• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.300-305
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• 2015

The glutamate decarboxylase gene (gadB) from Lactobacillus plantarum WCFS1 was cloned and expressed as an N-terminal hexa-histidine-tagged fusion protein in Escherichia coli BL21 (DE3) as the host strain. Purified glutamate decarboxylase (GAD) was immobilized onto porous silica beads by covalent coupling. The pH dependence of activity and stability of the immobilized GAD was significantly altered, when compared to those of the free enzyme. Immobilized GAD was stable in the range of pH 3.5 to 6.0. The resulting packed-bed reactor produced 41.7 g of γ-aminobutyric acid/l·h at 45℃.

• Mycobiology
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• v.42 no.3
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• pp.296-300
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• 2014

We selected Pleurotus ostreatus from among several edible mushrooms because it has high anti-gout xanthine oxidase (XOD) inhibitory activity. The maximal amount of XOD inhibitor was extracted when the Pleurotus ostreatus fruiting body was treated with distilled water at $40^{\circ}C$ for 48 hr. The XOD inhibitor thus obtained was purified by Sephadex G-50 gel permeation chromatography, ultrafiltration, $C_{18}$ solid phase extraction chromatography and reverse-phase high-performance liquid chromatography with 3% of solid yield, and its XOD inhibitory activity was 0.9 mg/mL of $IC_{50}$. The purified XOD inhibitor was a tripeptide with the amino acid sequence phenylalanine-cysteine-histidine and a molecular weight of 441.3 Da. The XOD inhibitor-containing ultrafiltrates from Pleurotus ostreatus demonstrated dose-dependent anti-gout effects in a Sprague-Dawley rat model of potassium oxonate-induced gout, as shown by decreased serum urated levels at doses of 500 and 1,000 mg/kg, although the effect was not as great as that achieved with the commercial anti-gout agent, allopurinol when administered at a dose of 50 mg/kg.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.560-563
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• 2003

During our previous studies on the relationship between nutrient requirements of S. hygroscopicus C9 and rapamycin biosynthesis, we developed chemically-defined media containing among other nutrients, aspartic acid, arginine, histidine, or ammonium sulfate. However, these media (“Cheng et al. medium” and “Lee et al. medium”) showed very slow growth characterized by a very long lag phase of growth. In an attempt to develop a chemically-defined or semi-defined medium to support more rapid growth and increased cell production, we have carried out studies to shorten the lag phase. Of the various additives tested, vitamin-free casein acid hydrolysate was the most significant by shortening the lag phase by 2-3 days. Mixtures of amino acids failed to replace casein acid hydrolysate. The active principle passed through an ultrafilter with a molecular weight cutoff of 1,000 and thus may be a peptide. The present work has yielded a semi-defined medium which should be useful for further growth studies on S. hygroscopicus C9.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.445-448
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• 2002

The Pseudomonas fluorescens KCTC 1767, a selected and identified as potential candidate for stereo-specific resolution of rac-ketoprofen ethyl ester, was systematically investigated in order to induce the high level expression and detailed characterization of the expressing enzyme esterase. We cloned the esterase gene from chromosomal DNA of Pseudomonas fluorescens KCTC 1767 by PCR with two synthetic primers that desinged for simple purification. The recombinant esterase from Pseudomonas fluorescens KCTC 1767 exibited a high conversion rate and enantioselectivity to the (S)-ketoprofen ethyl ester as expected. The enzyme was easily purified to homogeniety by using a metal chelating affinity chromatography as a protein with poly histidine taq, and thus obtained 0.6 mg of protein from a 100 mL culture broth in a single step. The purified enzyme was steadily stable at the pH range from 7.0 to 10. The activity was also retained to be about 70% after the preincubation at $40^{\circ}C$ but over $50^{\circ}C$ lost the activity completely. The molecular mass of the esterase was estimated to be about 43 kDa on SDS-PAGE, and an identical result was also shown in gel filteration chromatography. The specific activity was calculated 27 mM/mg-protein/min by using the rac-ketoprofen ethly ester as a substrate.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.1
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• pp.38-45
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• 1998

