• Korean Journal of Food Science and Technology
• /
• 제30권5호
• /
• pp.1214-1221
• /
• 1998

The study was carried out to investigate the antioxidant effects of ethanol, methanol, and water extracts or fractions of ethanol extract of Ligularia fischeri on low density lipoprotein(LDL) and ethanol extract was further fractionated. The methanol extract and ethyl acetate fraction showed strong antioxidant effect with 71.7% (13.36 nmol/mg) and 95% (1.35 nmol/mg) inhibition in the presence of $15\;{\mu}g/mL$, respectively. In the value of malondialdehyde(MDA) with oxidation time, ethanol, methanol and water extract in the presence of $25\;{\mu}g/mL$ inhibited the oxidation up to 4h incubation and ethyl acetate fraction showed strong antioxidant effect up to 8h incubation. Also, the ethanol, methanol and water extract showed antioxidant effects in the agarose gel electrophoresis test. The conjugated diene formation by lipid oxidation with addition of $Cu^{2+}$ in the Ligularia fischeri extracts and their fractions was decreased approximately 1.1 to 2.8 times and 2.2 to 3.2 times, respectively compared to control. In the degradation of apo B-100 by oxidation using SDS-PAGE, ethanol, methanol and water extract showed similar degradation band pattern compared to that of native LDL band. Apo B-100 contents using densitometer were 77.8, 92.5% and 82.3% in the ethanol, methanol and water extract, respectively, compared to 100% of native LDL. In the meanwhile, apo B-100 contents in hexane, ethyl acetate and water fraction were 38.8, 94.5 and 65.5% respectively. This results indicated that ethyl acetate fraction showed the strongest antioxidant effect on MDA value or apo B-100 contents of LDL.

• Korean Journal of Agricultural Science
• /
• 제22권2호
• /
• pp.163-169
• /
• 1995

This study was carried out to examine the genetic constitution of serum proteins and enzymes in Holstein Friesian cattle population. The genetic variants of post-transferrin-2(pTf-2), transferrin(Tf), post-albumin(pAlb), ceruloplasmin(Cp) and amylase-I(Am-I) were analyzed by using PAGE(polyacrylamide gel electrophoresis) and STAGE(starch gel electrophoresis). In serum proteins, the pTf-2 locus were observed to be controlled by codominant alleles designated F and S, and the distribution of genotypes were 76.34, 14.50 and 9.10% for pTf-2 FF, FS and SS types, respectively. The gene frequencies of the pTf-2 F and S allele were 0.836 and 0.164. The Tf locus were found to be controlled by four alleles, Tf A, D1, D2 and E at a single locus, and the distribution of genotypes were 6.11, 32.06, 19.08, 1.53, 10.69, 18.32, 9.92 and 2.29% for Tf AA, AD1, AD2, AE, D1D1, D1D2, D2D2 and D2E type, respectively. The gene frequencies of the Tf A, D1, D2 and E wee 0.321, 0.359, 0.298 and 0.019. The pAlb locus were identified to be genetically controlled by two alleles, pAlb F and S allele, and the distribution of genotypes were 32.06, 29.77 and 38.17% for pAlb FF, FS and SS types, respectively. The gene frequencies of the pAlb F and S allele were 0.461 and 0.531. The Alb locus were observed to be controlled by Alb A and B allele, and the gene frequencies of these were 0.996 and 0.004. In serum enzymes, the Cp locus were found to be controlled by F and S allele, and the distribution of genotypes were 46.57, 27.48 and 25.95% for Cp FF, FS and SS types, respectively. The gene frequencies of F and S allele were 0.603 and 0.394. The Am-I locus were observed to be controlled by Am-I B and C allele, and the distribution of genotypes were 39.69, 21.73 and 38.93% for Am-I BB, BC and CC types, the gene frequencies of Am-I B and C were 0.503 and 0.497, respectively.

