Showing papers in "Biotechnology and Bioprocess Engineering in 2004"
TL;DR: From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes and was co-amplified to solve the problem of malic acid accumulation.
Abstract: Apfl ldhA double mutantEscherichia coli strain NZN111 was used to produce succinic acid by overexpressing theE. coli malic enzyme gene (sfcA). This strain, however, produced a large amount of malic acid as well as succinic acid. After the analyses of the metabolic pathways, thefumB gene encoding the anaerobic fumarase ofE. coli was co-amplified to solve the problem of malic acid accumulation. A plasmid, pTrcMLFu, was constructed, which contains an artificial operon (sfcA-fumB) under the control of the inducibletrc promoter. From the batch culture of recombinantE. coli NZN111 harboring pTrcMLFu, 7 g/L of succinic acid was produced from 20 g/L of glucose, with no accumulation of malic acid. From the metabolic flux analysis the strain was found under reducing power limiting conditions by severe reorientation of metabolic fluxes.
61 citations
TL;DR: The nanoscale fabrication of biomolecular film and its application to bioelectronic devices and biochips is described and its applications to biodevices are described.
Abstract: Biodevices composed of biomolecular layer have been developed in various fields such as medical diagnosis, pharmaceutical screening, electronic device, photonic device, environmental pollution detection device, and etc. The biomolecules such as protein, DNA and pigment, and cells have been used to construct the biodevices such as biomolecular diode, biostorage device, bioelectroluminescence device, protein chip, DNA chip, and cell chip. Substantial interest has focused upon thin film fabrication or the formation of biomaterials mono- or multi-layers on the solid surfaces to construct the biodevices. Based on the development of nanotechnology, nanoscale fabrication technology for biofilm has been emerged and applied to biodevices due to the various advantages such as high density immobilization and orientation control of immobilized biomolecules. This review described the nanoscale fabrication of biomolecular film and its application to bioelectronic devices and biochips.
55 citations
TL;DR: The development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE.
Abstract: The supra molecular weight poly ([R]-3-hydroxybutyrate) (PHB), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da However, applications for this PHB have been hampered due to the difficulty of its production Reported here, is the development of a new metabolically engineeredEscherichia coli strain and its fermentation for high level production of supra molecular weight PHB RecombinantE coli strains, harboring plasmids of different copy numbers containing theAlcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared When the recombinantE coli XL 1-Blue, harboring a medium-copy-number pJC2 containing theA latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 60, supra molecular weight PHB could be produced at up to 898 g/L with a productivity of 207 g PHB/L-h The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced inRalstonia eutropha or recombinantE coli
49 citations
TL;DR: Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by the Pseudomonas aeruginosa, BYK-2 KCTC 18012P.
Abstract: The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).
46 citations
TL;DR: The conversion ratio of Cel+ cells toCel− mutants was strongly related to the production of bacterial cellulose independently from the cell growth, and the additions of organic acid and ethanol to a flask without slanted baffles induced the conversion of microbial cells from a wild type to Cel− mutants in a flask culture.
Abstract: The conversion of a cellulose-producing cell (Cel+) fromGluconacetobacter hansenii PJK (KCTC 10505 BP) to a non-cellulose-producing cell (Cel−) was investigated by measuring the colony forming unit (CFU). This was achieved in a shaking flask with three slanted baffles, which exerted a strong shear stress. The addition of organic acid, such as glutamic acid and acetic acid, induced the conversion of microbial cells from a wild type toCel− mutants in a flask culture. The supplementation of 1% ethanol to the medium containing an organic acid depressed the conversion of the microbial cells toCel− mutants in a conventional flask without slanted baffles. The addition of ethanol to the medium containing an organic acid; however, accelerated the conversion of microbial cells in the flask with slanted baffles. TheCel+ cells from the agitated culture were not easily converted intoCel−, mutants on the additions of organic acid and ethanol to a flask without slanted baffles, but some portion of theCel+ cells were converted toCel− mutants in a flask with slanted baffles. The conversion ratio ofCel+ cells toCel− mutants was strongly related to the production of bacterial cellulose independently from the cell growth.
39 citations
TL;DR: The results clearly demonstrate that the pectin microspheres that were prepared by the spray drying and crosslinking methods are potential carriers for colon-specific drug deliveries.
