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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal DOI :
Korean Society for Biotechnology and Bioengineering
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Volume & Issues
Volume 16, Issue 6 - Dec 2001
Volume 16, Issue 5 - Oct 2001
Volume 16, Issue 4 - Aug 2001
Volume 16, Issue 3 - Jun 2001
Volume 16, Issue 2 - Apr 2001
Volume 16, Issue 1 - Feb 2001
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Improved Technologies to Produce Heterologous Proteins in Recombinant Escherichia coli.
KSBB Journal, volume 16, issue 1, 2001, Pages 1~10
Escherichia coli has been used as an expression work horse for foreign genes. This article summarized recent development in genetic engineering techniques for overproduction of medical proteins and industrial enzymes. Special emphasis was placed upon research activities concerning folding and refolding of inclusion bodies at genetic and fermentation levels. Plasmid and mRNA stabilization, development of strong inducible promoters, modification of translational elements and reduction of rpoteolytic degradation were carried out to elevate an expression level of a target protein. Optimization of culture conditions, improvement of denaturation and renaturation steps and coexpression of molecular chaperones or foldase were accomplished to produce active proteins in soluble form. Fusion protein systems with selective separation and surface display technology were also performed in an effort to make the E. coli expression system more effective and versatile.
Improved Large-Scale Refolding Techniques for Inclusion Body Proteins
KSBB Journal, volume 16, issue 1, 2001, Pages 11~14
Techniques for protein refolding from inclusion body are discussed in view of its engineering application to large scale protein purification. Among the techniques, dilution and dialysis are mainly utilized due to simple operation. Membrane reactor, gel filtration chromatography, and continuous tank operation are emerging tools for their process-scale possibility in refolding. Reaction engineering approaches could be used to analyze the kinetic behaviour in the process scale refolding reactor. The kinetic analysis is helpful in the optimization of refolding yield in the refolding reactor.
Methylotrophic Yeasts as a New Host for Heterologous Protein Expression
KSBB Journal, volume 16, issue 1, 2001, Pages 15~23
The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.
In Vitro Performance Assessment of a Bioartificial Liver Packed with Polyurethane Foam
KSBB Journal, volume 16, issue 1, 2001, Pages 24~29
Recently hepatocyte-based bioartificial liver (BAL) and hepatocyte transplantation have been actively investigated to treat acute hepatic failure. The BAL acts as a bridge to provide patients with more time until a donor organ becomes available for transplantation or until their own liver can be regenerated. In this study, we manufactured a polyurethane foam (PUF) using 15% NCO-prepolymer with a pore opening that allows it to be used as a hepatocyte immobilizing material. Cubes of PUF (3 mm dim.) were seeded with rat primary hepatocytes at a density of 5.5
PUF by centrifuging them together. The cell laden PUF cubes were packed into a prototype reactor and perfused with a hormonally defined medium for a week. Hepatocytes in the pores of the PUF formed spheroids that showed stable ammonia removal and urea synthesis activities. The albumin production level was comparable to other BAL systems. The PUF packed hepatocyte bioreactor has the potential to be used as a BAL.
Growth Kinetics and Sporulation of Bacillus thuringiensis in High Cell Density Culture
KSBB Journal, volume 16, issue 1, 2001, Pages 30~35
High cell density culture of Bacillus thuringiensis was conducted in fed-batch culture and TCRC using a bioreactor incorporating ceramic membrane filter. Cell growth of B. thuringiensis in fed-batch culture increased linearly, which was well matched by the results of cell growth modeling. In spite of the slower growth rate during fed-batch culture, no spore formation was observed, which was contrary to the results of continuous culture. Changing culture mode to batch culture after fed-batch operation induced a 2.7
CFU/mL spore concentration using a 300 g/L glucose feed concentration. In TCRC operation incorporating ceramic filter within the bioreactor, the effect of glucose feed concentrations on the cell growth and spore formation of B. thuringiensis was determined. A maximum cell concentration of 1.8
CFU/ml, which corresponds to 82.6 g-cell/L, was obtained in the TCRC using a 50 g/L glucose feed concentration. In the TCRC, cell growth increased linearly and glucose concentration was limited, which agreed well with the results of cell growth modeling. No spore formation was observed except when 1 g/L of glucose was fed. Changing to batch culture induced a 1.2
CFU/mL of spore concentration, which was the highest spore concentration obtained among the various culture modes examined. The optimal glucose feed rate was found to be 0.55 g-glucose/h.
