• Proceedings of the Korean Society for Applied Microbiology Conference
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.505.1-505
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• 1986

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.

• Food Science of Animal Resources
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• 제37권3호
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• pp.402-409
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• 2017

A novel peptide having free radical scavenging activity was separated, using an on-line high-performance liquid chromatography (HPLC) - ABTS screening method, from bovine skim milk fermented by Lactococcus lactis SL6 (KCTC 11865BP). It was further purified using reverse phase-HPLC (RP-HPLC) and sequenced by RP-HPLC-tandem mass spectrometry. The amino acid sequence of the identified peptide was determined to be Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu-Trp (2,362 Da), which is corresponding to the C-terminal fragment of bovine ${\alpha}_{s1}$-casein (f179-199). The hydroxyl radicals scavenging activity ($IC_{50}$ $28.25{\pm}0.96{\mu}M$) of the peptide chemically synthesized based on the MS/MS data showed a slightly lower than that of the natural antioxidant Trolox ($IC_{50}$ $15.37{\pm}0.52{\mu}M$). Furthermore, derivatives of the antioxidant peptide were synthesized. The antioxidative activity of the derivatives whose all three proline residues replaced by alanine significantly decreased, whereas replacement of two proline residues in N-terminal region did not affect its antioxidative activity, indicating that $3^{rd}$ proline in C-terminal region is critical for the antioxidative activity of the peptide identified in this study. In addition, N-terminal region of the antioxidant peptide did not show its activity, whereas C-terminal region maintained antioxidative activity, suggesting that C-terminal region of the peptide is important for antioxidative activity.

• BMB Reports
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• 제29권4호
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• pp.353-358
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• 1996

Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow In ilk. fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl pH 7.5, and 1 mM $CaCl_2$ at $25^{\circ}C$. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. By contrast, the interaction of RNase A, a homologous protein to bAng, with actin results in about 10% quenching of the fluorescence. Fluorescence titration experiments were performed by adding increasing concentrations of bAng (0~1.0 ${\mu}M$) to a constant concentration of actin (0.1 ${\mu}M$), and the dissociation constant $K_d$ for the bAng/actin complex and the stoichiometry n were measured as $20{\pm}1$ nM and $1.0{\pm}0.1$ respectively. These results suggest that the interaction between bAng with actin is specific and that quenching of actin fluorescence has occurred in the bAng/actin complex. The bAng binding sites of actin are discussed in the results of this study, and we propose that Trp-80 in the small domain of bovine actin is responsible for the bAng/actin binding.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.822-829
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• 2007

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria(LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2,502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgarno sequence(aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7(LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate(TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase(GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32mg/400ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78mg/400ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The $K_M\;and\;V_{max}$ values for fructose-6-phosphate were $5.08{\pm}0.057mM(mean{\pm}SD)$ and $499.21{\pm}4.33{\mu}mol/min/mg$, respectively, and the optimum temperature and pH for the production of acetyl phosphate were $45^{\circ}C$ and 7.0, respectively.

• Korean Journal of Microbiology
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• 제36권1호
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• pp.26-32
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• 2000

Catechol 2,3-dioxygenase was purified from recombinant strain E. coli CNU312 carrying the tomB gene which was cloned from toluene-degrading Burkholderia cepacia G4. The purification of this enzyme was performed by acetone precipitation, Sephadex G-75 chromatography, electrophoresis and electro-elution. The molecular weight of native enzyme was about 140.4 kDa and its subunit was estimated to be 35 kDa by SDS-PAGE. It means that this enzyme consists of four identical subunits. This enzyme was specifically active to catechol, and$K_(m)$ value and $V_(max)$value of this enzyme were 372.6 $\mu$M and 39.27 U/mg. This enzyme was weakly active to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol, but rarely active to 2,3-DHBP. The optimal pH and temperature of the enzyme were pH 8.0 and $40^{\circ}C$. The enzyme was inhibited by $Co^(2+)$, $Mn^(2+)$, $Zn^(2+)$, $Fe^(2+)$, $Fe^(3+)$, and $Cu^(2+)$ ions, and was inactivated by adding the reagents such as N-bromosuccinimide, and $\rho$-diazobenzene sulfonic acid. The activity of catechol 2,3-dioxygenase was not stabilized by 10% concentration of organic solvents such as acetone, ethanol, isopropyl alcohol, ethyl acetate, and acetic acid, and by reducing agents such as 2-mercaptoethanol, dithiothreitol, and ascorbic acid. The enzyme was inactivated by the oxidizing agent $H_(2)$$O_(2)$, and by chelators such as EDTA, and ο-phenanthroline.