Abstract: Drug delivery systems that are based on pectin have been studied for colon specific delivery using the specific activity of colon microflora. The aim of this study was to design a novel method of manufacturing pectin microspheres without oils and surfactants and to investigate the potential use of the pectin microsphere as an oral colon-specific drug carrier. The pectin microspheres were successfully formed using the spray drying method and crosslinking with calcium chloride. From the crosslinked pectin microspheres, indomethancin (IND) release was more supressed than its release from non-crosslinked microspheres. In a low pH (pH 1.4) environment, the pectin microspheres released IND at an amount of about 18±2% of the total loaded weight for 24h while the release rate of IND was stimulated at neutral pH (pH 7.4). IND release from the pectin microspheres was increased by the addition of pectinase. The results clearly demonstrate that the pectin microspheres that were prepared by the spray drying and crosslinking methods are potential carriers for colon-specific drug deliveries.
38 citations
TL;DR: Results indicate that enzymatic decolorization of melanin has applications in the development of new cosmetic whitening agents.
Abstract: Melanin was decolorized by lignin peroxidase fromPhanerochaete chrysosporium. This decolorization reaction showed a Michaelis-Mentens type relationship between the decolorization rate and concentration of two substrates: melanin and hydrogen peroxide. Kinetic constants of the decolorization reaction were 0.1 OD475/min (V max) and 99.7 mg/L (K m) for melanin and 0.08 OD475/min (V max) and 504.9 μM (K m) for hydrogen peroxide, respectively. Depletion of hydrogen peroxide interrupted the decolorization reaction, indicating the essential requirement of hydrogen peroxide. Pulsewise feeding of hydrogen peroxide continued the decolorizing reaction catalyzed by lignin peroxidase. These results indicate that enzymatic decolorization of melanin has applications in the development of new cosmetic whitening agents.
37 citations
TL;DR: Ethanol extracts of PFF were the most prominent antitumor agents toward lung cancer cells (A549) and induced synergistic effects on the TRAIL-induced apoptosis in A549 cells, which were strongly resistant to TRAIL.
Abstract: When SiHa cells were incubated for varying periods of time with extracts of PFF and PFM, the cytotoxicity of the ethanol extracts of PFF was higher than those of the other extracts. These results indicated that the extracts from fruiting bodies ofP. ferulae contain antitumor substances. When A549, SiHa and HeLa cells were incubated with different concentrations of PFF and PFM extracts, the ethanol extracts of PFF showed strong cytotoxicity against A549 cells at concentrations over 10 μg/mL and against SiHa and HeLa cells at concentrations over 40 μg/mL. However, the differences in the cytotoxic effects of the hot water and ethanol extracts of PFM and the hot water extracts of PFF on all 3 cancer cells were not significant. Also, the PFF ethanol extracts induced synergistic effects on the TRAIL-induced apoptosis in A549 cells, which were strongly resistant to TRAIL. These results indicated that ethanol extracts of PFF were the most prominent antitumor agents toward lung cancer cells (A549).
33 citations
TL;DR: Observations indicated that C. militaris behaves as a bipolar heterothallic fungus and requires two mating compatible strains in order to produce regular clubshaped perithecial stromata, a fundamental requirement for its industrial cultivation.
Abstract: Interest inin vitro study of entomopathogenic fungi, includingCordyceps species, has been increasing due to their valuable bioactive compounds and biocontrol effects. AmongCordyceps species,in vitro stromata ofC. militaris has been successfully produced and cultivated for industrial purposes. However, genetic study onin vitro stromata formation ofC. militaris has not been carried out yet. Here, relationship between mating system and perithecial stromata formation ofC. militaris is reported. Mating system was determined by observing perithecial stromata formation from mono-ascospore cultures and their pair-wise combinations. Certain combinations of mono-ascospore strains produced perithecial club-shaped stromata, whereas other combinations produced either no stromata or only abnormal non-perithecial stromata. Similarly, monoascospore cultures without combination produced either no stromata or only abnormal nonperithecial stromata. Despite obvious heterothallism, self-fertility was occasionally observed in few strains ofC. militaris. These observations indicated thatC. militaris behaves as a bipolar heterothallic fungus and requires two mating compatible strains in order to produce regular clubshaped perithecial stromata, a fundamental requirement for its industrial cultivation.
31 citations
TL;DR: To confirm the chemical features of cellulose, various spectrophtometeric analysis were carried out using electron microscopy, X-ray diffractogram, and CP/MAS13C NMR, and the purified cellulose was found to be identical to that of Acetobacter xylinum.
