• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.375-379
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• 2002

Methyl mercaptan oxidase was successfully induced in Thiobacillus thioparus TK-m using methyl mercaptan gas, and was purified for the detection of mercaptans. The purification procedure Involved a DEAE (diethylaminoethyl) -Sephacel, or Superose 12, column chromatography with recovery yields of 47.5 and 48.5%, and specific activities of 374 and 1240.8 units/mg-protein, respectively, The molecular weight of the purified methyl mercaptan oxidase was 66.1kDa, as determined by SDS-PAGE. The extract, from gel filtration chromatography oxidizes methyl mercaptan, producing formaldehyde, which can be easily detected by the purpald-coloring method. The optimized temperature for activity was found to be at 55$\^{C}$. This enzyme was inhibited by both NH$_4$Cl and (NH$_4$)$_2$SO$_4$, but was unaffected by either KCl or NaCl at less than 200 mM. With K$_2$SO$_4$, the activity decreased at 20 mM, but recovered at 150 mM. In the presence of methanol, full activity was maintained, but decreased in the presence of glycerin, ethanol and acetone 43, 78 and 75%, respectively.

• 한국식품과학회지
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• 제18권4호
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• pp.288-293
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• 1986

유안염석과 column chromatography 및 gel여과 등으로 비활성이 45.2U/mg가 되는 정제된 Rhizopus oryzae의 생전분 분해효소를 16.2%의 수율로 얻었다. 정제효소는 전기영동상에서 그 순도가 인정되었고 분자량은 67000, Km값은 4.082mg/ml이었다. 정제효소는 $50^{\circ}C$, pH $4.0{\sim}5.0$에서 잘 작용하였으며 옥수수amylose가 가장 적합한 전분이었고 옥수수 생전분에 작용한 분해산물은 거의 glucose였다.

• 한국식품과학회지
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• 제19권4호
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• pp.295-299
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• 1987

유안염석과 column chromatography 및 gel여과등으로 비활성이 23.4U/mg이 되는 정제된 mungbean lipoxygenase를 12%의 수율로 얻었다. 정제효소의 작용최적 pH는 8.4이었으며 linoleic acid를 기질로 사용하였을 때 Km값은 0.25mM이었다. 정제효소는 pH $5.0{\sim}7.0$범위와 $50^{\circ}C$이하의 온도에서 비교적 안정하였다. 효소의 활성은 항산화제인 NDGA에 의해 현저히 저해되었고 chelating agents에 의해 저해되었다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.316.1-316.1
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• 2002

Dermatan sulfate (DS) was isolated from eel skin (Anguilla japonica) bv actinase and endonuclease digeslions followed by ${\beta}$-elimination reaction and DEAE-Sephacel chromatography. DS was a major glycosaminoglycan in eel skin with 88% of the total uronic acid. The content of IdoA2S$\alpha$1longrightarrow4GalNAc4S sequence in eel skin. which is known to be a binding site to heparin cofactor II. was two times higher than that of dermatan sulfate from porcine skin. The anti-lla activity of eel skin dermatan sulfate mediated through heparin cofactor ll(NCL) was 25 units/mg. whereas DS from porcine skin shows 23.2 units/mg. The average molecular weight was determined as 14 kDa by gel chromatography on a TSKgel G3000SWXL column. Based on H1 NMR spectroscopy. we suggest that 3-sulfated and/or 2.3-sulfated ldoA residues are present in the chain.

• 미생물학회지
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• 제35권4호
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• pp.315-321
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• 1999

핵전이 방법을 이용하여 효모에서 전분분해능이 향상된 새로운 우수 균주를 개발하고자 하였다. Saccharomycopsis fiburigera KCTC 7393과 Saccharomyces cerevisiae KCTC 7049에서 핵을 분리한 후 영양요구 돌연변이주인 S. cerevisiae의 안으로 전달시켜 잡종(MN-16)을 형성하여 전분 분해능이 증가된 잡종을 선별하였다. 세포 배양액으로 조효소 용액을 만든 후 ammonium sulfate 침전, DEE-Sephacel column chromatography, Sephacryl S-200 column chromatography 등의 정제과정을 통해 9.7%의 회수율로 약 10.6배 정제 된 효소를 얻을 수 있었다. 정제된 효소는 SDS-PAGE 전기영동을 통해 단일 band를 보여 주었으며, SDS-PAGE 와 Sephacryl S-200 column chromatography를 통해서 53kDa으로 나타났다. 정제효소의 최적 활성 온도는 40${\circ}C$이고, 안정성은 40~45${\circ}C$로 나타났다. 최적 활성 pH는 5.5이었고, pH 5.0~7.0 정도에서 pH안정성이 80%정도 유지되었다. 가용성 전분에 대한 $K_{m}$ 값은 2.5㎎/㎖이었다. 또한, 정제 효소의 금속 이온의 효과로 $Ca^{2+}, Co^{2+}, EDTA, Mg^{2+}, Mn^{2+}, Zn^{2+}$ 첨가시 활성이 촉진되었고, $Ca^{2+}$의 경우 가장 높은 반면 $Cu^{2+}, Fe^{2+}, Ni^{2+}$의 경우는 오히려 활성이 감소되었다.

