• 대한치과의사협회지
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• 제11권8호
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• pp.525-530
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• 1973

True collagenolytic enzymes in animal tissues were first demonstrated by Gross and Lapiere (1962), who showed the ability of such an enzyme in the culture medium of living explants of tadpole tissue to degrade a specific substrate of undenatured collagen under physiological conditions. Recently, tumor-associated collagenolytic activity has been demonstrated in human neoplasm and in ascites V Carcinoma. This investigation have been peforme to determine whether or not a collagen lytic enzyme could e found in isolated solid Tawa sarcoma of Donryu female rat obtained the culture medium. The results were as follows. 1. 11.5mg% of hydroxyproline contained in Donryu rat skin collagen, which was extracted by 0.5M acetic acid. 2. Cultivation of solid Tawa sarcoma tissues on reconstituted rat skin collagen gels showed lysis of adjacent gel after 18 hours, and much more extensive lysis after 5 days. 3. Collagen substrate was not attacked by the common proteolytic enzymes, trypsin, pepsin, and pronase.

• 한국미생물·생명공학회지
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• 제28권4호
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• pp.214-218
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• 2000

The inulinase producing microorganism was isolated from soil and tentatively identified as Arthrobacter protophormiae/ramosus. Inulinase was pruified by ethanol precipitation, DEAE-Sephadex ion exchange chromatography and Sephadex gel filtration chromatography. The molecular weight of the purified enzyme was 34 kDa. The specific activity, yield and purity were 31.5 Unit/mg, 19.5% and 18.5 fold, respectively. Optimal pH and temperature for reaction of the purified inulinase were 8.5 and $55^{\circ}C$, respectively. The enzyme was stable at pH 7.5, below$ 55^{\circ}C$, and the activity was stimulated Mg2+.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.76-76
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• 1995

Virus-encoded HIV-1 reverse transcriptase (RTase) is one of the major targets for the development of drugs for HIV-1 since it is an essential enzyme-for the replication cycle of HIV-1. We cloned the entire reverse trancriptase gene into an inducible expression vector with tac promotor= RTase was stably overexpressed and induced by IPTG and the highly-expressed RTase was purified partially by use of DEAE cellulose and Mono Q column. The partially purified enzyme (663kDa, 51kDa) as exhibited by SDS-PAGE showed the high specific activity (16,570U/mg) when the assay for the RTase activity was carried out using $^3$H-dTTP and poly(rA): oligo(dT)12-18 as the substrate.

• Journal of Microbiology and Biotechnology
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• 제7권6호
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• pp.435-437
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• 1997

The role of $Ca^{2+}$ in making functional malate synthase in Corynebacterium glutamicum was investigated using the cloned DNA coding for the enzyme. Introduction of cloned aceB into C. glutamicum overexpressed malate synthase as judged by SDS-PAGE. However, the increase in enzyme activity of the expressed malate synthase did not match the level of overexpression observed in SDS-PAGE. Addition of $Ca^{2+}$ to the growth medium specifically increased the activity. The malate synthase could be stained with ruthenium red in a $Ca^{2+}$-specific manner. This agrees with the previous observation which reported a potential $Ca^{2+}$-binding domain in the N-terminal region of the protein.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.552-559
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• 2008

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolisstrain (No.7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at $18^{\circ}C$. The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with mid-chain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.

• 한국식품영양과학회지
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• 제19권4호
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• pp.349-355
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• 1990

Actomyosine prepared from Yellowtail fish(seriola quinqueradiata) were stored at $0^{\circ}C$(ice-cooling) -3.5$^{\circ}C$(partial freeaing) and -2$0^{\circ}C$(freezing) Another actomyosin samples were prepared from the fish previously stored at the temperatures for a week as the maximum .Remaining activity of {{{{ {Ca }^{2+ } }}}}-and {{{{ {Mg }^{2+ } }}}}- dependent adenosine triphosphatase(ATPase) activity was measured fronm the actomyosin preparations. Specific activity of {{{{ {Mg }^{2+ } }}}}-ATPase of actomy-osin before storagew was 0.253$\mu$ mole pi/min/mg of protein and it was 1.5 times higher than that of {{{{ {Ca }^{2+ } }}}} -ATPase. The enzyme activities were markedly decreased during early period of storage. However no significant differences in the enzyme activity were revealed among the samples stored at different temperature. The enzyme of actomyosin prepared from the fish previously stored at the temperatures for a week revealed an acitivity of 2-3 times higher than that of freezing. Apparent denaturation constant of {{{{ {Mg }^{2+ } }}}} -ATPase of actomyosin was between 0.810-1.139 per day and it was about 1.5 times hgiher than that of {{{{ {Ca }^{2+ } }}}} -ATPase. But the constant of {{{{ {Mg }^{2+ } }}}} ATPase of actomyosin extracted from the fist stored for a week at each temperature was between 0.176-0.356 per day. This constant was 4 times higher than that of {{{{ {Ca }^{2+ } }}}}- ATPase in frozen stored fish. It was presumed from these results that denaturation of ATPase is largely accorded to the structural changes of actomyosin.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.230-237
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• 2004

