• Journal of Microbiology
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• 제44권6호
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.801-808
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• 2002

상업적 단백질 분해 효소에 0.02% $CaCl_2$를 첨가하여 응유 활성화를 시킨 탈지유에 대한 분해 작용의 결과를 요약하면 다음과 같다. 다양한 효소별 가수분해 시간에 따른 가수분해도는 미생물 유래 효소와 trypsin은 pepsin과 papain W-40보다 높은 분해도를 나타냈다. 12% TCA 용액에 가용성인 NPN의 양은 trypsin이 가장 높은 분해도를 나타내었고 rennet과 pepsin이 가장 낮은 분해도를 보였다. 전기영동에 있어서 trypsin과 protease S는 $\alpha$- lactalbumin을 분해하였고 papain w-40은 $\beta$- lactoglobulin을 미약하게 분해하였으며 neutrase 1.5는 90분 이후부터 $\alpha$-lactalbumin과 $\beta$-lactoglobulin을 분해하였다. Rennet과 비교한 전기영동상에서는 rennet에 의해 분해 되지 않은 ${\alpha}_s$- casein과 $\beta$-casein을 trypsin과 protease S가 다량 분해하였고 $\kappa$-casein은 rennet에 비해 papain W-40이 상당 수준의 분해상을 나타내었다. 이상의 결과 가수분해도 및 NPN 양은 trypsin, neutrase 1.5 및 protease S가 다른 효소에 비해 높게 나타났으며, 전기영동상에서는 pepsin과 neutrase 1.5가 rennet과 유사한 경향을 나타내었다.

• Toxicological Research
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• 제14권4호
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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.

• BMB Reports
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• 제35권4호
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• pp.403-408
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• 2002

A hypothetical 21.0 kDa protein (ORF O197) from Escherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197(termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP-and XTP-hydrolyzing enzyme that has been identified.

• BMB Reports
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• 제33권4호
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• pp.289-293
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• 2000

Annexin I is a 37 kDa member of the annexin family of calcium-dependent phospholipid binding proteins. Annexin I plays regulatory roles in various cellular processes including cell proliferation and differentiation. Recently we found that annexin I is a heat shock protein (HSP) and displays a chaperone-like function. In this paper we investigated the function of annexin I as an ATPase using 1 to 32 amino acids deleted annexin I (${\Delta}-annexin$ I). ${\Delta}-Annexin$ I hydrolyzed ATP as determined by thin layer chromatography. The ability of ATP hydrolysis was inhibited by ADP, GTP and GDP, but not by the AMP, GMP and cAMP. In view of the ATP hydrolyzing function of HSP, the results support the function of annexin I as a HSP.

• BMB Reports
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• 제28권5호
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• pp.443-450
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• 1995

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.

• 한국식품과학회지
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• 제47권6호
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• pp.751-756
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• 2015

소금의 과량 섭취가 건강에 유해한 영향을 미칠 수 있어 이를 해결하기 위한 다양한 연구가 진행되고 있다. 최근 우리나라의 전통 발효 식품인 간장, 액젓 등이 짠맛을 증진한다는 보고가 있어 이에 대한 연구가 활발히 이루어지고 있다. 본 연구에서는 어간장과 액젓의 주요 재료로 사용되는 멸치를 효소 가수분해한 eHAP의 짠맛 증진 효과에 대해 살펴보았다. 동일한 소금농도의 용액에 eHAP의 첨가량을 달리하였을 때 첨가량이 증가할수록 짠맛 증진효과도 증가하여, 50 mmol/L의 용액에 2%의 eHAP를 첨가하였을 경우 $67.79{\pm}6.35%$의 짠맛 강도를 인지하였으며, 용액의 소금농도가 높을수록 eHAP의 첨가에 따른 짠맛 증진효과가 더 크게 나타났다. 이러한 짠맛 증진 효과는 멸치 단백질 가수분해물에 다량 함유하고 있는 $\small{L}$-lysine과 $\small{L}$-arginine과 감칠맛을 나타내는 glutamic acid와 aspartic acid에 상호작용에 의한 것으로 보인다. 0.1%에서 2%까지 eHAP의 첨가량을 달리하였을 경우 최소 0.37%에서 최대 35.58%의 짠맛 증진 효과를 나타내어 멸치단백질 효소가수분해물이 염미 증진제로서 향후 활용 가능성이 있는 것으로 판단된다.

• 한국식품과학회지
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• 제26권6호
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• pp.781-786
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• 1994

HVP를 기본 원료로 하여 몇몇 반응 전구물질을 첨가하여 여러 가지 반응 조건에 따라 meatlike flavor를 제조하였다. pH와 온도는 반응액의 갈변화도에 큰 영향을 미쳐, pH와 온도가 높을수륵 갈변화 속도는 빨라졌다. 반응액에 잔존하는 환원당과 유리 아미노산은 반응 1시간까지 급격히 감소하였으나 반응 온도의 영향은 거의 받지 않았다. 각 아미노산의 경우, glutamic acid와cysteine은 반응 시간이 경과함에 따라 감소하나, glycine과 methionine은 거의 변화가 없었다. Maillard 반응을 통해 생성되는 meatlike flavor외 향기 성분은 49종이 통정되었으며, 이중에 3-methyl butanal, 2-methyl tetrahydrothiophen-3-one, 3,4-d imethyl thiophene, 2,4-dimethyl thiagole 둥의 잘 알려진 natural meat flavor 성분들도 포함되어 있었다. $120^{\circ}C$에서 제조된 반응액을 관능 평가한 결과, 반응 1시간대의 반웅액이 5% 유의수준에서 구수한 맛이 가장 강하였고, 이미가 가장 적었다. 이상의 결과에서, 본 반응 system에서는 반웅 1 시간내에 대부분의 Maillard 초기 반응이 일어나고, 그이후, 반응이 진행될수록 구수한 맛을 내는 성분은 줄어들고, 이미를 나타내는 성분이 증가한다고 추정되었다.

• 한국식품영양과학회지
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• 제18권2호
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• pp.167-174
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• 1989

우리나라 연안에서 일시에 대량으로 어획되고, 영양적으로 우수하나 원료학적 특성 및 가공적성이 좋지 못해 일부는 비식용사료로 이용되고 있는 멸치를 신속하게 대량처리하여 보다 효율적으로 이용하기 위한 방안의 하나로 속성멸치간장엑스분의 제조를 시도하였다. 마쇄한 멸치의 최적가수분해조건은 자가소화구와 코오지첨가구 모두 $50^{\circ}C$에서 최대활성을 나타내었고, 분해시간은 6시간이 가장 적합하였다. pH는 자가소화구의 경우 pH 8.0부근에서 코오지의 첨가구의 경우 pH 7.0이 가장 좋았고, 코오지농도는 마쇄한 멸치에 대해 10%가 가장 적합하였으며, 식염은 가수분해 이후의 공정에서 첨가하는 것이 바람직하였다. 실제 제품화 할 경우 수율증가를 감안하면 pH조절은 필요하지 않으리라 생각된다. 저장성과 독특한 맛을 고려하여 최종공정에서 첨가하는 가장 적합한 식염농도는 농축액에 대하 15%이었고, 쓴맛교정을 위하여 첨가하는 분리대두단백질의 최적조건은 마쇄한 멸치에 대해 5% 이었다. 구명된 최적조건하에서 가수분해시킨 제품(C), (A) 및 (B)의 가수분해율은 각각 58.4%, 32.1%, 86.2% 이었다.

• BMB Reports
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• 제29권1호
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• pp.11-16
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• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.