• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• The Korean Journal of Ecology
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• v.26 no.1
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• pp.5-12
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• 2003

Seed germination rate and seedling growth were measured on 6 different species(Phytolacca americana, Eupatorium rugosum, Rumex acetocella, Echinochloa crusgalli, Cassia mimosoides var. nomame, Setaria viridis) treated with leaf extract of E. rugosum. Total phenolic compound and heavy metal were analyzed on leaf and soil with and without E. rugosum. The growth of P. americana seedlings were stimulated by 10% and 25% of E. rugosum water extract treatment. The content of total phenolic compounds in soil was lower than that of leaf extract, and 25% was confirmed as threshold concentration in natural systems because the total phenolic compounds were not significantly different between the control soils and the soil treated with 10%, and 25% extract. Total phenolic compound concentrations of the leaf extracts were highest (1.66 mg/l) with E. rugosum grown under the Quercus forest canopy and lowest (1.09 mg/l) for the plant grown in the mixed forest edge. Leaf extracts of plants selected in different sampling sites (Forest interior, Forest edge, under Pinus Canopy and Quercus Canopy) were significant, while soil extracts were not. Seed germination of R. acetocella and S. viridis were significantly inhibited at over 50% concentrations of E. rugosum, but C. mimosoides var. nomame was not affected at any concentration. The radicle and shoot growth of the native species group were reduced two times more than those of the exotic species group by the treatment of extracts. Especially, the seed germination percentage and dry weight of E. rugosum were greater than those of the control group by treatments with extracts of 10% and 25%. Analysis of aqueous extracts from E. rugosum by HPLC identified 6 phenolic compounds: caffeic acid (460.9 mg/l), benzoic acid (109.7 mg/l), protocatechuic acid (7.3 mg/l), ρ-hydroquinone (6.0 mg/l), cinnamic acid (2.7 mg/l) and hydroquinone (0.23 mg/l). The seed germination of P. americana was also inhibited dramatically by protocatechuic acid and cinnamic acid even though the content of caffeic acid (460.9 mg/l) was the highest among analyzed phenolic compounds. The heavy metal content of soil without A. altissima was higher than that of soil with E. rugosum. Particularly, Al, Fe and Mn was considerably high and most of the heavy metal were accumulated in leaves where a high level of total phenolic compounds was found.

• KSBB Journal
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• v.18 no.1
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• pp.55-61
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• 2003

To isolate and purify the antimicrobial and antitumor agents in Xanthium strumarium L. hydrothermal extract. The crude extract was extracted in ether or ethylacetate under neutral, acidic, and alkali conditions. The antimicrobial activity of each extract was tested against 16 strains of bacteria, 2 strains of yeast, and 2 strains of fungus. The ether neutral extract (XE-N) exhibited the strongest growth inhibition upon the 8 strains of gram-positive bacteria, 6 strains of gram-negative bacteria and Cryptococcus neoformans. Fluorescein diacetate (FDA) testing of XE-N and XEA-N showed growth inhibition of the 3 strains of E. coli, S. aureus and C. albicans even at 30 ng/mL, with the exception of p. aeruginosa. XE-N-S1 and XE-N-S3 from neutral ether extract (XE-N), XE-N-S3 from the acidic ether extract (XE-A), and XEA-N-S1 from ethylacetate (XEA-N) were purified as antimicrobial and antitumor agents. However all purified compounds decomposed with the exception of XE-N-S1. The results upon the antitumor activities of the crude extract and of its purified compounds, showed that XE-N-S1 had the best antitumor activity against HeLa cells. In terms of antitumor activity against HepG2 cells, XE-N-S1 and XE-N-S3 were superior, and against HT29 cells XE-N and XE-N-Sl were good, against Saos2, NCI H522, NCI H1703, Clone M3 cells XE-N-51 was very good, and against LN CAP cells XE-N-S3 was the best. Comparing of cellular toxicities various extracts and purified compounds with the existing antitumor agents, XE-A, XEA-A and XEA-B had the lowest toxicity, and XE-B had a lower toxicity than etoposide. XE-N-S1 and XE-N-S3 showed higher toxicities than etoposide, and the toxicity of XE-A-S3 was higher than that of etoposide, and lower than that of csplatin.