균주 Pseudomonas sp. HK-6로부터 2,4,6-trinitrotoluene (TNT)의 대사 과정에서 유도되는 nitroreductase (NTR)를 분리 및 정제하여 다양한 특성조사를 실시하였다. NTR은 균주 HK-6의 세포추출물로부터 ammonium sulfate 침전, DEAE-sepharose, 그리고 Q-sepharose chromatography의 일련의 과정을 통하여 분리되었고, NTR의 활성을 가지는 세개의 다른 fractions를 확인하였다. 균주 HK-6의 NTR fractions I, II, 그리고 III의 비 활성은 각각 4.85 unit/mg, 5.47 unit/mg, 5.01 unit/mg으로 측정되었으며, 세포추출물에 비해 각각 9.0배, 10.1배, 9.3배 농축된 것으로 나타났다. SDS-PAGE에서 측정된 균주 HK-6의 NTR fractions I, II 그리고 III의 분자량은 모두 약 27 kDa으로 확인되었다. 정제된 NTR의 활성에 온도, pH, 금속 이온, 억제 물질의 효과와 기질 특이성 등에 대한 물리화학적 특성 조사를 실시하였다. 균주 HK-6의 NTR fractions I, II, 그리고 III의 적정온도는 $25～35^{\circ}C$ 확인되었고, 모두 $30^{\circ}C$에서 최대 활성을 나타내었으며, 이들 효소 활성의 적정 pH는 7.0～8.0이었고, 최적 pH는 7.5로 확인되었다. TNT에 대한 HK-6의 NTR fractions I, II, 그리고 III의 활성은 금속 이온 $Ag^{＋}$ , $Cu^{2＋}$ , 그리고 $Hg^{V}$ 에 의해서 약 70%이상 저해되었으며, $Mn^{2+}$ 또는 $Ca^{2＋}$ 에 의해서 약 20～50%로 활성이 억제되었다. 그러나 $Fe^{2＋}$ /첨가 시에는 효소의 활성에 크게 영향을 미치지 않는 것으로 나타났다. NTR의 활성에 대한 억제물질의 영향은 $\beta$-mercaptoethanol 첨가 시에 효소의 활성이 모두 억제되었고, dithiothreitol, EDTA, 그리고 NaCl 첨가시에도 활성이 감소하는 것으로 확인되었다. TNT와 그 유사기질을 이용하여 HK-6에서 분리된 NTR의 기질 특이성을 조사한 결과, TNT, nitrobenzene, 그리고 RDX에 대해서는 비교적 활성이 높게 나타났으나 2,6-DNT와 2,4-DNT에서는 낮은 활성을 나타내는 것으로 확인되었다.

• 한국식품과학회지
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• 제34권4호
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• pp.552-558
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• 2002

팽나무버섯(Flammulina velutipes)에서 polyphenol oxidase가 황산암모늄 침전법, Superdex G-75 겔여과크로마토그래피, Phenyl superose 친화크로마토그래피, Mono-Q 이온교환수지 크로마토그래피, 그리고 Superdex S-200 겔크로마토그래피 등의 과정으로 정제되었으며, 특성화 되었다. 정제된 효소의 비활성도는 199.1 units/mg으로 나타났으며, 이 효소는 40 kDa의 단일폴리펩티드 사슬로 구성되어 있음이 밝혀졌다. 효소반응의 최적 pH와 온도는 각각 6.0과 $25^{\circ}C$,이었으며, pH 3과 5 사이의 산성조건과, pH 8과 10 사이의 알카리 조건에서는 활성이 감소되거나 상실되었다. 이 효소는 L-DOPA와 caffeic acid 등의 o-diphenols류에 대하여 높은 효소활성을 나타내었으며, L-DOPA와 caffeic acid에 대한 Km 값은 각각 3.97mM과 1.78mM로 계산되었다. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA와 $Mg^{2+}$은 팽나무버섯 pholyphenol oxidase의 효소활성을 감소시켰으며, $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ 등은 효소의 활성을 촉진하는 인자로 밝혀졌다. 정제된 효소는 $-70^{\circ}C$에서 3개월, 그리고 $-20^{\circ}C$에서 1개월간 활성의 손실 없이 저장이 가능하였다.