To extract insoluble proteins and to improve funtional properties of abolished proteins, an phytase producing Aspergillus sp. SM-15 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. Phytase production reached to maximum when the wheat bran medium containing 1% mannose, 1% yeast extract, 1% (NH4)2HPO4 and 0.2% calcium chloride was cultured for 4 days. Phytase was purified 17.1 fold and specific activity was 244.32unit/mg by a sequencial process of ammonium sulfate fraction, ion exchange chromatography and gel filtrations Pruified enzyme was confirmed as a single band by the polyacrylamide gel electro-phoresis. The molecular weight of phytase was estimated to be 46,000. The optimum pH and temperature for the phytase activity were 5.5 and 5$0^{\circ}C$. The enzyme is stable in pH 4.5~5.5, 6$0^{\circ}C$. The activity of purified enzyme was inhibited by Hg2+ whereas activited by Pb2+ and Fe2+. The activity of phytase was inhibited by the treatment with iodine. The result indicate the possible involvement of histidine at active site. Km and Vmax of the puridied phytase were 37.037mM/L and 159.87umol/min, respectively.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.46-51
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• 1987

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

• KSBB Journal
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• v.14 no.2
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• pp.188-191
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• 1999

We have investigated the fortifying effect of amino acids on the cell growth and productivity during the perfusion culture of hybridoma vR8 cells in serum-free media. Through the quantitative analysis of amino acids and metabolites in perfusion culture, we found that many amino acids(glutamine, histidine, arginine, methionine, isoleucine, leucine, phenylalanine, tryptophane) were heavily consumed at cell density of $1.06{\times}10^7$cells/mL. Due to amino acid depletion, cells died suddenly. So we supplemented the media with those amino acids by 30-170%. As a result, were could increase maximum cell density by 270%, average specific productivity by 175%, and average volumetric productivity by 560% in this fortified media, GC-HY-S2.

• Journal of Applied Biological Chemistry
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• v.54 no.4
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• pp.231-237
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• 2011

A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring $6{\times}histidine$ tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.988-992
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• 2006

The ptsHI operon from Leuconostoc mesenteroides ssp. mesenteroides SY1 (L. mesenteroides SY1), a strain isolated from kimchi, was cloned and characterized. The ptsH open reading frame (ORF) was 273 bp in size, which can encode a protein of 90 amino acid residues with a molecular weight of 9,212 Da. The pfsI ORF was 1,719 bp in size, which was capable of encoding a protein of 572 amino acids with a molecular mass of 62,549 Da. ptsH and pfsI genes were transcribed as a single transcript of 2.0 kb in size regardless of carbon sources, supporting the operon structure. Although the deduced amino acid sequences of the HPr and EI were highly homologous with those of other Gram-positive bacteria, an additional amino acid (glutamine at the $3^{rd}$ amino acid) was present in HPr from L. mesenteroides SY1. Phosphorylation sites of HPr included the histidine residue ($16^{th}$) and serine residue ($47^{th}$). Mutant HPrs, in which each phosphorylation site was mutated into alanine, were obtained, and phosphorylation with HPr and mutated HPrs showed that HPr was phosphorylated at the serine residue ($47^{th}$) by HPr kinaseiphosphorylase (HPr K/P).

• Journal of Life Science
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• v.14 no.1
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• pp.26-31
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• 2004

Redox factor-1 (Ref-1), known as a redox regulator, controls the DNA binding of AP-1 and is activated in HT29 colon cancer cells by hypoxia in vitro. REF-1 also increases tile DNA binding affinity of Hypoxia-inducible Factor-lalpha$ (HIF-lalpha$), HIF-like Factor (HLF) and early growth response-1 (Egr-1) which induce expression of the genes involved in angiogenesis, so that we speculate that REF-1 may play a role in hypoxia-induced angiogenesis. In this research we tried to detect novel proteins interacting with REF-1 using Yeast two-hybrid system using full-length REF-1 cDNA as bait. As result of such screening we detected 3 positive clones. DNA sequencing and GeneBank search revealed that one of the clones contained the same sequences as M.musculus cDNA for tioredoxin.