• Asian-Australasian Journal of Animal Sciences
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• 제25권7호
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• pp.1029-1037
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• 2012

The present study was designed to investigate the effect of ${\alpha}$-melanocyte-stimulating hormone (${\alpha}$-MSH), nitric oxide (NO) and L-cysteine on melanin production and expression of related genes MC1R, Tyr, Tyrp-1 and Tyrp-2 in muzzle melanocytes of differently colored three native Hanwoo cattle. Muzzle samples were taken from black, brindle and brown Hanwoo and purified melanocytes were cultured with ${\alpha}$-MSH, nitric oxide and L-cysteine at 100 nM, $50{\mu}M$ and 0.07 mg/ml of media respectively. The amounts of total melanin, eumelanin and mRNA expression at Tyr, Tyrp-1, Tyrp-2 and MC1R levels were quantified. ${\alpha}$-MSH and nitric oxide significantly increased (p<0.05) the amount of total melanin in black and brindle whereas eumelanin production in brown Hanwoo muzzle melanocytes. On the contrary, L-cysteine greatly (p<0.05) depressed the eumelanin production in black color but increased in brown. Simultaneously, up regulation of Tyr by nitric oxide and ${\alpha}$-MSH and down regulation of Tyr, Tyrp-2 and MC1R genes by L-cysteine were observed in muzzle melanocytes of all three phenotypes. The results of this study revealed nitric oxide and ${\alpha}$-MSH contribute hyper-pigmentation by enhancing eumelanogenesis whereas L-cysteine contributes to pheomelanin production in different colored Hanwoo muzzle melanocytes.

• Korean Journal of Microbiology
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• 제32권2호
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• pp.132-138
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• 1994

L-Ascorbic acid-producing enzyme in Neurospora crassa was found to exist in mitochondria and the activity of this enzyme was increased by the addition of D-fluconno-${\gamma}$-lactone or L-gulono-${\gamma}$-lactone in the media. L-Ascorbic acid-producin enzyme in N. crassa has been purified with ammonium sulfate precipitation. DEAE Sepharose CL-6B ion exchange chromatography. Sephacryl S-200 gel filtration chromatography and Reactive yellow 3-agarose dye affinity column chromatography. The specific activity of this enzyme was increased to 239.6 fold and the yield was 2.1%. The molecular weight of the native enzyme was 150.000 dalton when it was estimated with Sephacryl S-200 gel filtration chromatography. Its molecular weight appeared as 75.000 dalton by SDS-polyacrylamide gel electrophoresis. which suggested that this enzyme was consisted with two identical subunits. The optimal pH for this enzyme was 9.0 and the $K_m$ value for D-galactono-${\gamma}$-lactone was 0.073 mM.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1753-1759
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• 2006

Paenibacillus sp. HY-8 isolated from the digestive tracts of the longicorn beetle, Moechotypa diphysis, produced an extracellular endoxylanase with a molecular weight of 20 kDa estimated by SDS-PAGE. The xylanase was purified to near electrophoretic homogeneity from the culture supernatant after ammonium sulfate precipitation, gel filtration, and ionexchange chromatography. The purified xylanase exhibited the highest activities at pH 6.0 and $50^{\circ}C$. The $K_m\;and\;V_{max}$ values were 7.2 mg/ml and 16.3 U/mg, respectively, for birchwood xylan as the substrate. Nucleotide sequence of the PCR-cloned gene was determined to have the open reading frame encoding a polypeptide of 212 amino acids. The N-terminal amino acid sequence and the nucleotide sequence analyses predicted that the precursor xylanase contained a signal peptide composed of 28 amino acids and a catalytically active 19.9-kDa peptide fragment. The deduced amino acid sequence shared extensive similarity with those of the glycoside hydrolase family 11 of xylanases from other bacteria. The predicted amino acid sequence contained two glutamate residues, previously identified as essential and conserved for active sites in other xylanases of the glycoside hydrolase family 11.

• BMB Reports
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• 제31권5호
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• pp.508-514
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• 1998

We studied the effects of ASC-17D rat Sertoli cell-conditioned media (rSCCM) on the proliferation of the DU145 prostate cancer cells. rSCCM was prepared from ASC-17D cells cultured in DMEM/F-12 serum-free media at a nonpermissive temperature of $40^{\circ}C$, which is the condition for the high expression of c1usterin. We found that rSCCM could inhibit the proliferation of DU145 cells by arresting the cell cycle in the G1 phase in a dose-dependent manner. This growth arresting activity was abolished by boiling rSCCM for 5 min. The G1 growth-inhibiting activity of rSCCM was also detected in other prostate-originated cancer cells examined (i.e., LNCaP and PC-3) but not in other cells (ASC-17D, HepG2, SK-N-SH, and NIH3T3). Western blot analysis of partially purified growth inhibiting fractions with the clusterin antibody showed that the cytostatic factor in rSCCM was not c1usterin. This cytostatic factor was semi purified by DEAE-Sepharose, ammonium sulfate precipitation, and Phenyl-Sepharose column chromatography, and was estimated to have a molecular weight of 88 kDa by Sephacryl S-300 gel filtration.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1653-1663
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• 2010

The first gene (${\alpha}$-gal1) encoding an extracellular ${\alpha}$-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The ${\alpha}$-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal ${\alpha}$-galactosidases belonging to glycosyl hydrolase family 27. The ${\alpha}$-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant ${\alpha}$-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at $70^{\circ}C$, pH 4.5, and lost no activity over 10 days at $50^{\circ}C$. ${\alpha}$-Gal1 followed Michaelis-Menten kinetics ($V_{max}\;of\;240.3{\mu}M/min/mg,\;K_m\;of\;0.294 mM$) and was inhibited competitively by galactose ($K_m{^{obs}}$ of 0.57 mM, $K_i$ of 2.77 mM). The recombinant T. emersonii ${\alpha}$-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the ${\alpha}$-galactosidic linkage. Owing to its substrate preference and noteworthy stability, ${\alpha}$-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.