• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1281-1287
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• 2017

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.

• Applied Biological Chemistry
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• v.30 no.1
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• pp.77-87
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• 1987

The mutant with high productivity, X. malvacearum SNUF 560-6, was acquired from the X. malvacearum SNUF 560 with low productivity by UV-light irradiation. It was preserved is lyophilized stock culture and it was transferred to PDA slant to maintain viability fortnightly. Fermentations were started by retransfering to MY agar slant from PDA stok culture. The experiments for optimal xanthan gum production were studied in a chemically defined medium. Of the carbon and nitrogen sources tested, 0.4% sucrose medium and 10mM glutamic acid medium yielded the highest xanthan gun production respectively. The addition of 10g/l succinic acid stimulated xanthan gum production. Also 65mM $PO_4\;^{-3}\;(12.6g/l\;KH_2PO_4)$ was effective on xanthan gum production. Finally, medium 1 and medium 2 which have high xanthan gum production potencies were achieved in this stud. The components of medium 1 and medium 2 were as follows: Medium 1 : sucrose 40g/l glutamate 10mM $PO_4\;^{-3}\;54mM\;(KH_2PO_4\;12.65g/l)$ Citrate 2g/l $MgSO_4{\cdot}7H_2O\;0.2g/l$ $H_3BO_3\;0.005/l$ ZnO 0.006/l $FeCl_2{\cdot}6H_2O\;0.0024g/l$ $CaCO_3\;0.02g/l$ Medium 2 : $Sucrose\;40g/l\;(NH_4)_2SO_4\;2g/l$ $PO_4\;^{-3}\;65mM\;(KH_2PO_4\;12.65g/l)$ Succinate 10g/l $MgSO_4{\cdot}7H_2O\;0.02g/l$ $H_3BO_3\;0.06g/l$ ZnO 0.006g/l $FeCl_2{\cdot}6H_2O\;0.0024g/l$ $CaCO_3\;0.02g/l$.

• KSBB Journal
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• v.29 no.6
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• pp.443-447
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• 2014

Biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) was examined in recombinant Escherichia coli W strain from sucrose. The Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene, which encode engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) and engineered C. propionicum propionyl-CoA transferase ($Pct_{Cp}$), respectively, were expressed in E. coli W to construct key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli W expressing the phaC1437 gene and the pct540 gene could synthesize P(3HB-co-13mol%LA) up to the polymer content of 31.3 wt% when it was cultured in chemically defined MR medium containing 20 g/L of sucrose and 2 g/L of sodium 3-hydroxybutyrate. When Ralstonia eutropha phaAB genes were additionally expressed to provide 3-hydroxybutyrate-CoA (3HB-CoA) from sucrose, P(3HB-co-16mol%LA) could be synthesized from sucrose as a sole carbon source without supplement of sodium 3-hydroxybutyrate in culture medium, but the PHA content was decreased to 12.2 wt%. The molecular weight of P(3HB-co-16 mol%LA) synthesized in E. coli W using sucrose as carbon source were $1.53{\times}10^4$ ($M_n$) and $2.78{\times}10^4$ ($M_w$), respectively, which are not different from those that have previously been reported by other recombinant E. coli strains. Engineered E. coli strains developed in this study should be useful for the production of lactate-containing PHAs from sucrose, one of the most abundant and least expensive carbon sources.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.297-304
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• 2017

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG ($45.3{\pm}6.2%$) compared to control ($25.6{\pm}3.4%$). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG ($89.3{\pm}1.9%$) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG ($94.6{\pm}1.1%$). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation ($27.8{\pm}4.9%$) compared to no treatment ($41.4{\pm}1.9%$) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM ($37.4{\pm}3.4%$ and $34.4{\pm}2.6%$, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

• The Korean Journal of Mycology
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• v.18 no.3
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• pp.158-163
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• 1990

