Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.
The putative EPA synthesis gene cluster was mined from the entire genome sequence of Shewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO1600 (acyl transferase, AT), ORFSO1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO1597 (enoyl reductase, ER), ORFSO1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified from S. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genes orfSO1597 and orfSO1604. The DNA fragment was cloned into Escherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in four E. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in the E. coli clone was affected by cultivation temperature, showing maximum yield at $20^{\circ}C$ and no production at $30^{\circ}C$ or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.
Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.
Hyean-Yeol Park;Sun Hyu Kim;Jeong-Hoon Yu;Ji Eun Kwon;Ji Yang Lim;Si Won Choi;Jong-Sung Yu;Yongju Jung
Journal of Electrochemical Science and Technology
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v.15
no.1
/
pp.198-206
/
2024
Given the high theoretical capacity (1,675 mAh g-1) and the inherent affordability and ubiquity of elemental sulfur, it stands out as a prominent cathode material for advanced lithium metal batteries. Traditionally, sulfur was sequestered within conductive porous carbons, rooted in the understanding that their inherent conductivity could offset sulfur's non-conductive nature. This study, however, pivots toward a transformative approach by utilizing diatom shell (DS, diatomite)-a naturally abundant and economically viable siliceous mineral-as a sulfur host. This approach enabled the development of a sulfurlayered diatomite/S composite (DS/S) for cathodic applications. Even in the face of the insulating nature of both diatomite and sulfur, the DS/S composite displayed vigorous participation in the electrochemical conversion process. Furthermore, this composite substantially curbed the loss of soluble polysulfides and minimized structural wear during cycling. As a testament to its efficacy, our Li-S battery, integrating this composite, exhibited an excellent cycling performance: a specific capacity of 732 mAh g-1 after 100 cycles and a robust 77% capacity retention. These findings challenge the erstwhile conviction of requiring a conductive host for sulfur. Owing to diatomite's hierarchical porous architecture, eco-friendliness, and accessibility, the DS/S electrode boasts optimal sulfur utilization, elevated specific capacity, enhanced rate capabilities at intensified C rates, and steadfast cycling stability that underscore its vast commercial promise.
This study was conducted for searching the functional feeding groups, and community stability of the macroinvertebrate on forest fire area of Uljin-gun. The samples were collected from 2 sites of control area and 2 sites of experimental area during 2007 to 2009. The identified species were 89 belonged to 47 families, 16 order, 6 class, and 4 phylum in control area. And 84 belonged to 43 families, 16 order, 6 class, and 4 phylum were identified in experimental area. As a result of changes in species and individuals of E.P.T. taxa investigated in experimental area by year, Ephemeroptera was 21 species, $2,434.6inds./m^2$, Plecoptera was 3 species, $199.8inds./m^2$, and Trichoptera 14 species, $540.2inds./m^2$ in 2007. And in 2009, Ephemeroptera was 9 species, $296inds./m^2$, Trichoptera was 4 species, $44.4inds./m^2$, and Plecoptera was none, showing that species and individuals belonging to E.P.T. taxa decrease rapidly every year. Community analysis by year, in 2008 when the water system started to be influenced by the fire directly, it showed a trend that H' and RI decreased in the experimental area. Functional feeding group by year, it showed a trend that species and individuals of GC type which is a functional group picking up and eating FPOM (fine particulate organic matter) from deposits in the bottom of water or benthic areas and performs an important function of material circulation in ecosystem decrease every year. Community stability by year, an environment of water system in forest fire area started to be somewhat destroyed, from 2008, it is shown that both species in I area which have great ability of resistance and recovery and species in III area which live in relatively stable water system decreased a little.
The Muryangsa temple five-storied stone pagoda (Treasure No. 185) was geographically located in the area of the Baekje Kingdom. The architectural style of the Muryangsa temple five-storied stone pagoda is the pagoda at the early Goryeo Dynasty that was succeeded technique of the Baekje Kingdom and form of the Shilla Kingdom. Because this pagoda is located outside during old time that it received serious petrological and biological weathering in rock blocks and occurred the center subsidence in the upper capstone. This study executed ground stability interpretation in order to know what central subsidence in the upper capstone occurred for soft ground. The ground stability interpretation used seismic survey, electrical resistivity survey and GPR survey by non-destructive method. As the result, the ground appeared in the condition which is good. Specially, high resistance zone appeared from electric resistivity survey which come to seem with ground reinforcement harden. Consequently, central subsidence condition in the upper capstone is not by the instability of ground, and is judged with the thing by the structure instability in rock blocks over the upper capstone. This will be applied basic data with the long-term monitoring or preservation countermeasure of the pagoda.
Choi, Hae Won;Park, Young Seok;Chung, Shin Hye;Jung, Min Ho;Moon, Won;Rhee, Sang Hoon
The korean journal of orthodontics
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v.47
no.4
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pp.229-237
/
2017
Objective: The aim of this study was to compare the initial stability as insertion and removal torque and the clinical applicability of novel orthodontic zirconia micro-implants made using a powder injection molding (PIM) technique with those parameters in conventional titanium micro-implants. Methods: Sixty zirconia and 60 titanium micro-implants of similar design (diameter, 1.6 mm; length, 8.0 mm) were inserted perpendicularly in solid polyurethane foam with varying densities of 20 pounds per cubic foot (pcf), 30 pcf, and 40 pcf. Primary stability was measured as maximum insertion torque (MIT) and maximum removal torque (MRT). To investigate clinical applicability, compressive and tensile forces were recorded at 0.01, 0.02, and 0.03 mm displacement of the implants at angles of $0^{\circ}$, $10^{\circ}$, $20^{\circ}$, $30^{\circ}$, and $40^{\circ}$. The biocompatibility of zirconia micro-implants was assessed via an experimental animal study. Results: There were no statistically significant differences between zirconia micro-implants and titanium alloy implants with regard to MIT, MRT, or the amount of movement in the angulated lateral displacement test. As angulation increased, the mean compressive and tensile forces required to displace both types of micro-implants increased substantially at all distances. The average bone-to-implant contact ratio of prototype zirconia micro-implants was $56.88{\pm}6.72%$. Conclusions: Zirconia micro-implants showed initial stability and clinical applicability for diverse orthodontic treatments comparable to that of titanium micro-implants under compressive and tensile forces.
