• 한국미생물·생명공학회지
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• 제2권1호
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• pp.13-17
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• 1974

토양에서 분리한 Streptomyces 속 중에 금속을 함유하고 있는 protease를 강하게 분필하는 균일 주를 선별하여 그 효소학적 성질 멸가지를 알아본 결과는 다음과 같다. 1 본 효소의 작용 최적 pH는 중성부근이며 최적온도는 37$^{\circ}C$ 부근이다. 2. 본 효소는 중성부근에서 비교적 안정하며 열에는 비교적 불안정하며 37$^{\circ}C$에서 100분간 열처리했을때 약 50% 가량 실활하였다. 3. Hg$^{＋＋}$, Cu$^{＋＋}$, Cd$^{＋＋}$, Ag$^{＋}$ 등에 의하여 강하게 조해되며, Mn$^{＋＋}$, $Mg^{＋＋}$, $Ca^{＋＋}$, Pb$^{＋＋}$, $Ba^{＋＋}$ 등에는 별 영향을 받지 않는다. 4. oxalate, citrate, 2-4-dinitrophenol, $\varepsilon$-amino caproic acid, thiourea, cysteine에 의해서는 강하게 조해되었다.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.745-748
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• 2005

A phytase from Aeromonas sp. LIK 1-5 was partially purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. Its molecular weight was 44 kDa according to SDS-PAGE gel. Enzyme activity was optimal at pH 7 and at $50^{\circ}C$. The purified enzyme was strongly inhibited by 2 mM EDTA, $Zn^{2+},\;Co^{2+},\;or\;Mn^{2+}$, and activated by 2 mM $Ca^{2+}$. The K_m value for sodium phytate was 0.23 mM, and the enzyme was resistant to trypsin. The N-terminal amino acid sequence of the phytase was similar to that of other known alkaline phytases. The phytase was specific for ATP and sodium phytate, which is different from other known alkaline phytases. Based on the substrate specificity, the phytase may therefore be a novel alkaline phytase.

• Journal of Microbiology
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• 제34권2호
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• pp.198-205
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• 1996

Two proteases were partially characterized from culture filtrate of Mycobacterium, tuberculosis KIT110. Their molecular weights were approximately 200 and 180 kDa, respectively and they exhibited similar enzymatic characteristics. These enzymes were inhibited significantly by EDTA and to some extent by EGTA. Their activity was enhanced by $Ca^{2+}$ and $Mg^{2+}$ to some degree. However, $Cu^{2+}$ and $Ag^{2+}$ completely inhibited the enzyme activity at the concentration of 2.5 and 5 mM, respectively. The optimal pH was 7.0 and optimal temperature was around $40^{\circ}C$. These enzymes were rapidly inactivated at $80^{\circ}C$. Therefore, they were heat-labile, neutral metalloproteases. These enzymes exhibited antigenicity shown by their reacting with sera from the partients with pulmonary tuberculosis. These enzymes were able to degrade serum proteins including hemoglobin, bovine serum albumin, lysozyme and immunoglobulin G and structural matrix protein such as type I collagen. Therefore, these enzymes may be thought to contribute to tissue necrosis and pathogenesis during infection.

• 한국수정란이식학회지
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• 제12권3호
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• The Korean Journal of Physiology and Pharmacology
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• 제8권5호
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• pp.259-264
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• 2004

Cytolysin produced by Vibrio vulnificus has been incriminated as one of the important virulence determinants in V. vulnificus infection. Ion selectivity of cytolysin-induced pores was examined in a CPAE cell, a cell line of pulmonary endothelial cell, using inside-out patch clamp techniques. In symmetrical NaCl concentration (140 mM), intracellular or extracellular application of cytolysin formed ion-permeable pores with a single channel conductance of $37.5{\pm}4.0$ pS. The pore currents were consistently maintained after washout of cytolysin. Replacement of $Na^+$ in bath solution with monovalent ions $(K^+,\;Cs^+\;or\;TEA^+)$ or with divalent ions $(Mg^{2+},\;Ca^{2+})$ did not affect the pore currents. When the NaCl concentration in bath solution was lowered from 140 to 60 and 20 mM, the reversal potential shifted from 0 to -11.8 and -28.2 mV, respectively. The relative permeability of the cytolysin pores to anions measured at $-40\;mV\;was\;Cl^-\;=\;NO_2^-\;{\geq}\;Br^-\;=\;I^-\;> \;SCN^-\;>\;acetate^-\;>\;isethionate^-\;>\;ascorbic acid^-\;>\;EDTA^{2-},$ in descending order. The cytolysin-induced pore current was blocked by $CI^-$ channel blockers or nucleotides. These results indicate that V. vulnificus cytolysin forms anion-selective pores in CPAE cells.