• Journal of Animal Science and Technology
• /
• 제47권6호
• /
• pp.1025-1032
• /
• 2005

This study was carried out to separate ovotransferrin in chicken egg white by gel chromatography and heparin affinity chromatography. In gel filtration which was performed with 50mM Phosphate buffer (pH 7.2, 0.15M salt) at a flow rate of 2.0 ml/min, ovotransferrin and ovalbumin were eluted together in fraction number 11-16. In order to separate pure ovotransferrin, fraction No. 12-14 of them which have high concentration of ovotransferrin were concentrated and rechromatographed. However, the ovotransferrin did not separated clearly. In heparin affinity chromatography, the separation was performed with 50mM ethylaminetetraacetic acid (EDTA, pH7.2) and 50mM Phosphate buffer (pH 7.2, 0.15M salt contained) on ferrous and ferric ion saturated column at as same flow rate as gel filtration system's. Ovotransferrin and albumin were eluted together at 10-15min (fraction No.3) and 15-20min (fraction No.4), respectively. However, purified ovotransferrin was eluted at 156-165min and 165-175min (tube No.32-33) with 50 mM phosphate buffer (pH 7.2, 0.15M salt free), respectively. Heparin affinity chromatography with ferric ion saturated column was resulted in the best separation of ovotransferrin rather than separation by gel chromatography and ferrous ion saturated heparin affinity chromatography.

• Microbiology and Biotechnology Letters
• /
• 제39권1호
• /
• pp.9-19
• /
• 2011

The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

• Journal of the Korean Chemical Society
• /
• 제47권4호
• /
• pp.363-369
• /
• 2003

Selenized yeast (Se yeast) containing $0.1{\%}$(w/w) of selenium was obtained when the yeast was incubated at a selenium concentration of 1$1.14{\times}10_-3 M$ in rich medium. After washing several times, the inorganic selenium on the cell wall was confirmed with MBRT. There was no indication of inorganic selenium on the cell wall when the blue color in MBRT was stayed for 15 minutes. The selenized yeast was sonicated, then the selenium contained protein was obtained after salting out by ammonium sulfate at the concentration $80{\%}$ saturation. The seven protein bands were seperated by SDS-PAGE and the selenium concentration in protein was measured by ICP-AES. Analytical data showed that the large expressed protein band contained a relatively large amount of selenium. The proteins of the 47kDa was contained the concentrations of 69.5 ${\mu}$ Se/g of most many content. The protein (47 kDa) was seperated from PVDF membrane by tank-electroblotting. The isolated protein was hydrolyzed under acid condition and reacted with PITC. The derivatives of amino acids were analyzed by HPLC and compared with the data obtained from regular yeast. The resulting selenium-yeast was analyzed with the selenomethionine concentration of $2{\%}$ comparaed with general amino acids. The goal of this study is to analyze the selenium concentration in protein bands and measure the degree of biotransformation of selenomethionine in a specific protein.

• Microbiology and Biotechnology Letters
• /
• 제26권6호
• /
• pp.483-489
• /
• 1998

We carried out the expression and characterization of yeast thioredoxin system including thioredexin 1 (Trx1), Trx2, thioredoxin reductase (TR), and a novel thioredoxin (Trx3), which was reported in the data base of Saccharomyces genome. The Trx1, 2 and TR were expressed as soluble proteins in E. coli and the sizes of purified proteins were equal to the reported their molecular weights. The expressed Trx3 was found in both soluble fraction and precipitate. The size of Trx3 purified from soluble fraction of E. coli crude extracts was estimated as 14 kDa on SDS-PAGE instead of 18 kDa for Trx3 in precipitate. N-terminal amino acid sequence of the small size of purified Trx3 from soluble fraction was analyzed as FQSSYTS which is correspond to the sequence from 20 to 26 for Trx3. Trx3 together with thioredoxin reductase and NADPH was able to reduce the disulfide bridge of insulin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Trx3 stimulated the antioxidant effect of thioredoxin peroxidase 1 (TPx1) which inhibited inactivation of glutamine synthetase (GS) in dithiothreitol (DTT) containing metal catalyzed oxidation system. The stimulation effect of Trx3 was 10% of the effect of either Trx1 or Trx2. In addition, Trx3 could reduce the disulfide of TPx to thiol, so that the TPx had thioredoxin dependant peroxidase activity. In western blotting analysis, antibodies against purified Trx3 did not cross-react with crude extracts of yeast, purified Trx1, and Trx2 proteins. But, in PCR reaction using the cDNA library of yeast as a template, gene encoding of trx3 was amplified.

