• 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.

• 미생물학회지
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• 제30권4호
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• pp.299-304
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• 1992

An intracellular protease form cells of Pseudomonas carboxydovorans DSM 1227 grown on carbon monoxide was purified 57-fold in six steps to homogeneity with a yield of 4.3% using azocoll as a substrate. The molecular weight of the enzyme was determined to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed the purified enzyme to be a dimer with two identical subunits of molecular weight 72,000. The enzyme was stimulated by $Mg^{2+}$ but was inhibited completely by $Cd^{2+}$ $Fe^{2+}$ $Hg^{2+}$, and $^Zn{2+}$ The enzyme activity was also inhibited by EDTA, EGTA, phenylmethylsulfonyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl fluoride, and phenyl glyoxal, but was increased by 1-ethyl-3(dimethyl aminopropyl)carbodiimide, iodoacetamide and dithiothereitol. The optimal pH and temperature for the enzyme reaction were found to be 7-8 and 50.deg.C, respectively. Casein and bovine serum albumin were hydrolyzed by the enzyme, but carbon monoxide dehydrogenase was not.

• 미생물학회지
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• 제29권3호
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• pp.167-171
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• 1991

An intracellular protease from cells of Pseudomonas carboxydohydrogena grown on nutrient broth was purified to better than 95% homogeneity in five steps using azocaseine as a substrate. The molecular weight of the native enzyme was determined to be 125, 000. Sodium dodecyl sulfate-gel electrophoresis revealedat least two non-identical subunits of molecular weight 70, 000 and 56, 000. The enzyme activity was completely ingibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The enzyme was also inhibited by $Mg^{2+}$ , $Zn^{2+}$ , $Cd^{2+}$, $Cu^{2+}$ , and $Fe^{2+}$ , but was stimulated by iodoacetamide. Maximal reaction rate of the enzyme was observed at pH8.0 and 30.deg.C. The isoelectric point of the enzyme was found to be 7.5. The enzyme was unable to hydrolyze carbon monoxide dehydrogenase.

• 미생물학회지
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• 제27권3호
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• pp.237-244
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• 1989

Azocasein을 기질로 사용하여 nutrient broth에서 성장한 Pseudomonas carboxydovorans로부터 다섯 단계의 순화 과정을 거쳐 68배 순화된 세포내 가용성 단백질 가수 분해 효소를 얻었다. Pyruvate나 succinate, acetate, 또는 일산화탄소를 이용하여 성장한 세균들은 이 효소의 활성을 나타내지 않았다. 순화된 효소의 크기는 약 53,000이었고, 한개의 polypeptide로 구성되어 있었다. 이 효소는 serinerp통의 단백질 가수분해 효소로 $Cd^{2+}, Cu^{2+}, Hg^{2+}$, 등의 2가 양이온과 EGTA에 의해 활성이 완전히 억제되었고 iodoacetamide에 의해 활성이 증가되었다. 이 효소는 pH 8.0과 $50^{\circ}C$에서 최대 활성을 나타내었으며, 일산화탄소 산화효소를 가수분해 시키지 못하였다.

• Mycobiology
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• 제37권2호
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• pp.121-127
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• 2009

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

• Applied Biological Chemistry
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• 제30권2호
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• pp.169-178
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• 1987

Kluyveromyces marxianus로 부터 inulase를 생산하고 정제하며 intra 및 extracellular inulase의 성질을 조사하였다. 본 균주는 stationary phase인 24시간째 intra 및 extracelullar enzyme의 생산이 최고에 달했으며 유기 질소원으로 YNB를 사용하고 배양 중 pH를 조절해 줌으로써 효소 생산을 향상시킬 수 있었다. 조효소는 DEAE-cellulose에 의해 intra 및 extracellular inulase 모두 2개의 fraction으로 분리되었고 각 fraction의 전기영동 양상은 비슷하여 주 band를 비롯 모두 3개의 glycoprotein band가 관찰되었으며 이중 주 band만 inulase 및 invertase activity를 보유하고 있었다. 정제 효소의 inulase 및 invertase의 최적 pH는 각각 5.0과 4.5였고 intra가 extracellular enzyme 에 비해 다소 넓은 범위의 pH에서 높은 활성을 나타내었다. 모든 fraction의 최적 온도는 inulase가 $40^{\circ}C$, invertase가 $50^{\circ}C$였으며 intracellular enzyme이 더 넓은 범위의 온도에서 안정하였고 열에 대한 안정성도 intracellular inulase가 extracellular inulase보다 높게 나타났다. Km value는 intra가 $16{\sim}19mM$, extracellular inulase가 $9{\sim}11mM$로써 extracellular inulase가 inulin에 대한 친화력이 더 높았으나 모두 exo-type의 inulase로 판명되었다.