• KSBB Journal
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• v.21 no.2
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• pp.110-117
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• 2006

During routine maintenance, animal cell lines are commonly cryopreserved in growth medium containing serum with 10% DMSO. But, in case of bioprocess under the serum-free conditions, including cultivation of cell lines and producing of pharmaceuticals, the cryopreservation should be executed without serum to prevent a cross-contamination. This experiments were performed to investigate the effects of the serum-free cryopreservation on the CHO cells. To improve the survival rates of the cryopreserved CHO cells in serum-free condition, first, the effects of permeable and non-permeable additives for substitute serum on cell viability were investigated. The combination of 10% DMSO and 0.03 M raffinose in MEM-${\alpha}$ without serum indicated 76% of cell viability. However, it did not reach the survival rates(more than 95%) of the conventional cryopreservation. In the second, to evaluate the cryopreservative ability of the serum-free medium(SFM) we compared viability of the CHO cells cryopreserved in the SFMs(Sigma C5467, C4726, and C1707, JBI SF486 and PF486), the cryoprotectant(Genenmed CAN-1000) and the MEM-${\alpha}$ with serum. All solution contained 10% DMSO. As a result of the comparison, cryopreserved cells in the SFMs showed over 95% of viability and appeared predominant viability better than cryoprotectant CAN-1000. Finally, we assessed the stability of the CHO cells in the long-term cryopreservation(LTC) using SFM. Every three months, the cryopreserved CHO cells were thawed to estimate the cell viability and the recovery rates. Then, real-time RT-PCR analyzed the inserted CHO DHFR gene. All results for the LTC appeared the same stability as the serum containing cryopreservation. In the conclusion, it could be seen that the LTC in the SFM can substitute for serum using methods in the bioprocess proceeded by CHO cells for more than 18 months.

• KSBB Journal
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• v.23 no.6
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• pp.513-518
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• 2008

This paper described the extraction/purification of $\beta$-carotene from recombinant E.coli and evaluation of anti-wrinkle activity of purified $\beta$-carotene. No significant differences in extraction yields were observed when hexane or isobutyl acetate was used. However, extraction from wet-cell cake resulted in 2-fold higher amount of $\beta$-carotene than that from dry cells. Disruption of 5 g-wet cells by ultrasonic homogenizer, acetone dehydration, extraction with isobutyl acetate resulted in 36 mg of $\beta$-carotene corresponding to 61.2% of recovery. The formation and separation of $\beta$-carotene crystal improved the purity. 633 mg of $\beta$-carotene crystal with 93% purity was obtained from 223 g/L of wet-cell cake harvested from 2.5-L fed-batch culture broth. The cultures of normal human primary fibroblast were performed to investigate the effect of $\beta$-carotene on cytotoxicity as MTT assay and anti-wrinkle activity as collagen synthesis assays. $1.7{\mu}M$ of $\beta$-carotene was found to be optimal concentration at which 1.4-fold higher amount of collagen was synthesized than that in absence of $\beta$-carotene. This indicates that highly purified $\beta$-carotene can be obtained from recombinant E.coli by applying simple method with less toxic solvent and can be used in functional cosmetics as anti-wrinkle agent.

• KSBB Journal
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• v.23 no.6
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• pp.519-524
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• 2008