• 한국미생물·생명공학회지
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• 제23권4호
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• pp.432-438
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• 1995

The intracellular alginase from Vibrio sp. AL-145 was purified by ion chromatography on DEAE-Cellulose column, Q-Sepharose column, and gel filtration on Sephadex G-100 column. The optimum pH and temperature for the activity of the purified intracellular enzyme were 8.0 and 37$\circ$C, respectively. The enzyme was stable at the pH range of 7.5-8.5, and at 30$\circ$C for 30 min. The molecular weight of the intracellular enzyme was estimated to be about 23, 000 daltons by SDS-polyacrylamide gel electrophoresis. NaCl was required for enzyme activity and the optimum concentration was 0.5 M. The activity of intracellular enzyme was inhibited by Co$^{2+}$, Hg$^{2+}$, Zn$^{2+}$, 0-phenanthroline, $\rho$-CMB, EDTA and iodoacetate, and stimulated by Ca$^{2+}$, L-cysteine and 2-mercaptoethanol. This enzyme was an alginase specifically degrading alginic acid.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

• Parasites, Hosts and Diseases
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• 제41권3호
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• pp.165-169
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• 2003

This study describes the characterization of 80 kDa pretense showing gelationlytic property among three pretenses in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The pretense activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This pretense was active only in the presence of calcium ion but not other divalent cationic ions such as $Cu^{2+},{\;}Zn^{2+},{\;}Mg^{2+},{\;}and{\;}$Mn^{2+}$, implying that $Ca^{2+}$ is critical factor for the activation of the protease. The 80 kDa pretense was optimally active at pH 7.5. Its gelatinolytic activity was maximal at $37^{\circ}C$, and significant level of enzyme activity of the pretense remained after heat treatment at $56^{\circ}C$ for 30 min or $100^{\circ}C$ for 10 min, This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 11 10-phenanthroline. Thus, the 80 kDa pretense in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.

• Preventive Nutrition and Food Science
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• 제19권3호
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• pp.213-219
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• 2014

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than $60^{\circ}C$. Fibrinolytic activity was increased by 5 mM $MgCl_2$ and 5 mM $CaCl_2$ but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the ${\alpha}$ and ${\beta}$ chains of fibrinogen but was unable to degrade the ${\gamma}$ chain.

• 미생물학회지
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• 제31권4호
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• pp.318-325
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• 1993

아닐린 분해균주 Flavimonas oryzihabitans KU21 의 원형질체 생성, 재생, 그리고 융합 등의 최적 조건을 조사하였다. 세포를 37.deg.C 로 prewarming 시킨 0.2 M Tris-Hcl(pH8.0) 완총액으로 현탁시킹후 0.5% 이상 원형질체로 전환되었으며, 이때 효소처리는 37.deg.C 에서 진탕하지 않고 처리했을 때가 가장 효과적이었다. MgCl/sub 2/ 와 CaCl/sub 2/ 수용액은 원형질체의 안정성을 높였고 완층액에 0.8% BSA 를 첨가함으로써 상온에서 4시간 까지 원형질체의 생존력을 80% 까지 유지할 수 있었다. 원형질체의 재생은 overlaying 방법이 가장 효과적이어서 3.8% 의 재생을 보였다. Rich regeneration medium 에 20 mM CaCl/sub 2/ 를 첨가하였을 때 재생율이 약 3.5배 증가하였고 완충액에 0.8% BSA 를 첨가했을 때는 4.5배의 증가율을 나타내었다. 삼투안정제로 0.5 M sucrose 를 첨가한 rich regeneration medium 에서 F. oryzihabitans 의 원형질체는 top plating 하여 6시나 이후부터 재생되기 시작하여 11 시간까지 80% 의 재생이 이루어졌다. 융합원으로 40.deg. PEC6000 과 CaCl/sub 2/ 를 사용하여 F, oryzihabitans 의 종내 원형질체 융합을 유도하였을때 recombinants 의 생성율은 2.0 * 10/sup -5/-3.6 * oryzihabitans 의 종내 원형질체 융합을 유도하였을때 recombinants 의 셍성율은 2.0 * 10/sup -5/-3.6 * 10/sup -5/ 이었으며 recombinants 들은 여러 세대 후에도 분리되지 않고 안정하였다.