• Journal of Applied Biological Chemistry
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• 제50권4호
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• pp.196-201
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• 2007

An intracellular invertase was purified to homogeneity from the cell extract of an alkalophilic and thermophilic Bacillus sp. TA-11, which was classified as a new species belonging to Bacillus cereus based on chemotaxanomic and phylogenetic analyses. The purified enzyme with a recovery of 26.6% was determined to be a monomeric protein with a molecular weight of 23 kDa by SDS-PAGE and 26 kDa by gel filtration. The maximum enzyme activity was observed at pH 7.0 and $50^{\circ}C$, and the purified enzyme was stable at the pH range of 5.0 to 8.0 and below $60^{\circ}C$. $K_m$ and $V_{max}$ values of the enzyme for sucrose were 370 mM and 3.0 ${\mu}M$ per min, respectively. The enzyme activity was significantly inhibited by bivalent metal ions ($Hg^{2+}$, $Cd^{2+}$ and $Cu^{2+}$) and sugars (glucose and fructose).

• Journal of Life Science
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• 제9권1호
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• pp.64-68
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• 1999

The properties of purified intracellular adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) of Nocardioides sp. J-326TK isolated from soil have been studied. The enzyme deaminated adenosine and 2`-deoxyadenosine and the respective {TEX}$K_{M}${/TEX} values were 4.0×{TEX}$10^{-4}${/TEX} M and 5.0× {TEX}$10^{-4}${/TEX} M, but the enzyme was not active on 8-bromoadenosine, 6-methylaminopurine riboside, ATP, ADP, 2`-AMP, 3`-AMP, 5`-AMP, dAMP, cAMP, NAD, FAD, NADP and adenine. The enzyme activity was strongly inhibited by the addition of {TEX}$Hg^{2+}${/TEX} and {TEX}$Ag^{+}${/TEX}, {TEX}$Cu^{2+}${/TEX}, {TEX}$Co^{2+}${/TEX} and {TEX}$Mn^{2+}${/TEX} also inhibited the activity but much less extent. The effect of alkyl reagents, metal chelating reagents and certain other compounds on the enzyme activity were also examined. No reagent activated the enzyme. On the contrary, the enzyme reaction was slightly inhibited by o-phenanthroline and 6-benzyladenosine.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.66-66
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• 2003

Flavonoids are phenolic compounds widely distributed in wide variety of edible plants including leafy vegetables, fruits, beverages. Quercetin is one of bioflavonoid compounds and has anti-tumor effect by suppressing tumor growth in vitro and in vivo, including multiple biological effects by antioxidant and effective anti-inflammatory agent. The present study investigated whether quercetin can enhance antioxidant enzyme activities (glutathione peroxidase: GPX, superoxide dismutase : SOD, catalase: CAT) and intracellular reactive oxygen intermediate (ROI) levels in the presence of taurine on B16F10 murine melanoma cells. From this result, the antioxidant enzyme activities of quercetin in the presence of taurine was enhanced. In addition, the same treatments decreased intracellular reactive oxygen intermediate levels on B16F10 murine melanoma cells. Taken together, these results demonstrate that the antioxidant effect of quercetin can enhance in the presence of taurine and it might play an important role in anti-tumor effect.

• 미생물학회지
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• 제38권4호
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.