• Journal of Applied Biological Chemistry
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• v.52 no.1
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• pp.15-20
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• 2009

Poplar contains various flavonoids including naringenin, kaempferol, myricetin, apigenin, luteolin, rhamnetin, and quercetin. These flavonoids are synthesized from naringenin with various enzymes. However, none of genes from poplar involved in flavonoid biosynthesis have been biochemically characterized. We cloned PFNS I-1 from Populus deltoids by RT-PCR method. The open reading frame of PFNS I-1 consisted of 1,017-bp and it showed high similarity with other FNS genes. The purified recombinant PFNS I-1, expressed in Escherichia coli, catalyzed the reaction from flavanone (naringenin) to flavone (apigenin). The reaction of PFNS I-1 was enhanced by cofactors such as oxoglutarate, $Fe^{2+}$, ascorbate and catalase. Thus, it is concluded that PFNS N-1 encodes a flavone synthase I.

• Journal of the Korean Chemical Society
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• v.41 no.4
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• pp.205-209
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• 1997

The site specificity of dephosphorylation of myelin basic protein(MBP) was studied in vitro. To assign amino acid site of dephosphorylation, MBP was phosphorylated by protein kinase C(PKC) and dephosphorylated by protein phosphatase PP2A. The phosphorylated MBP was digested by trypsine and the digested peptides were separated by a reverse phase HPLC chromatography. The radioactivity of each fraction was counted and partially sequenced. Seven radioactive peptides were observed and $Ser^{55}$ in the second peak($P_2$) shows the best susceptibility for the phosphorylation. However in the dephosphorylation, the fifth peak($P_5$) appeared to release it's phosphate group most rapidly. This result demonstrates that MBP is a suitable substrate for protein phosphatase.

• Analytical Science and Technology
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• v.17 no.1
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• pp.37-44
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• 2004

An alcohol oxidase from Hansenula polymorpha was strongly induced when cells were grown with 0.5% methanol supplementation as the carbon source. The induced Hansenula polymorpha alcohol oxidase was purified to electrophoretic homogeneity by using DEAE-Sephacel and Mono Q column chromatographys. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol and methanol. The activity of the enzyme optimally proceeded at pH 8.5 and $50^{\circ}C$. The midpoint of the temperature-stability curve for the enzyme was approximately $52^{\circ}C$ and the enzyme was not completely inactivated even at $65^{\circ}C$ temperature. The enzyme showed resistance toward detergents and highly stable over 7 weeks of storage condition. This Hansenula polymorpha alcohol oxidase may be useful for the enzymatic determination of alcohol and for the industrial production of alcohols and aldehydes.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.237-241
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• 1997

Lipase (EC 3.1.1.3) in the culture filtrate of Streptomyces coelicolor A3(2) was active on ${\alpha}$-naphthyl-butyrate as well as on various triacylglycerols with different lengths of acyl chains. The extracellular lipase was purified 15-fold by ammonium sulfate fractionation, Sephadex G-100, DEAE-Cellulose and Phenyl-Sepharose CL4B column chromatography with overall yield of 16%. It showed an molecular weight of 34.7 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme activity with tributyrin as substrate was optimal at pH 8.0~9.0 and at $37^{\circ}C$. The enzyme activity decreased when the chain length of acyl group of triacyglycerol increased. A-factor, a hormone-like regulator of Streptomyces differentiation inhibited the lipase activity, which might corelate with the low enzyme activity in early exponential growth phase.

• Korean journal of applied entomology
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• v.36 no.4
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• pp.323-330
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• 1997

The apple leafminer, Phyllonorycrer ringoniella (Lepidoptera: Gracillariidae), is the most important pest on apple leaves and is not easy to be controlled with insecticides because of leafmining habit of its larvae. This study was undertaken to develop an artificial diet for mass rearing of the moth, and to look at their reproductive behavior. For developing an artificial diet the problem of diet contamination with microorganisms and how to supply artificial diet to first instar larvae have to be solved first. Artificial oviposition trial was successful with shadowy bottomside of various papers sprayed with apple leaf extracts. The highest activity of both calling and mating was observed within 30 min. after the onset of photophase and 3- to 4-day -old adults showed the highest mating rate. A ring-shaped sex pheromone gland was found at the intersegmental membrane between the 8th and 9th abdominal segments of the female. (E, 2)-4,lOtetradecadienyl acetate was identified as the major component from the femall gland extracts by the gas chromatography (GC) and GC-mass spectrometry. Additionally it was thought that (a-10-tetradecenyl acetate could be the second component of their sex pheromone.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.297-302
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• 1998

$\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.564-569
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• 1992