Monacolin-K is a strong anti-hypercholesterolemic agent produced by Monascus sp. via polyketide pathway. High-yielding mutants of monacolin-K were developed through rational screening strategies adopted based on understanding of monacolin-K biosynthetic pathway. Through the experiments for investigating various amino acids as putative precursors for the monacolin-K biosynthesis, it was found that production level of monacolin-K was remarkably increased when optimum amount of cysteine was supplemented into the production medium. We suggested that these phenomena might be related to the special roles of SAM (S-adenosyl methionine), a putative methyl group donor in the biosynthetic pathway of monacolin-K, demonstrating close interrelationship between SAM-synthesizing primary metabolism and monacolin-K synthesizing secondary metabolism. Namely, increase in the intracellular amount of SAM derived from the putative precursor, cysteine which was extracellularly supplemented into the production medium might contribute to the significant enhancement in the monacolin-K biosynthetic capability of the highly mutated producers. On the basis of these assumptions derived from the above fermentation results, we decided to construct efficient expression vectors harboring SAM synthetase gene (metK) cloned from A. nidulans, with the hope that increased intracellular level of SAM could lead to further enhancement in the monacolin-K production through overcoming a rate-limiting step associated with monacolin-K biosynthesis. Hence, in order to overcome the plausible rate-limiting step associated with monacolin-K biosynthesis by increasing intracellular level of SAM, we transformed the producer mutants with an efficient expression vector harboring gpdA promoter of the producer microorganism, and metK gene. Notably, from the resulting various transformants, we were able to screen a very high-yielding transformant which showed approximately 3.3 fold higher monacolin-K productivity than the parallel nontransformed mutants in shake flask cultures performed under the identical fermentation conditions.

• KSBB Journal
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• v.23 no.6
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• pp.463-469
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• 2008

During the last decade, monacolin-K biosynthesized by fermentation of red yeast rice (Monascus strains) was proved to have an efficient cholesterol lowering capability, leading to rapid increase in the market demand for the functional red yeast rice. In this study, the production medium composition and components were optimized on a shake flask scale for monacolin-K production by Monascus pilosus (KCCM 60160). The effect of three different soybean flours on the monacolin-K production were studied in order to replace the nitrogen sources of basic production medium (yeast extract, malt extract and beef extract). Among the several experiments, the production medium with dietary soybean flour to replace a half of yeast extract was very good for monacolin-K production. Plackett-Burman experimental design was used to determine the key factors which are critical to produce the biological products in the fermentation. According to the result of Plackett-Burman experimental design, a second order response surface design was applied using yeast extract, beef extract and $(NH_4)_2SO_4$ as factors. Applying this model, the optimum concentration of the three variables was obtained. The maximum monacolin-K production (369.6 mg/L) predicted by model agrees well with the experimental value (418 mg/L) obtained from the experimental verification at the optimal medium. The yield of monacolin-K was increased by 67% as compared to that obtained with basic production medium in shake flasks.

• Korean Chemical Engineering Research
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• v.44 no.6
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• pp.650-658
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• 2006

The recycling of TDA from solid waste of TDI plant(TDI-R) by near-critical hydrolysis reaction had been studied by means of a statistical design of experiment. The main and interaction effects of process variables had been defined from the experiments in a batch reactor and the correlation equation with process variables for TDA yield had been obtained from the experiments in a continuous pilot plant. It was confirmed that the effects of reaction temperature, catalyst type and concentration, and the weight ratio of water to TDI-R(WR) on TDA yield were significant. TDA yield decreased with increases in reaction temperature and catalyst concentration, and increased with an increase in WR. As a catalyst, NaOH was more effective than $Na_2CO_3$ for TDA yield. The interaction effects between catalyst concentration and temperature, WR and temperature, catalyst type and reaction time on TDA yield had been defined as significant. Although the effect of catalyst concentration on TDA yield at $300^{\circ}C$ as subcritical water was insignificant, the TDA yield decreased with increasing catalyst concentration at $400^{\circ}C$ as supercritical water. On the other hand, the yield increased with an increase in WR at $300^{\circ}C$ but showed negligible effect with WR at $400^{\circ}C$. The optimization of process variables for TDA yield has been explored with a pilot plant for scale-up. The catalyst concentration and WR were selected as process variables with respect to economic feasibility and efficiency. The effects of process variables on TDA yield had been explored by means of central composite design. The TDA yield increased with an increase in catalyst concentration. It showed maximum value at below 2.5 of WR and then decreased with an increase in WR. However, the ratio at which the TDA yield showed a maximum value increased with increasing catalyst concentration. The correlation equation of a quadratic model with catalyst concentration and WR had been obtained by the regression analysis of experimental results in a pilot plant.