To investigate symbiotic relationship of 'Chunma (Gastrodia elata) and the rhizomorph of Armillaria mellea, volatile organic acids and alcoholic compounds which were considered to be contained in Gastrodia elata were tested to determine stimulatory effects on rhizomorph growth on a chemically defined medium. Also, volatile organic acids were isolated from Gastrodia elata and analyzed by gas chromatography. The growth of rhizomorph was stimulated by the presence of alcohols and volatile organic acids, but acetic acid and methanol were ineffective. In the presence of valeric acid and ethanol, Armillaria mellea produced abundant rhizomorph at concentrations of 0.1 and 1%, respectively. Ethanol and valeric acid supplemented at regular intervals of 3 days as lower concentrations in the medium stimulated the growth of Armillaria mellea. The concentrations of ethanol and valeric acid as low at 0.01% added 3 days intervals for 15 days were more effective than initial concentrations of 0.1 and 1% in stimulating rhizomorph development of Armillaria mellea. Eight kinds of volatile organic acids were identified and quantified by gas chromatography. The major compounds were n-propionic, valeric, iso-carproic and caproic acids, and the minor compounds were iso-butyric, butyric, iso-valeric and hepatanoic acids. Valeric acid was the most abundant among them.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.133-142
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• 1997

The chemical media composition and culture conditions were optimized for mycelial growth of Phellinus igniarius 26005. The method of solid-state fermentation, cultivation of basidiomycetal strains in various grains, was developed. Media composition for optimal growth of Phellinus igniarius 26005 was made of 7.0% malt extract, 0.3% bacto soytone, and 0.2% yeast extract. The optimum condition for mycelial growth was $28^{\circ}C$ and pH 7.0, respectively. For the mass cultivation of mycelia, the hydrated grains with cold water, were put into the plastic bottle. The mycelial growth rate in the bottled grains was high in the early stage with inoculation of homogenized mycelium. The activity of mycelium was maintained by adding sterilized water in the middle of cultivation. The glucosamine content which determins the mycelial growth rate in solid material was in the order of job's tears>barley>black soybean>wheat>malt soybean>brown rice>sorghum>glutinous rice.

• KSBB Journal
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• v.31 no.1
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• pp.27-32
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• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• Development and Reproduction
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• v.12 no.1
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• pp.19-30
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• 2008

Proper development of fertilized oocyte to blastocyst is a key step in mammalian development to implantation. During development of preimplantation embryos, the mammalian embryo needs supply the energy substrate for keep viability. Usually mammalian oocyte get substrate especially energy substrate from oviduct and uterus, because it does not store much substrate into cytoplasm during oogenesis. Carbohydrates are known as a main energy substrate for preimplantation stage embryos. Glucose, lactate and pyruvate are essential component in preimplantation embryo culture media and there are stage specific preferences to them. Glucose transporter and $H^+$-monocarboxylate cotransporter are a main mediator for carbohydrate transport and those expression levels are primarily under the control of intrinsic or extrinsic factors like insulin and glucose. Other organic substances, amino acids, lipids and nucleotides are used as energy substance and cellular regulation factor. Though since 1960s, successful development of fertilized embryo to blastocyst has been accomplished with chemically defined medium for example BWW and give rise to normal offspring in mammals, the role of metabolites and the regulation of intermediary metabolism are still poorly understood. Glucose may permit expression of metabolic enzymes and transporters in compacting morula, capable of generating the energy required for blastocyst formation. In addition, it has been suggested that the cytokines can modulate the metabolic rate of carbohydrate in embryos and regulate the preimplantation embryonic development through control the metabolic rate. Recently we showed that lactate can be used as a mediator for preimplantation embryonic development. Those observations indicate that metabolites of carbohydrate are required by the early embryo, not only as an energy source, but also as a key substrate for other regulatory and biosynthetic pathways. In addition metabolites of carbohydrate may involve in cellular activity during development of preimplantation embryos. It is suggested that through these regulation and with other regulation mechanisms, embryo and uterus can prepare the embryo implantation and further development, properly.