• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.107-107
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• 2018

양파는 백합과에 속하는 여러해살이풀로 서남아시아의 페르시아가 원산인데 세계 각국의 온대지방에서 많이 재배하고 있다. 양파의 생약명은 옥총(玉憁)으로 피 속의 콜레스테롤을 떨어뜨리고 심장혈관의 혈류량을 증가시켜 성인병 예방에 좋다. 철원지역은 내륙지방이면서도 고도가 높아 기온차가 큰 대륙성 기후의 성격이 강하기 때문에 이 지역에서 재배한 양파는 남부지방에서 재배된 양파보다 당도가 높고 맛도 좋다고 알려져 있다. 인산질 성분은 특히 근의 발육을 촉진시키는 중요한 성분이다. 하지만 토양부의 인산성분이 수확 시까지 다량 지속되면 양분의 경합으로 뿌리부의 부패를 초래하기 쉽다. 따라서 수확기에 환원형 인산질을 중심으로 엽면시비 방법으로 영양균형을 유도하여 양파의 품질과 저장성을 향상시키는 것이 중요하다. 본 연구는 아인산(H3PO3)과 수산화칼륨(KOH)을 희석하여 산도조절 후, 아인산염으로 철원양파에 엽면 처리한 후 성분을 비교분석하였다. 본 연구에서 아인산염의 농도를 500, 1000, 1500배 희석하여 철원양파에 2018년 5월에 분무기를 이용하여 3회 엽면살포 한 후 6월 말경 수확한 후 HPLC를 이용하여 페놀화합물을 분석하였다. 그 결과 양파에서 중요한 물질 중의 하나인 큐에르세틴(quercetin)함량은 대조구(96.39 ug/g)에 비해 500배(179.70 ug/g), 1000배(150.27 ug/g), 1500배(105.95 ug/g) 처리구에서 높은 함량을 보였다. 또한 caffeic acid, p-coumaric acid, ferulic acid, rutin, kaempferol, 당도 함량은 처리구에서 대조구보다 높은 함량을 보였다. 따라서 아인산염 처리는 철원양파에서 페놀화합물의 함량을 높이는데 중요한 역할을 한다는 것을 알 수 있었다. 본 연구를 통하여 아인산염과 같은 환원형 엽면시비법을 이용하여 재배한다면 고품질의 기능성 양파를 생산할 수 있을 것이라 생각한다.

• Molecules and Cells
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• v.37 no.2
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• pp.172-177
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• 2014

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-${\alpha}$-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4' and 7 hydroxyl groups, but not at the $5^{th}$ hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-${\beta}$-D-glucoside, genistein 7-O-${\beta}$-D-glucoside (genistin), genistein 4',7-O-${\beta}$-D-diglucoside, biochanin A-7-O-${\beta}$-D-glucoside (sissotrin), daidzein 4'-O-${\beta}$-D-glucoside, daidzein 7-O-${\beta}$-D-glucoside (daidzin), daidzein 4', 7-O-${\beta}$-D-diglucoside, and formononetin 7-O-${\beta}$-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli $BL21(DE3)/{\Delta}pgi{\Delta}zwf{\Delta}ushA$ overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of $200{\mu}M$ of supplemented isoflavonoids, without any additional UDP-${\alpha}$-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.

• Biomedical Science Letters
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• v.24 no.1
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• pp.30-34
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• 2018