• 미생물학회지
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• 제45권2호
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• pp.200-207
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• 2009

Erythritol 생합성에 중요한 효소인 erythritol 4-phosphate dehydrogenase를 Penicillium sp. KJ81로부터 분리 정제하여 효소의 특성을 조사한 결과 다음과 같은 결론을 얻었다. Erythritol 4-phosphate dehydrogenase의 최적 생산 조건은 30% sucrose, 0.5% yeast extract, 0.5% $(NH_4)_2SO_4$, 0.1% $KH_2PO_4$ 그리고 0.05% $MgCl_2$ (pH 7.0) 배지에서 1 vvm aeration, 교반속도 200 rpm, $37^{\circ}C$로 Penicillium sp. KJ81을 배양한 결과 8일 배양 시 최대의 생산량을 보였다. Penicillium sp. KJ81주의 균체 추출액으로부터 ultrafiltration, 조제용 disc gel electrophoresis를 이용하여 erythritol 4-phosphate dehydrogenase를 분리 정제하였다. 최종 수율은 33%이었으며 정제배수는 39.5였다. 정제된 효소의 pI값은 4.6으로, erythrose 4-phosphate에 특이적으로 반응하였으며, Km값은 1.07mM이었다. Native-PAGE에서 single band를 보인 효소의 분자량은 약 1,500 kDa이었다. 효소활성의 최적 pH와 온도는 pH 7.0, $30^{\circ}C$였으며, 효소는 pH 4.0~9.0, 그리고 $30^{\circ}C$까지 안정하였다. 다양한 금속이온 중 $Cu^{2+}$, $Zn^{2+}$에 의하여 효소의 활성이 저해되었다. 다양한 아미노산 반응 잔기 변형시약 중 iodine과 NBS에 의해 효소의 활성이 저해되는 것을 확인하였다.

• Journal of Nutrition and Health
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• 제19권6호
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• pp.363-373
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• 1986

This study was devised to elucidate whether soused shrimp exhibits a digestive action on boiled pork meats. and the mechanism by which sousing with a high concentration of sodium chloride preserves nutrients in foods for a prolonged pe\ulcornerriod. Protease was isolated from soused shrimp using a combination of ammonium sulfate fractionation. DEAE - cellulose ion exchange chromatography and gel filtra\ulcornertion. The isolated protease had specific activity of 1.560 units. 210 purification fo\ulcornerld with an yield of 38%. Its optimum pH and temperature were 8.0 and $43^{\circ}C$ respectively. The molecular weight of the enzyme was 35.000. The Km value of the enzyme for casein was 1.6 x $10^{-6}$ M The e=yme required the presence of cu\ulcornerpric ion to exhibit its full activity. Eighty eight percent of the enzyme activity was in\ulcornerhibited by 3.5M NaCI showing a reversibly linear decrease of the enzyme activity as NaCI concentration increased. The nature of the inhibition by NaCl was rever\ulcornersible and noncompetitive. The protease activity in soused shrimp was well preser\ulcornerved with the elapse of time at least in part due to NaCI induced suppression of autodigestion. The enzyme was denatured by acid easily. i.e. 1% of the original activity remained after staying at pH 2 for 10 minutes. which is within the norm\ulcorneral range of pH of the human stomach. Soused shrimp was observed to be one of those containing the highest protease activity compared with the other soused foo\ulcornerds such as soused oyster. squid. clam. and Pollack intestine with respect to spec\ulcornerific activities of dialized 1:4 whole homogenates(w/v) in 5 mM sodium phospha\ulcornerte - 2.4 mM j3 - mercaptoethanol buffer. pH 8.0. Casein and boiled meats including pork, beef, and chicken appeared to be the good substrates for the protease. Casein was the best. Therefore. the ingestion of boiled meats including pork together with soused sh\ulcornerrimp would help digestion of boiled pork in human not only by increasing appe\ulcornertite also by the direct proteolytic digestion of boiled meats by soused shrimp to\ulcorner some extent. And a high concentration of sodium chloride inhibited the protease activity reversibly in a remarkable degree, which ensued in a significant retardat\ulcornerion of autodigestion of protein in foods by proteases, and hereby contributed to the preservation of foods for an extended period.