• Applied Biological Chemistry
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• 제41권7호
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• pp.522-526
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• 1998

콩 유용성분 탐색의 일환으로 그리고 향후 콩 ferritin 항체 및 유전자 확보를 목표로 발아된 콩으로부터 ferritin을 분리 정제하여 그 몇가지 특성을 조사하였다. 72시간 발아된 콩으로부터 ammonium sulfate 침전(0.55 saturation), DEAE-cellulose, Sephacryl S-300, Bio-Scale Q2 column chromatographies를 통하여 ferritin을 분리하였다. 정제된 콩 ferritin은 SDS-PAGE 분석에서 21 kDa의 크기를 나타냈으며, Sephacry S-300을 통한 겔거르기 chromatography와 non-denaturing 폴리아크릴아마이드 전기영동 분석에서 $510{\sim}560\;kDa$의 크기로 측정 되었다. 또한, immunodiffusion test에서 anti-soybean ferritin antiserum과 상호 반응하였다. 원자흡광광도계와 표준 철 용액을 이용한 정제된 콩 ferritin 중의 철 함유량은 833 mol Fe/mol protein 이었으며, 이는 호박씨로부터 분리한 ferritin보다 31배 더 많은 양의 철 함유량 이었다. 정제된 콩 ferritin중의 철은 horse spleen ferritin 중의 철과 유사하게 iron staining 되었다.

• BMB Reports
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• 제34권3호
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• pp.219-224
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• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.327-330
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• 1996

An amino acid antimetabolite named YS-460 was isolated from the culture filtrate of a newly isolated Actinomycetes identified as Streptomyces sp. Fermentation was carried out in the synthetic medium at 30$\circ$C for 5 days. Purification was done by ion exchange resin, active carbon, silica gel column chromatography and obtained 38 mg of pure active substance per liter of the broth. YS-460, an amino acid like substance, has the molecular formula of C$_{7}$H$_{11}$NO$_{3}$- Its structure determined to be furanomycin by spectral analysis. It is active against some bacteria on a chemically defined medium and reversed competitively by L-isoleucine and non-competitively by L-leucine and L-valine.

• 한국식물병리학회지
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• 제11권4호
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• pp.312-317
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• 1995

The polygalacturonase (PG) production in rotten apples by Botryosphaeria dothidea was purified by using gel filtration and ion exchange column chromatography, and the biochemical characters of PG were investigated. The purified PG appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with approximate molecular weight of 49 kilodalton (kDa). The molecular weight was equal to the native molecular weight estimated by gel filtration. The Km and Vmax values of PG were 0.51 mg/ml and 90.9 $\mu$M/min/ml, respectively. Optimum pH was 4.0~5.0, and the PG activity was stable from pH 5.0~10.0. Optimum temperature of the enzyme activity was 4$0^{\circ}C$. The PG activity was relatively stable at 2$0^{\circ}C$, but it was reduced 45% at 4$0^{\circ}C$ and completely inactivated at 8$0^{\circ}C$. The PG activity was considerably inhibited by Cu2+, Zn2+, SDS and EDTA, whereas it was not effected by Ca2+, K+, Mg2+ or Na+ ions.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.202-205
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• 2005

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.161-161
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• 1998

To achieve the aim of this investigation, the extracellular protease was isolated from bacteria BAC-4, a strain was cultivated in the medium for the production of penicillin acilase in a period of 32 hours. The enzyme was first purified by aceton precipitation method, followed by ion exchange chromatography on DEAE-sephacel column. The highest specific activity of the aceton fraction was found to be 2.19 unit per mg, with degree of purification of 13 times. Further purification of the enzyme on DEAE -sephacel had a specific activity of 58.6 unit per mg and degree of purification of 344 times compared to its crude extract. The optimum pH of the enzyme was 8.4, and the potimum temparature was 37$^{\circ}C$. The K$\_$M/ and $V_{max}$ calculated at experiment conditions were found to be 0.66%(W/V) and 3.61 unit per mL respectively.

• 미생물학회지
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• 제29권6호
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• pp.345-352
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• 1991

A serine proteinase of molecular weight 60 kd was purified from culture supernatant of P. aeruginosa using DEAE-Trisacryl M ion-exchange and AcA 54 gel filtration column chromatography, and the properties of serine proteinase were characterized. By means of SDS-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was 55 kd. The optimal pH for the activity of purified enzyme was 7.5. The activity of the purified enzyme was completely inhibited by Di-isopropylfluorophosphate(DFP) and N-.alpha.-p-tosyl-L-lysine choloromethyl detone(TLCK) but not by other proteinase inhibitors such as E-64, pepstatin A, 1, 10-phenanthroline. The purified enzyme was capable of degrading type I and type IV collagen. Antisera obtained from hymans infected with Pseudomonas aeruginosa reacted to the purified serine proteinase in immunoblots. These results indicate that the purified enzyme is trypsin-like serine proteinase and this enzyme of P. aeruginosa may play an important role in tissue damage as a spreading factor and may be useful for serodiagnosis of Pseudomonas infections.

• Archives of Pharmacal Research
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• 제23권4호
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• pp.407-412
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• 2000

The presence of the nitric oxide synthase (NOS) enzyme from Salmonella typhimurium (S. typhimurium) was identified by measuring radiolabeled L-$[^3H]$citrulline and NO, and Western blot analysis. NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially. The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis. The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between $37^{\circ}C$ and $43^{\circ}C$. The activity was inhibited by NOS inhibitors such as aminoguanidine and $N^{G}$ $N^{G}$-dimethyl-L-arginine. taken together, partially purified NOS in S. typhimurium is assumed to be a different isoform of mammalian NOSs.OSs.

• Bulletin of the Korean Chemical Society
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• 제17권2호
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• pp.143-146
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• 1996

A continuous potential pulse is applied to a silver-working electrode on a pulsed amperometric detector (PAD) for detection of free cyanide and sulfide. The moving phase is 0.1 M sodium hydroxide, 0.5 M sodium acetate and 5% (v/v) ethylenediamine mixture, and the flow rate is 0.7 mL/min. Optimized pulse conditions include a -200 mV (vs. Ag/AgCl reference electrode) detection potential(Ed) for 60 msec and 50 mV cleaning potential (Ec) for 120 msec. The silver working electrode surface is not poisoned by cyanide or sulfide, and the PAD maintains long-term stability without loss of sensitivity and reproducibility at these pulse conditions. The detection limit of cyanide and sulfide separated by ion chromatography using an anion exchange column is 0.1 ppm and 0.05 ppm, respectively.

• 한국동물학회지
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• 제30권2호
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• pp.167-176
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• 1987

배추흰나비의 전용기 혈림프에서 $\beta$-N-Acetylglucosaminidase를 DEAE-cellulose ion exchange 및 Sephadex G-200 column chromatography 법을 이용하여 정제하였다. 정제된 효소는 7% acrylamide gel 전기영동에서 단일 band를 나타내었으며, 당을 함유한 복합단백질이었다. 최적 pH는 5.0, 최적반응온도는 6$0^{\circ}C$이었으며, 각 온도에서 10분간 incubation한 열안정성에 있어서는 6$0^{\circ}C$까지 비교적 높은 활성을 나타내었지만 7$0^{\circ}C$ 이후에는 거의 활성을 나타내지 않았다. 또한 Hg$^2$+ 처리는 심한 효소활성의 저해 현상을 보였지만, Mn$^2$+, $Mg^2$+ 등은 활성의 증가를 나타내었으며, Km 값은 6.67$\times$10-$^3$M, pl 값은 5.8, 분자량은 4.1$\times$105 daltons 이었다.