Silver (Ag) has been widely used in commercial products and medical fields since ancient times because of its antibacterial effect. It is harmless and non-toxic to the human body. For this reason, recent research has actively evaluated antimicrobial activity using silver (Ag). In this study, we investigated the inhibitory effect of a silver-based compound, silver phosphate ($Ag_3PO_4$) on the growth of Staphylococcus aureus and the activation of human immunity. First, the inhibitory effect of $Ag_3PO_4$ on the growth of Staphylococcus aureus was confirmed by a growth curve and a colonyounting method. As a result, the growth inhibitory effect increased as the concentration of $Ag_3PO_4$ increased. Specifically, treatment with $5{\mu}g/mL$ of $Ag_3PO_4$ resulted in no bacteria growth, and the colony-counting method showed a remarkable inhibition. In addition, the expression of cytokine IL-8 by $Ag_3PO_4$ was examined to investigate the cellular immune system activation by $Ag_3PO_4$. After pretreatment of Staphylococcus aureus for 1 hour with $50{\mu}g/mL$ $Ag_3PO_4$, an increased IL-8 mRNA expression resulted. In cells treated with $Ag_3PO_4$, we found that the expression of IL-8 was enhanced in a time-dependent fashion compared to non-treated cells. These results indicate that $Ag_3PO_4$ induces antimicrobial activity against Staphylococcus aureus and activates human immunity. These results are expected to contribute to the future study of the mechanism of silver (Ag) and silver-based compounds in relation to antibacterial activity.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.36 no.5
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• pp.545-551
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• 2012

We investigated the diffusive transport of bupivacaine HCl through the microchannels of microfluidic drug delivery devices. In the biodegradable microfluidic drug delivery devices developed in this research, the drug release rate can be controlled by simply modulating the geometrical parameters of the microchannels, such as the length, number, and cross-sectional area of the microchannels, when the microchannels are used as paths for drug release. However, the hydrophobic nature of a biodegradable polymer, 85/15 poly(lactic-co-glycolic acid), hinders the infiltration of a release medium (phosphate-buffered saline) through the microchannels into the reservoir of a device that contains bupivacaine HCl, at the early stage of drug release. This can have an adverse effect on the early stage release of local analgesic compounds from the device. In this study, microfluidic channels were surface-treated with surfactants such as PEG600 and Tween80, and the effects of the surfactants on the release performance are presented and analyzed.

• Journal of Plant Biology
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• v.12 no.2
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• pp.7-14
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• 1969

Dunaliella tertiolecta did not show any increase in respiration rate when supplied with glucose, glycerol, sucrose, L-alanine, acetate, pyruvate and succinate. This was in contrast to Chlorella pyrenoidosa, which, under identical conditions, showed significant increase when supplied with glucose or acetate but not with the other compounds. Production of 14CO2 from added 14C-glucose in D. tertiolecta was lower than the other 14C-labelled substrates: L-alinine, glycerol, succinate, but higher than 14C-sucrose addition. And it was also lower than C. pyrenoidosa experiments which was added 14C-glucose as a substrate. Light reduced amounts of labelled carbon dioxide from 14C-glucose or 14C-acetate and increased incorporation of 14C from the substrates to cell materials in either D. tertiolecta or C. pyrenoidosa. The contribution of 14C from 14C-glucose to 14CO2 in cell-free system of D. tertiolecta were much higher than in whole cell suspension. It was contrast to C. pyrenoidosa which were showed reduction of 14CO2 production in cell-free systems than whole cell suspensions. When cell-free systems of D. tertiolecta and C. pyrenoidosa were supplied with ATP, NAD, NADP or/and hexokinase, it was remarkably increased production of 14CO2 from the substrates than the control. It was concluded that the low ability of D. tertiolecta to metabolize glucose were caused by the impermeability of the cell membrane to glucose and were not due to deficiencies of enzyme systems concerning glucose metabolism. In the cell-free systems, it seemed to be more active pentose phosphate pathway than glycolytic pathway in D. tertiolecta.