• Korean Chemical Engineering Research
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• v.44 no.6
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• pp.588-596
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• 2006

The surface of polystyrene membrane treated by Ar, $O_2$ plasma, and the effects were observed before and after the treatment and permeability of $CO_2$, $N_2$ and selectivity of $CO_2$ relative to $N_2$ was measured using continuous flow gas permeation analyzer (GPA). The mole ratio of O over C in the surface was increased from 0 to 0.179 with Ar plasma treatment and route mean square of surface was increased from $15.86{\AA}$ to $71.64{\AA}$. Therefore the contact angle was decreased from $89.16^{\circ}$ to $18.1^{\circ}$. Thus Plasma treatments made surface of membrane tend to be highly hydrophilic. The optimum condition for the $CO_2$ permeability and ideal selectivity of the plasma treated membrane was as follows: the measurement of Ar (60 W, 2 min, $70^{\circ}C$) plasma treatment was $1.14{\times}10^{-12}[m^3(STP){\cdot}m/m^2{\cdot}sec{\cdot}atm]$ and 4.22. In the case of $O_2$ plasma treatment, the contact angle was decreased at $13.56^{\circ}$ with increase of O/C ratio ($0.189{\AA}$) and route mean square of surface ($57.10{\AA}$). The optimum condition for the $CO_2$ permeability and ideal selectivity of the plasma treated membrane was as follows: the measurement of $O_2$ (90 W, 2 min, $70^{\circ}C$) plasma treatment was $7.1{\times}10^{-12}[m^3(STP){\cdot}m/m^2{\cdot}sec{\cdot}atm]$ and 11.5. After plasma treatment, the changes of membrane surface were all subtly linked with both cross-linking and etching effects. Finally, it was confirmed that the gas permeation capacity and selectivity of the modified membrane with plasma could be improved by an appropriate control of the plasma conditions such as treatment time, the power input and sort of plasma gas.

• Korean Chemical Engineering Research
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• v.44 no.6
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• pp.659-668
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• 2006

The optimization of supercritical water oxidation (SCWO) process for decomposing nitromethane was studied by means of a design of experiments. The optimum operating region for the SCWO process to minimize COD and T-N of treated water was obtained in a lab scale unit. The authors had compared the results from a SCWO pilot plant with those from a lab scale system to explore the problems of scale-up of SCWO process. The COD and T-N in treated waters were selected as key process output variables (KPOV) for optimization, and the reaction temperature (Temp) and the mole ratio of nitromethane to ammonium hydroxide (NAR) were selected as key process input variables (KPIV) through the preliminary tests. The central composite design as a statistical design of experiments was applied to the optimization, and the experimental results were analyzed by means of the response surface method. From the main effects analysis, it was declared that COD of treated water steeply decreased with increasing Temp but slightly decreased with an increase in NAR, and T-N decreased with increasing both Temp and NAR. At lower Temp as $420{\sim}430^{\circ}C$, the T-N steeply decreased with an increase in NAR, however its variation was negligible at higher Temp above $450^{\circ}C$. The regression equations for COD and T-N were obtained as quadratic models with coded Temp and NAR, and they were confirmed with coefficient of determination ($r^2$) and normality of standardized residuals. The optimum operating region was defined as Temp $450-460^{\circ}C$ and NAR 1.03-1.08 by the intersection area of COD < 2 mg/L and T-N < 40 mg/L with regression equations and considering corrosion prevention. To confirm the optimization results and investigate the scale-up problems of SCWO process, the nitromethane was decomposed in a pilot plant. The experimental results from a SCWO pilot plant were compared with regression equations of COD and T-N, respectively. The results of COD and T-N from a pilot plant could be predicted well with regression equations which were derived in a lab scale SCWO system, although the errors of pilot plant data were larger than lab ones. The predictabilities were confirmed by the parity plots and the normality analyses of standardized residuals.