A green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum NCIB 8327, was grown in modified Pfennig's medium including glu1amate as a nitrogen source. Glutamine synthetase was isolated through a series of ultracentrifugation. DEAE-Sepharose CL-6B ion exchange chromatography. Sephacryl S-300 gel permeation chromatography, and preparative HPLC. The recovery and purification fold of the enzyme were 2% and 46.3. respectively. The isolated enzyme was homogeneous on UV-Visible spectrum and polyacrylamide gel electrophoretogram. The relative molecular mass of the native enzyme was estimated to be 280,000 by gel permeation chromatography. The enzyme consisted of ten subunits with relative similar molecular mass. 30.000. which was estimated by SDS-polyacrylamide gel electrophoresis. The optimal temperature and pH of the enzyme were $30^{\circ}C$ and 7.0. Km values were 27.9 mM for L-glutamine and 0.92 mM for hydroxylamine-HCr. The enzyme activity was inhibited by alanine. glycine. and tryptophan considerably, but was not affected by asparagine, lysine. leucine. and valine.

• Korean Chemical Engineering Research
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• v.49 no.6
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• pp.840-845
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• 2011

Degradation products of agarose are biologically active and thus used as an ingredient in pharmaceuticals or functional cosmetics. Furthermore, it has been strongly considered as a substrate for bio-ethanol fermentation. Recently, we isolated new agarase-producing microorganism, Pseudoalteromonas sp. from south sea of Korea. In this study, we aimed to separate and purify the agarase from culture broth of this strain. Separation of agarase was performed by ion- exchange chromatography on DEAE-Sepharose resin. Equilibrium pH and volume ratio of resin to the amount of protein were optimized for the efficient adsorption of protein. 410 ${\mu}g$ of protein was completely adsorbed to 3 mL of resin at pH 7.5. The total amount of eluted protein increased as NaCl concentration increased to 400 mM at isocratic elution. Agarase was separated by linear gradient elution of NaCl (0~1,000 mM). Three major protein peaks were observed and the presence or absence of agarase in these eluted proteins was measured by Lugol's staining technique. Only six eluted protein fractions showed strong agarase activity.

• Journal of Life Science
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• v.17 no.5 s.85
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• pp.625-633
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• 2007

We isolated a new extracellular alginate lyase-producing microorganism, which displayed alginate-depolymerizing activity in plate assays, from coastal soils in Wando, Jeollanam-do, Korea. This alginate-depolymerizing bacterium belonged to the genus Streptomyces and it was named Streptomyces sp. MET 0515. An extracellular alginate lyase(ALY1) secreted by Streptomyces sp. MET 0515, was purified to homogeneity by a combination of acetone precipitation, anion-exchange chromatography (Q-Sepharose and DEAE-Sepharose) and Sephacryl S-200 HR gel filtration chromatography. Its molecular mass was 26 kDa as determined by SDS-PACE analysis. The enzyme had an optimal temperature of $70^{\circ}C$ for its activity, and was most active at pH 7.5. The thermal and pH stability were $0-50^{\circ}C$, and pH 6.0-9.0, respectively. The enzyme activity was stimulated by 1mM $Mn^{2+}$, and inhibited by 1mM $Fe^{3+}$, 1mM EDTA and 1mM $Zn^{2+}$. Preliminary analysis of substrate specificity showed that this alginate lyase had activity on both poly-alpha 1,4-L-guluronate and poly-beta 1,4-D-mannuronate in the alginate molecule.

• Korean journal of applied entomology
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• v.34 no.3
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• pp.181-190
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• 1995

Malate dehydrogenase in the mosquito ovary after a blood meal, Aedes aegypti, was purified and characterized. MDH purification steps involved DEAE-Sepharose, S-Sepharose and Cibacron blue affinity chromatography. The purified MDH was 70,000 daltons in molecular weight and was a homodimer consisting of tow identical subunits. Optimal activity of purified MDH was obtained pH 9.0-9.2 in malate-oxaloacetate reaction and pH 9.8-10.2, in oxaloactate-malate reaction. With obtained pH 9.0-92 in malate-oxaloacetate reaction and pH 9.8-10.2, in oxaloactate-malate reaction. With malate as substrate, purified mitochondrial MDH (1.28$\times$${10}^{-4}$ M) had lower Km value than cytoplasmic MDH (8.92x${10}^{-3}$ M). MDH activity was inhibited by citrate, $\alpha$-ketoglutarate, and ATP. Inhibition of MDH activity by ATP and citrate was less in malate-oxaloacetate reaction and in oxaloacetate-malate reaction. MDH activity was completely inhibited by ATP in oxaloacetate-malate reaction and not inhibited by citrate in malate-oxaloacetate reaction. Temporal activity change of MDH is similar to that of isocitrate dehydrogenase in the ovary after blood feeding; their activities in the ovary began to rise at 18 hours after a blood meal, and reached at the maximal level at 48 hours.