• Journal of Korean Society on Water Environment
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• v.24 no.6
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• pp.682-689
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• 2008

The purpose of this study was to find out the variation between molecular size distribution (MSD) of natural organic matter (NOM) in raw waters after different water treatment processes like conventional process (coagulation, flocculation, filtration) followed by advanced oxidation process (ozonation, GAC adsorption). The MSD of NOM of Suji pilot plant were determined by Liquid Chromatography-Organic Carbon Detection (LC-OCD) which is a kine of high-performance size-exclusion chromatography (HPSEC) with nondispersive infrared (NDIR) detector and $UV_{254}$ detector. Five distinct fractions were generally separated from water samples with the Toyopearl HW-50S column, using 28 mmol phosphate buffer at pH 6.58 as an eluent. Large and intermediate humic fractions were the most dominant fractions in surface water. High molecular weight (HMW) matter was clearly easier to remove in coagulation and clarification than low molecular weight (LMW) matter. Water treatment processes removed the two largest fractions almost completely shifting the MSD towards smaller molecular size in DW. No more distinct variation of MSD was observed by ozone process after sand filtration but the SUVA value were obviously reduced during increase of the ozone doses. UVD results and HS-Diagram demonstrate that ozone induce not the variation of molecular size of humic substance but change the bond structure from aromatic rings or double bonds to single bond. Granular activated carbon (GAC) filtration removed 8~9% of organic compounds and showed better adsorption property for small MSD than large one.

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.5
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• pp.565-569
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• 1993

Antioxidative activity of browning product(BP) fractionated from fermented soybean sauce(SS) was studied during the oxidation process of linoleic acid mixture system. SSBP was a powder type product prepared from fermented soybean sauce by the fractionation through the Sephadex G-10 column and freeze drying of collected fraction. The aqueous model systems were used for the evaluation of antioxidative activity of SSBP during the oxidative reaction at $50^{\circ}C$ by the determination of peroxider and conjugated dienoic acid compounds. The linoleic acid mixture for the aqueous model systems was consisted of linoleic acid(64.6%), oleic acid(27.4%), and other acids in ethanolic phosphate buffer solution(pH 7.0). SSBP had a considerable antioxidative activity with the inhibition of formation of peroxides and conjugated dienoic acids during the autoxidation of linoleic acid mixtures in aqueous model systems. Antioxidative activity of SSBP was relatively higher than SS, however, lower than ${\alpha}-tocopherol$ and butylated hydroxyanisol. The antioxidative effect of SSBP was increased by the its concentrations from 0.05% to 0.5% in the oxidation reactions of aqueous model systems. Therefore, SSBP was considered as one of the potential natural antioxidants for the use of food products.

• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.23-36
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• 2009

In the present work, the metabolic network of primary metabolism of the slow-growing myxobacterium Sorangium cellulosum was reconstructed from the annotated genome sequence of the type strain So ce56. During growth on glucose as the carbon source and asparagine as the nitrogen source, So ce56 showed a very low growth rate of $0.23\;d^{-1}$, equivalent to a doubling time of 3 days. Based on a complete stoichiometric and isotopomer model of the central metabolism, $^{13}C$ metabolic flux analysis was carried out for growth with glucose as carbon and asparagine as nitrogen sources. Normalized to the uptake flux for glucose (100%), cells recruited glycolysis (51%) and the pentose phosphate pathway (48%) as major catabolic pathways. The Entner-Doudoroff pathway and glyoxylate shunt were not active. A high flux through the TCA cycle (118%) enabled a strong formation of ATP, but cells revealed a rather low yield for biomass. Inspection of fluxes linked to energy metabolism revealed that S. cellulosum utilized only 10% of the ATP formed for growth, whereas 90% is required for maintenance. This explains the apparent discrepancy between the relatively low biomass yield and the high flux through the energy-delivering TCA cycle. The total flux of NADPH supply (216%) was higher than the demand for anabolism (156%), indicating additional reactions for balancing of NADPH. The cells further exhibited a highly active metabolic cycle, interconverting $C_3$ and $C_4$ metabolites of glycolysis and the TCA cycle. The present work provides the first insight into fluxes of the primary metabolism of myxobacteria, especially for future investigation on the supply of cofactors, building blocks, and energy in myxobacteria, producing natural compounds of biotechnological interest.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.