• Journal of Life Science
• /
• 제14권3호
• /
• pp.435-440
• /
• 2004

The gene encoding a extremely thermostable 4-$\alpha$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis, was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence of the enzyme was distantly related to other functionally-related ones, such as D-enzymes. The enzyme is of industrial interest because of a novel activity of producing cycloamylose and is also important for fundamental studies of protein, sugar-metabolizing enzymes. In this paper, the overexpression of 4-$\alpha$-glucanotransferase in E. coli was carried out expression vector system with lac and T7 promoters. The enzyme was successfully overexpressed, and purified by the heat treatment of a cell-free extract, successive Butyl-Toyopearl and Mono Q chromatographies. The purified recombinant enzyme showed the same specific activity and the same mobility in SDS-PAGE as natural enzyme.

• Journal of Life Science
• /
• 제16권2호
• /
• pp.245-252
• /
• 2006

A bacterial strain, identified as Staphylococcus aureus JJ-11, producing collagenase was isolated out of 40 persons having skin troubles. S. aureus JJ-11 produced collagenase optimally in the media containing 1.5%(w/v) gelatin, 1%(w/v) yeast extract, 0.4%(w/v) $K_2HPO_4$, 0.005%(w/v) $NiSO_4{\cdot}6H_2O$ at $37^{\circ}C$ for 18 hrs. The collagenase produced by Staphylococcus aureus JJ-11 was purified at 6.66-folds purity through application of chromatography with Amberlite IRA-900 and Sephacryl S-300 HR columns. The molecular weight of the partially purified enzyme was estimated to be 62 kDa by SDS-PAGE. The protein exhibited optimum enzymatic activity at pH 7.0, and showed a stable activity at pH 4-8. The optimum temperature for collagenase was at $37^{\circ}C$, and activity was maintained upto $40^{\circ}C$. The enzyme activity was slightly elevated in the presence of divalents such as, $Fe^{2+},\;Co^{2+}\;and\;Ba^{2+}$ However, the activity was inhibited in the presence of $Sr^{2+}\;or\;Hg^{2+}$. The inhibition of activity by O-phenanthroline and EDTA suggested that the enzyme may contain metal which is required for activity. The enzyme showed the highest activity when insoluble collagen (type I) was, used as a substrate.

• KSBB Journal
• /
• 제19권4호
• /
• pp.301-307
• /
• 2004

The characteristics of the a-amylase of Aspergillus coreanus NR 15-1 isolated from traditional Korean Nuruk have been carried out. The a-amylase of A. coreanus NR 15-1 was purified by ammonium sulfate precipitation followed by column chromatographies on CM-cellulose, DEAE-cellulose, Sephadex G-100 gel filtration and hydroxyapatite. The a-amylase was purified 78-fold with a yield of 8.7%. The molecular weight of the a-amylase was estimated to be 49 kDa by Sephadex G-100 gel filtration and 51 kDa by SDS-polyacrylamide gel eletrophoresis. These experimental results suggested that the purified enzyme might be monomer. The enzyme was stable between pH 4 and 11. The optimum pH was 5.0. The optimum temperature for enzyme was 45$^{\circ}C$ and the enzyme was stable up to 50$^{\circ}C$. The enzyme was significantly inhibited by 1 mM N-bromosuccinimide. These results suggested that tryptophan residue was involved in the active site of a-amylase. The enzyme was identified as a-amylase because the reaction products of soluble starch hydrolyzed by the purified enzyme was oligosaccharide by thin layer chromatography.

• KSBB Journal
• /
• 제23권5호
• /
• pp.403-407
• /
• 2008

Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.