Cloning and Nucleotide Sequencing of a Partial Glutamate Decarboxylase Gene from Arabidopsis thaliana cDNA Library
KSBB Journal, volume 16, issue 1, 2001, Pages 36~40
In order to study the molecular mechanism of
-aminobutyric acid (GABA) production in plants, we cloned and sequenced a partial glutamate decarboxylase (GAD) cDNA from the Arabidopsis thaliana cDNA library, using primers targeted at highly conserved sequences of the petunia GAD gene. The cDNA fragment was inserted into TA cloning vector with T7 promoter and the recombinant plasmid obtained was used to transform E. coli. The plasmid DNA purified from the transformed E. coli was digested with EcoRI and the presence of the insert was confirmed. Nucleotide sequence analysis showed that the fragment is a partial Arabidopsis thaliana GAD gene and that the sequence showed 98% and 78% identity to the region of the putative Arabidopsis thaliana GAD sequences deposited in GenBank, Accession nos: U46665 and U10034, respectively. The amino acid sequence deduced from the partial Arabidopsis thaliana GAD gene showed 99% and 91% identities to the GAD sequences deduced from the genes of the U46665 and U10034, respectively. The partial cDNA sequence determined may facilitate the study of the molecular mechanism of GABA metabolism in plants.
Studies on Adherance Inhibition and Detachment of Helicobacter pylori Using Egg Yolk IgY and Additives
KSBB Journal, volume 16, issue 1, 2001, Pages 41~47
H. pylori is known to be a key pathogen of chronic gastric and duodenal ulcers. Bacterial adhesion to hosts is an essential step for bacterial infection and the inhibition of this adhesion provides a possible method for the treatment of the infection. The inhibitory effect of antibody lgY, produced from immunized hens with H. pylori antigen, was studied in vitro. The inhibition of H. pylori adhesion to AGS was as high as 90% using 0.5mg/ml of lgY, and almost 80% of the detachmentwas also achieved. The inhibitory effect of adhesion-inhibition candidates was investigated. Additives in combination with lgY increased the adhesion-inhibiting effect by about 30-50%. However, the adhesion molecules of H. pylori were varied and complex, therefore the further studies are necessary to develop an adhesion inhibitor and effective enough to be employed for the treatment of H.pylori, in vivo.
Solubilization of BSA into AOT Reverse Micelles Using the Phase-Transfer Method: Effects of pH and Salts
KSBB Journal, volume 16, issue 1, 2001, Pages 48~53
Bovine serum albumin(BSA) was solubilized into the reverse micellar phase consisting of sodium bis(2-ethylhexyl) sulfosuccinate(AOT) and isooctane using the phase transfer method. Of particular interest in this study were the effects of pH and the added salt type and concentration on the solubilization efficiency. When univalent or divalent salts such as KCl, NaCl,
were added to the aqueous phase at a concentration of 0.1 M, maximum solubilization efficiency was attained at a pH ranging from 5 to 7, depending on the added salt type. Increased salt concentration up to 1 M resulted in an increased solubilization efficiency for
and NaCl, while the addition of
beyond 0.1 M showed an anomalous trend. Further, it was noteworthy that too a large extent the protein precipitated in the interface between the organic and aqueous phases at lower pHs and lower salt concentrations. The size of the reverse micelle water pool was estimated by measuring the molar ratio of the surfactant to the water,
. Irrespective of pH in the aqueous phase, the resulting value of
was almost constant, eg., 20 for
. However, the value of
decreased with increased salt concentration in the cases of KCl and
Effect of Metal Ions on Iron Oxidation Rate of Thiobacillus ferrooxidans Used in a Bioleaching Process
KSBB Journal, volume 16, issue 1, 2001, Pages 54~60
The activity of microorganisms is an important factor that determines the efficiency of the bacterial recovery of precious metals from low-grade ore. Metal-leaching microorganisms must have a tolerance, within the concentration levels encountered to leached metals. In this study, the tolerance levels of Thiobacillus ferroxidans to the single and mixed metal ions systems, composed of
were investigated. When single metal ions of
(1~6 g/L), or
(1~6 g/L) were added to the growth medium of T. ferrooxidans, the iron oxidation rate of this bacterium was not significantly inhibited. The maximum inhibition percentage observed on the iron oxidation rate of T. ferrooxidans was approximately 50% in the medium supplemented with two or three mixed metal ions of
. However, when
was also added to the medium with the other metal ions, the inhibitory effect on the iron oxidation activity of T. ferroxidans was remarkably increased.