Abstract: Gluconobacter oxydans that produces the cellulose was isolated. In order to confirm the chemical features of cellulose, various spectrophtometeric analysis were carried out using electron microscopy, X-ray diffractogram, and CP/MAS13C NMR. The purified cellulose was found to be identical to that ofAcetobacter xylinum. For effective production of cellulose, the various carbon and nitrogen sources, mixture of calcium and magnesium ions, and biotin concentration were investigated in flask cultures. Among the various carbon sources, glucose and sucrose were found to be best for the production of cellulose, with maximum concentration of 2.41 g/L obtained when a mixture of 10 g/L of each glucose and sucrose were used. With regard to the nitrogen sources, when 20 g/L of yeast extract was used, the maximum concentration of bacterial cellulose was reached. The concentration of cellulose was increased with mixture of 2 mM of each Ca2+ and Mg2+. The optimum biotin concentration for the production of cellulose was in the range of 15 to 20 mg/L. At higher biotin concentration (25–35 mg/L), the bacterial cellulose production was lower.
31 citations
TL;DR: In this paper, a predispersed solvent extraction (PDSE) of succinic acid with Tri-n-octylamine (TOA) dissolved in 1-octanol was examined.
Abstract: Predispersed solvent extraction (PDSE) of succinic acid with Tri-n-octylamine (TOA) dissolved in 1-octanol from aqueous solutions of 50 g/L succinic acid was examined. It was found that the equilibrium data in PDSE was equal to that in conventional solvent extraction in spite of the lack of mechanical mixing in PDSE. The influence of salts on succinic acid extraction and the stability of colloidal liquid aphrons (CLAs) were also investigated. Results indicated that in the presence of sodium chloride, less succinic acid was extracted by CLAs and the stability of CLAs decreased. However, the stability of CLAs was sufficient to make PDSE practically applicable to real fermentation broth, considering the concentration range of salts in the fermentation process for succinic acid.
TL;DR: In this article, the three known compounds sargahydroquinoic acid (1), sargaquinoic acids (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine.
Abstract: Peroxynitrite formationin vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products for their treatment. The three components (1–3) isolated fromSargassum thunbergii as well as the organic solventsoluble fractions and the aqueous layer ofS. thunbergii were evaluated for their potential to scavenge authentic ONOO and ONOO derived from 3-morpholinosydnonimine (SIN-1). The antioxidant activity of the individual fractions was in the order of 85% aquaous (aq.) MeOH>n-BuOH>n-hexane> H2O. The three known compounds sargahydroquinoic acid (1), sargaquinoic acid (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine. These results showed a possible antioxidant activity in major constituents ofS. thunbergii.
TL;DR: Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics.
Abstract: In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.
TL;DR: In this paper, a low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay.
Abstract: A low-cost, simple strip reader system using a linear movement mechanism of CD-ROM deck has been developed to characterize a lateral flow membrane-based immunochromatographic assay. The test strip reader was assembled by a CD-ROM deck and home-made optical head especially designed for immunoassays. The optical head for detecting reflected light from the test strip surface consists of green light-emitting diode, large area silicon photodiode, and anodized aluminum mounting block providing a slit structure for cutting light from the LED. The stepping motor of the deck was operated in the full step mode, whose distance of each reading point is about 0.15 mm. The performance of the strip reader was tested by analysis of HBV (hepatitis B virus) antigen test kit. This strip reader can be useful for inexpensive, disposable, and membrane-based assays that provide visual evidence of the presence of an analyte in a liquid sample.
TL;DR: In this paper, the authors formulated a sustained release system for indomethacin (IND) with rosin gum obtained from a pine tree. But, they did not investigate the influence of solvents on the formation of colloidal microparitcles.
Abstract: The aim of this study was to formulate a sustained release system for indomethacin (IND) with rosin gum obtained from a pine tree. Rosin microparticles were prepared by a dispersion and dialysis method without the addition of surfactant. In order to investigate the influence of solvents on the formation of colloidal microparitcles, various solvents like ethanol, DMF, DMAc, and acetone were used. The rosin microparticles containing IND were characterized by X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). The morphologies of rosin microparticles observed by scanning electron microscopy (SEM) were spherical. The solvents used to dissolve rosin significantly affected the drug content and drug release rate of IND. The release behaviors of IND from the rosin microparticles were dependent on the drug content and size of the particles. Rosin microparticles with a higher drug content and of a larger particle size had a slower drug release rate. Also, the IND release rate from the rosin microparticles could be regulated by the rosin content in the microparticles. From these results, rosin microparticles have the potential of being used as a sustained release system of IND.