• 대한화학회지
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• 제39권6호
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• pp.459-465
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• 1995

효모 acetolactate synthase를 분리 정제하여 기본적인 생화학적 성질에 대한 연구를 수행하였다. 효모를 0.5% glucose, 51 mM $K_2HPO_4$, 22 mM $KH_2PO_4$, 8mM$(NH_4)2SO_4,\;0.4\;m M\;MgSO_4$를 포함하는 최소 배지에서 37$^{\circ}C$로 18시간 동안 배양하였다. 배양된 세포들을 원심분리법으로 수학해 0.1 mM TPP, 0.5 mM DTT, 1${\mu}M$ FAD와 1mM MgCl_2$를 포함하는 20 mM phosphate 완충용액(pH 7.0)에 현탁시켜 하룻밤 동안 방치하였다. 효모를 파쇄한 후 이것의 $10,000{\times}g$ 상충액을 모아 ammonium sulfate 분별 침전법과 DEAE-Se-phacel 그리고 leucine-agarose chromatography법을 이용하여 부분 정제하였다. 단백질의 농도, 시간, 온도, pH, 기질농도 등의 영향을 조사하였으며 측정한 결과 최적온도는 50$^{\circ}C$이고, pH 8.0∼8.5 사이에서 최고 값을 나타냈다. $K_m$과 $V_{max}$값은 각각 8.4 mM과 17.9 nmol/mg/min으로 얻어졌다. 효소의 안정성은 ethylene glycol과 glycerol의 존재하에서 크게 향상됨이 관찰되었다. Feedback inhibition 연구 결과 Val에 의해 가장 많은 영향을 받았고 Leu에는 거의 영향을 받지 않았다.

• BMB Reports
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• 제32권3호
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• pp.299-305
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• 1999

Protein carboxyl O-methyltransferase (PCMT) catalyzes the transfer of a methyl group from Sadenosyl-L-methionine to free carboxyl groups of methyl-accepting substrate proteins. Two isozymes were separated by DEAE-Sephacel chromatography from porcine brain cytosol and designated PCMT I and II. Isozymes I and II were further purified by adenosyl homocysteine-Sepharose 4B and Superose HR 12 chromatography. The molecular weights of the purified PCMT I and II were determined by mass spectrometry to be 20,138 Da and 25,574 Da, respectively. The two enzymes displayed different isoelectric points; 7.9 for PCMT I and 5.3 for PCMT II. Isozymes I and II exhibited similar substrate specificities when tested with various methyl-accepting proteins. Myelin basic protein, a component of myelinated neurons, was found to be an excellent methyl-accepting substrate for both PCMT isozymes with different $K_m$ values, $21.1\;{\mu}M$ for PCMT I and $10.6\;{\mu}M$ for PCMT II. The PCMT activity and methyl-accepting capacity displayed similar distribution in the various brain regions with an exception of the lower values in the cerebellum. The overall distribution may relate to a general function of protein repair by PCMT in the brain.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.700-707
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• 2010

A novel antioxidative peptide (APVPH I, antioxidative peptides from venison protein hydrolysates I) was purified from venison by enzymatic hydrolysis, column chromatography of DEAE-Sephacel, and high-performance liquid chromatography. The molecular mass of the purified peptide was found to be 9,853 Da and the amino acid sequences of the purified peptide was Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly. The purpose of this study was to evaluate the effects of APVPH I against $H_2O_2$-induced neuronal cells damage in PC-12 cells. Antioxidative enzyme levels in cultured neuronal cells were increased in the presence of the peptide. In addition, APVPH I inhibited productions of nitric oxide (NO), reactive oxygen species (ROS), malondialdehyde (MDA), and cell death against $H_2O_2$-induced neuronal cell damage in PC-12 cells. It was presumed to be APVPH I involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that APVPH I substantially contributes to antioxidative properties in neuronal cells.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.897-905
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• 2003

Phospholipase D (PLD) and ADP-ribosylation factor (ARF) were partially purified on a series of column chromatography, and their biochemical properties were characterized to understand the regulatory mechanism of PLD activation by ARF protein in the antigen-induced immune responses in guinea pigs. Heparin Sepharose and high-Q Sepharose column chromatographies were used for the purification of PLD, and Sephadex G-25, DEAE Sephacel, Source 15 PHE (HIC), Superdex-75, and Uno-Q column chromatographies were used for the purification of ARF. The purified PLD and ARF proteins were identified with anti-rabbit PLD- or ARF-specific antibodies, showing about 64 or 85 kDa for the molecular mass of PLD and 29 or 35 kDa for the sizes of ARF. Partial cDNA of ARF3 was cloned by RT-PCR in guinea pig lung tissue and its nucleotides and amino acids were sequenced. Guinea pig ARF3 showed 92% of nucleotides sequence identity and 100% of amino acid sequence homology with human ARF3. The ARF-regulated PLD activity was measured in the oleate or ARFs-containing mixed lipid vesicles. The purified and recombinant ARF (rARF) activities were assessed with the $GTP{\gamma}S$ binding assay. The PLD activity was induced by oleate in a dose-dependent manner. The purified ARF and recombinant ARF3 increased PLD activity in guinea pig lung tissues. These data show that the activity of membrane-bound PLD can be regulated by the cytosolic ARF proteins, suggesting that ARF proteins in guinea pig lung can act as a regulatory factor in controlling the PLD activity in allergic reaction.