and 0.6 mM Na-benzoyl-L-arginine-p-nitroanilide (BAPNA) with a pH of 8.0 at $40^{\circ}C$ for 60 min. The activities of papain and four commercial proteases were investigated after mixing with 100 ug/uL enzymes and 0.3% casein with a pH of 8.0 at $40^{\circ}C$ for 60 min. We performed a screening assay to detect the inhibitory activity (%) of crude extracts from eight species of fish eggs against the target proteases trypsin and papain. The assay revealed a wide distribution of trypsin and papain inhibitors in fish eggs. The specific inhibitory activities (11.6.28.6 U/mg) of crude extracts from fish eggs against trypsin and BAPNA substrate were higher than that (0.64 U/mg) of egg whites, used as a commercial inhibitor. The inhibitory activities of crude extracts from fish eggs against trypsin, and of egg whites against casein substrate (1.94.4.51 U/mg), were higher than those of papain (0.24.1.57 U/mg) and commercial protease (0.04.0.32 U/mg). The extracts from fish eggs were rich in protease inhibitors that exhibited strong inhibitory activity against trypsin, a serine protease, and papain, a cysteine protease.

• 대한수의학회지
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• 제55권3호
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• pp.175-179
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• 2015

Proteases play important roles in parasite development and host parasite interactions. The protease of Kudoa spp. has been recognized as a key factor of severe proteolysis of fish muscle post-mortem; however, there is little information available regarding the protease of Kudoa (K.) septempunctata, which was recently identified as a cause of food poisoning in humans. The present study was conducted to isolate and characterize proteases to elucidate the type of protease contained in the parasite and determine the optimal pH for protease activity. We confirmed the cysteine protease and metalloprotease produced by K. septempunctata. While the cysteine protease showed optimal activity at pH 5 that decreased rapidly with increasing pH, the optimal activity of metalloprotease was pH 7, and it remained stable from pH 6 to pH 8. These results indicate that the pH of cysteine protease is not proper for fish muscle postmortem, and that metalloprotease can act in human intestines. Overall, the present study provides important information that improves our understanding of the role of protease physiology and the subsequent food poisoning caused by K. septempunctata.

• 한국수산과학회지
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• 제37권4호
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• pp.259-268
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• 2004

Keeping qualities of crude proteases (CP) and fractionated proteases (FP) sedimenting with $30\~80{\%}$ ammonium sulfate from four kinds of fish viscera as a seafood processing waste were examined. Azocaseinolytic activlties (pH 6 and 8) of CP from anchovy (Engraulis japonica), mackerel (Scomber japonicus), bastard flatfish (Pararlichthys olivaceus) and red sea bream (Chysorphys major) were stable without activity loss at $4^{\circ}C$ for 7 months. Activities of NaCP (CP containing $30{\%}$ sodium chloride) on azocasein were approximately $30{\%}$ lower than those of CP. FP activities Increased 3.4-16.1 folds compared to those of CP and NaCP Powdered crude protease (PCP) and fractionated and powdered protease (FPP) containing various sugars (lactose, sucrose, glucose and dextrin) were prepared by freeze drying. Activities of PCP and FPP containing sucrose were higher and more stable than those of PCP and FPP containing other sugars at $30^{\circ}C$ for whole keeping periods. PCP and FPP from mackerel viscera showed the highest proteolytic activity among four kind of fish vlsceras. The Optimum conditions and stabilities of FPP from mackerel viscera were pH 9 and $50^{\circ}C$, and pH 5-10 and $20-45^{\circ}C$, respectively. The results of this study suggest that FPP from seafood processing waste may be used as processing aids.

• 한국식품영양과학회지
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• 제30권2호
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• pp.222-227
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• 2001

The members of the genus Vibrio include harmless aquatic strain as well as strains capable of causing infections in human and fish. Pathogenic mechanisms are only understood for Vibrio cholerae O1 and O139 and not for the majority of Vibrio species. Twelve clinical and nonclinical strains were examined by in vitro and in vivo experiments for the importance of extracellular enzymes as a virulence determinant of Vibrio species. In vivo cytotoxicity assay was performed by injecting approximately $10^{8}$ cells/mL into mice (BALB/c). V. harvyi and V. vulnificus showed 100% lethality within 3hr after bacterial injection. V. fluvialis and four strains of V. parahaemolyticus showed 50% lethality within 4hr. V. mimicus, V. alginolyticus and V. furnissii revealed 30% lethality within 9hr. Nonclinical strains, V. campbellii and V. ordalii, did not show any lethality. In vitro protease and hemolytic activities were also good indicators for clinical and nonclinical strains of Vibrio species. The clinical strains showed much higher activities than nonclinical strains. The activity of some clinical strains of re-isolates was evidently increased. Most clinical strains had $\beta$ hemolytic activity. The results demonstrate that the prevalent distribution of extracellular proteases in pathogenic Vibrio sp. implies their importance as a virulence determinant.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.423-428
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• 2000

Bacteriocins are classified as proteins which are produced by heterogeneous groups of bacteria, having and antimicrobial effect of the closely related organisms. Recently, bacteriocins derived from lactic acid bacteria and other food-related organisms have been the subject of much research on potential food biopreservatives. The goal of this study was to screen and characterize the bacteriocinogenic lactic acid bacteria from Jeot-gal(commercial fermented fish foods). All bacteriocinogenic isolates were identified as lactic acid bacteria. Isolates NK24, NK34, and SA72 were tentatively identified as Lactobacillus brevis, according to the API 50 CHL kit database. All antimicrobial substances produced from four lactic acid bacteria isolates completely lost their antibacterial activity after being treated with some proteases, indicating to their proteinaceous nature. The bacteriocin produced from isolates NK24, NK34, and SA72 showed a broad spectrum of activity when compared to those produced from isolate SA131. All bacteriocins isolated during the course of this study showed a bactericidal mode of inhibition.

• 한국수산과학회지
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• 제32권4호
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• pp.458-465
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• 1999

Distribution of the proteolytic activities of crude pretense extracted from the viscera of ten kinds of fish was examined. Their proteolytic activities on proteinous substrates (azocasein, hemoglobin, and casein) from the viscera of anchovy, bastard flatfish, mackerel and red sea bream were higher than those of other fishes, and the crude pretenses were further fractoinated with acetone or ammonium sulfate. Optimum concentrations for pretenses fractionation were $0\~55\%$ for acetone and $30\~70\%$ for ammonium sulfate. The fractionated viscera pretense of mackerel showed the highest proteolytic activity among four kinds of fishes. Activities of cathepsin D- and pepsin-like enzymes at pH 3.0, cathepsin L-, B-, H- and G-like enzyme at pH 6,0, and Hypsin- and chymotrypsin- like enzymes (pH 8.0) were detected in the fractionated viscera pretense, whereas activities of cathepsin L- and chymoeypsin-like enzyme were observed in commercial pretenses. Proteolytic activities of Alcalase, Protamex, and Aroase AP-10 for azocasein were slightly higher than the fractionated viscera pretenses, but their amidolytic activities at pH 6.0 and 8.0 toward synthetic substrates were lower than counterpart. The fractionated pretenses from fish viscera would be utilized as commercial pretenses.