TL;DR: This study reviews the methods and strategies for the development of DNA microarray for pathogen detection, with the focus on probe design.
Abstract: Pathogens pose a significant threat to humans, animals, and plants. Consequently, a considerable effort has been devoted to developing rapid, convenient, and accurate assays for the detection of these unfavorable organisms. Recently, DNA-microarray based technology is receiving much attention as a powerful tool for pathogen detection. After the target gene is first selected for the unique identification of microorganisms, species-specific probes are designed through bioinformatic analysis of the sequences, which uses the information present in the databases. DNA samples, which were obtained from reference and/or clinical isolates, are properly processed and hybridized with species-specific probes that are immobilized on the surface of the microarray for fluorescent detection. In this study, we review the methods and strategies for the development of DNA microarray for pathogen detection, with the focus on probe design.
TL;DR: The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was investigated using recombinant Pichia pastoris with the enantiOSElective epoxide hydrolase of Rhodotorula glutinis and the rate was enhanced by the addition of 5% (v/v) Tween 20.
Abstract: The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was investigated using recombinantPichia pastoris with the enantioselective epoxide hydrolase ofRhodotorula glutinis The rate of the recombinant epoxide hydrolase-catalyzed enantioselective hydrolysis of racemic epichlorohydrins was enhanced by the addition of 5% (v/v) Tween 20 Enantiopure (R)-epichlorohydrins with an enantiopurity of 100%ee and a yield of 26% were obtained within 5 min from 50 mM racemates
TL;DR: The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate at pH 7 and lowest growth at pH 5.927 h−1.
Abstract: The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0927 h−1 The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0927 h−1) at pH 7 and lowest growth at pH 5 The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5 There is about 28% different of the protein yield for theE coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C
TL;DR: In this paper, a 50% yield of xylitol was obtained by using the beech wood hydrolyzed solution with the addition of 1% yeast extract and 1% glucose at an initial concentration.
Abstract: Agricultural waste products, beech wood and walnut shells, were hydrolyzed at 40°C using mixed crude enzymes produced byPenicillium sp. AHT-1 andRhizomucor pusillus HHT-1.d-xylose, 4.1 g and 15.1 g was produced from the hydrolysis of 100 g of beech wood and walnut shells, respectively. For xylitol production,Candida tropicalis IFO0618 and the waste product hydrolyzed solutions were used. The effects on xylitol production, of adding glucose as a NADPH source,d-xylose and yeast extract, were examined. Finally, a 50% yield of xylitol was obtained by using the beech wood hydrolyzed solution with the addition of 1% yeast extract and 1% glucose at an initial concentration.
TL;DR: In the case of scCO2 modified with 70% aqueous methanol, the licorice tissue obtained after extraction was found to be severely degraded by excessive swelling, and the absolute density of the Licorice residues was observed to be the highest.
Abstract: Optimal conditions for the supercritical carbon dioxide (scCO2) extraction of glycyrrhizin from licorice (Glycyrrhiza glabra) were investigated, with an emphasis on the types and levels of modifiers. The morphology of the licorice tissue remaining after the scCO2 extraction of glycyrrhizin was examined by scanning electron microscopy, coupled with measurements of absolute density. Conventional organic solvent extraction was also carried out for purpose of quantitative comparison. At 50 MPa and 60°C glycyrrhizin could not be extracted with pure scCO2, while a considerable amount of glycyrrhizin was extracted when water was added to scCO2 as a modifier. The highest recovery was found to be about 97% when 70% aqueous methanol was added to scCO2 at a concentration of 15%. The optimal pressure and temperature for the supercritical fluid extraction of glycyrrhizin were observed to be 30 MPa and 60°C, respectively. Under these conditions, the percentage recovery of glycyrrhizin attained a maximum value of 102.67±1.13% within 60 min. Furthermore, in the case of scCO2 modified with 70% aqueous methanol, the licorice tissue obtained after extraction was found to be severely degraded by excessive swelling, and the absolute density of the licorice residues was observed to be the highest.
TL;DR: The aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite) and identify the microorganisms involved as CandidatusB.