KIM, HWA-JIN;CHO, YOUNG-HEE;BAE, EUN-KYUNG;SHIN, TAEK-SU;CHOI, SUNG-WON;CHOI, KEE-HYUN;PARK, JI-YONG
Journal of Microbiology and Biotechnology
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v.15
no.1
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pp.29-34
/
2005
W/O/W (water-in-oil-in-water) type multiple emulsion was applied to improve the storage stability of an antagonistic microorganism, Burkholderia gladioli. Encapsulation of microorganism into a W/O/W emulsion was conducted by using a two-step emulsification method. W/O/W emulsion was prepared by the incorporation of B. gladioli into rapeseed oil and the addition of polyglycerin polyriconolate (PGPR) and castor oil polyoxyethylene (COG 25) as the primary and secondary emulsifier, respectively. Microcrystalline cellulose was used as an emulsion stabilizer. To evaluate the usefulness of W/O/W emulsion formulation as a microbial pesticide for controlling the bacterial wilt pathogen (Ralstonia solanacearum), the storage stability and antagonistic activity of emulsion formulation were tested in vitro. The storage stability test revealed that the viability of formulated cells in emulsion was higher than that of unformulated cells in culture broth. At $4^{\circ}C$, the viabilities of formulated cells and unformulated cells at the end of 20 weeks decreased to about 2 and 5 log cycles, respectively. At $37^{\circ}C$, the viability of formulated cells decreased to only 2 log cycles at the end of storage. On the other hand, the viable cells in culture broth were not detected after 13 weeks. In activity test, formulated cells in emulsion were more effective in inhibiting the growth of pathogen than unformulated cells in culture broth. Unformulated cells completely lost their antagonistic activity during storage under similar conditions. The W/O/W multiple emulsion formulation was shown to be useful as the novel liquid formulation for biological control.
Objective: The aim of this study was to investigate the periodicity of the lower extremity joint flexion/extension angle to compare the local stability between young and elderly women during walking on a treadmill. Method: Eighteen young women (mean $age=21.2{\pm}1.6y$; mean $mass=57.1{\pm}6.1kg$; mean $height=1.61{\pm}0.04m$) and 18 elderly women (mean $age=66.4{\pm}1.2y$; mean $mass=55.4{\pm}8.3kg$; mean $height=1.56{\pm}0.04m$) participated in this study. Approximate entropy (ApEn) was used to determine the periodicity in the lower limb joint angles. Results: The ApEn values of the two groups were statistically greater in the surrogate data test than in the original time series data (p<.05). The periodicity of the hip and ankle flexion/extension angles decreased in the elderly women group compared with the young women group (p<.05). The periodicity of the lower extremity joint flexion/extension angle showed that the ankle joint increased dominatingly in both groups (p<.05); the hip joint decreased compared with the knee joint in the young women group; and the knee joint decreased compared with the hip joint in the elderly women group (p<.05). Conclusion: These results suggest that the lower extremity joint flexion/extension angles of the young and elderly women during walking contained random noises as well as biological signals. In addition, the differences in the periodicity in the lower extremity joint between the young and elderly women may provide some insight in predicting potential falls and be used as a characteristic indicator for determining local stability in elderly women during walking.
Kim, Sun-Hyung;Ji, Yeun-Sun;Lee, Eui-Seok;Hong, Soon-Taek
Preventive Nutrition and Food Science
/
v.21
no.3
/
pp.289-295
/
2016
Curcumin is a flavonoid found in the rhizome of the turmeric plant (Curcuma longa L.) and has recently attracted interest because it has numerous biological functions and therapeutic properties. In the present study, we attempted to incorporate curcumin into medium-chain triglyceride (MCT) nanoemulsions (0.15 wt% curcumin, 10 wt% MCT oil, and 10 wt% emulsifiers) with various emulsifiers [polyoxyethylene (20) sorbitan monolaurate (Tween-20), sorbitan monooleate (SM), and soy lecithin (SL)]. The physicochemical properties of the nanoemulsions including the Ostwald ripening stability were investigated. The initial droplet size was found to be 89.08 nm for the nanoemulsion with 10 wt% Tween-20 (control), and when Tween-20 was partially replaced with SM and SL, the size decreased: 73.43 nm with 4 wt% SM+6 wt% Tween-20 and 67.68 nm with 4 wt% SL+6 wt% Tween-20 (prepared at 15,000 psi). When the nanoemulsions were stored for 28 days at room temperature, the droplet size increased as the storage time increased. The largest increase was observed for the control nanoemulsion, followed by the 4 wt% SL+6 wt% Tween-20 and 4 wt% SM+6 wt% Tween-20 systems. The Turbiscan dispersion stability results strongly supported the relationship between droplet size and storage time. The time-dependent increase in droplet size was attributed to the Ostwald ripening phenomenon. Thus, the Ostwald ripening stability of curcumin-loaded MCT nanoemulsions with Tween-20 was considerably improved by partially replacing the Tween-20 with SM or SL. In addition, curcumin may have acted as an Ostwald ripening inhibitor.
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