Abstract: We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH4/+-N/L) and different nitrite concentrations (50∼150 mg NO2-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.
TL;DR: In this paper, a simulated moving bed (SMB) chromatography system was used to separate a racemic mixture of ketoprofen in to its enantiomers, achieving a purity of 96% under the calculated operating conditions.
Abstract: A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.
TL;DR: In this paper, the authors investigated the total Oligomeric proanthocyanidin contents and total antioxidant activity of wild grape seeds and developed an efficient extraction process with various temperatures, solvent compositions and times.
Abstract: The Oligomeric proanthocyanidin (OPC) in green and black tea, grape seeds, grapes and wine has raised much attention but that OPC in wild grape seed remains to be intensively investigated. This study investigated the total OPC contents and total antioxidant activity of wild grape seeds and developed an efficient extraction process with various temperatures, solvent compositions and times. Also, a chromatography column packed with the Dia-ion HP-20 resin was used for further purification of the OPC. The total OPC contents were determined with the Folin-Ciocalteu reagent, and the antioxidant activity using total antioxidant potential (TAP) and 1,1-diphenyl-2picrylhydrazyl (DPPH). The yield of final purified OPC was 1.78 (+)-catechin equivalent (CE) g/100 g, with IC50 activities of TAP and DPPH of 31.60 and 15.70 μg/mL. These activities of the final purified OPC were about two times higher than that of the BHA used as a reference sample.
TL;DR: The effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient were examined.
Abstract: Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andKhGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andKhGM-CSF of 7.64 after 2 h of incubation at room temperature.
TL;DR: The A. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen.
Abstract: AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×106 conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.
TL;DR: In this paper, the separation of two amino acids, phenylalanine and tryptophan, was carried out using laboratory simulated moving bed (SMB) chromatography.
Abstract: The separation of two amino acids, phenylalanine and tryptophan, was carried out using laboratory simulated moving bed (SMB) chromatography. The SMB process consisted of four zones, with each zone having 2 columns. The triangle theory was used to obtain the operating conditions for the SMB. The mass transfer coefficients of the two amino acids were obtained from the best-fit values by comparing simulated and experimental pulse data. The competitive adsorption isotherms of the two amino acids were obtained by single and binary frontal analyses, taking into consideration the competition between the two components. A competitive Langmuir isotherm, obtained from single-component frontal chromatography, was used in the first run, and the isotherm from binary frontal chromatography in the second, with the flow rate of zone I modified to improve the purity. Compared to the first and second runs, the competitive Langmuir isotherm from the binary frontal chromatography showed good agreement with the experimental results. Also, adjusting the flow rate in zone I increased the purity of the products. The purities of the phenylalanine in the raffinate and the tryptophan in the extract were 99.84 and 99.99%, respectively.
TL;DR: Using the WSM (water-soluble fraction of a methanol extract from Ulva lactuca) to inhibit 50% of the human leukemia cell line U937 in growth, while splenocyte growth was stimulated up to a concentration of 100 μg/mL.
Abstract: This is the first report on the antitumor and immunostimulating activities ofUlva lactuca Using the WSM (water-soluble fraction of a methanol extract fromUlva lactuca), a concentration of 140 g/mL was found to inhibit 50% of the human leukemia cell line U937 in growth, while splenocyte growth was stimulated up to a concentration of 100 μg/mL In addition, NO production by a macrophage cell line (RAW 2647) and alkaline phosphatase activity in mouse splenocytes were both stimulated with 10 μg/mL of WSM Dose-dependent patterns were observed on all three cell-lines Accordingly, the current results indicate thatUlva lactuca may be useful as a natural antitumor and immunostimulating agent
TL;DR: The results suggest that this strategy, which consists of a combination of μμCP and cSA-fused proteins, is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications, one such being the use in protein-protein assays.
Abstract: In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (μCP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene ofStreptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of μμCP and cSA-fused proteins, is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications, one such being the use in protein-protein assays.
TL;DR: In this article, a bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory and identified as Paracoccus sp. WJ98 based on its 16S rRNA sequence analysis, morphological and physiological characteristics.
Abstract: A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K2HPO4, 0.04% KH2PO4, and 0.01% MgCl2·6H2O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn2+ and Hg2+. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.
TL;DR: The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures of Ginkgo biloba were investigated and callus induced from the leaf petioles exhibited distinct morphological and physiological responses.
Abstract: The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.