Degradation of BTX by Aerobic Microbial Consortium
KSBB Journal, volume 16, issue 1, 2001, Pages 61~65
In this study, a BTX degrading microbial consortium was obtained from the activated sludges of a BTX releasing sewage water and city sewage water treatment plant. The MY microbial consortium was developed for benzene and toluene degradation, whereas the MA microbial consortium was developed for xylene isomers. The major microorganism of the MA consortium was identified as Rhodococcus ruber DSM 43338T, whereas that of the MY consortium was Rhodococcus sp. In terms of the degradation of a single component, the removal rate of benzene was fastest and decreased in order; toluene, o-xylene, p-xylene and m-xylene. For degradation of mixed BTX, most BTX were degraded within 108 hours and the degradation rate showed either stimulatory or inhibitory effects depending on the composition. MA and MY microbial consortium obtained in this study may be used effectively to remove BTX biologically.
Extraction of Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata
KSBB Journal, volume 16, issue 1, 2001, Pages 66~70
Factors affecting the supercritical carbon dioxide extraction of camptothecin(CPT) and 10-hydroxycamptothecin(HCPT) from the dried powder of Cmptotheca acuminata were studied. Only a few amount of CPT and HCPT was extracted with pure supercritical carbon dioxide. Methanol and ethanol were efficient modifiers to extract CPT and HCPT. At
, 250 bar, 1 mL/min flow rate, 41% of CPT and 35% of HCPT were extracted with supercritical carbon dioxide modified with 16% of methanol. The diffusion effect of HCPT on extraction efficiency was studied in this solid-fluid system. Round matrix hot-ball model assumption revealed that the value of D/
which was higher than that of solvent extraction with methanol.
Extraction and Purification of Acanthoside-D from Acanthopanax chilsanensis
KSBB Journal, volume 16, issue 1, 2001, Pages 71~75
Acanthoside-D, contained in Acanthopanax chilsanensis is known as a ginseng-like substance. This work was focused to set up analytical and preparative conditions for Acanthoside-D purification. The ethanol extract from the powder of the trunk of Acanthopanax chilsanensis was partitioned with hexane. A
) column was used to separate Acanthoside-D from the trunk of Acanthopanax chilsanensis. From the experimental results, the mobile phase used for isolating Acanthoside-D from the extract was water/acetonitrile/methanol=80/14/6 %(v/v). The flow rate of mobile phase was 1.0 mL/min, and UV wavelength was fixed at 210 nm. Finally on a semi-preparative column (3.9
, Lichrospher 100RP-18) with the same mobile phase composition, the allowable maximum injection volume increased to 250
Synthesis of Stigmastanol as a Serum Cholesterol-lowering substance Using Pd Catalyst
KSBB Journal, volume 16, issue 1, 2001, Pages 76~81
Stigmastanol, a functional agent of cholesterol-lowering in humans, was synthesized from stigmasterol. To investigate the usability as a raw material, the contents of sterol in vegetable oils and extract of soybean chaff were analyzed. The total sterol contents showed high values of 213.7 and 209.8 mg/100g in corn and soybean oils respectively. The extract of soybean chaff has played a good role as a raw material with high sterol contents. The kinetics of hydrogenation of stigmasterol was studied using a 5% Pd/AC catalyst in the temperature range of 30~
. Increasing temperature showed a prominent decrease in conversion. The optimum temperature was
for high yield of stigmastanol. The effects of
pressure, agitation speed, catalyst loading, and stigmasterol concentration on reaction rate profile were also examined. From the power law model analysis using the initial rates of reaction, the reaction order was calculated as 0.705 for stigmasterol concentration and 0.147 for hydrogen pressure.
Enzymatic Glycosylation of Acrylic Acid and Methacrylic Acid
KSBB Journal, volume 16, issue 1, 2001, Pages 82~86
Glyco-acrylate and methacrylate were synthesized by lipase-catalyzed glycosylation of acrylic acid, methacrylic acid and their vinyl esters with
-methyl fructoside and glycerol in t-butanol as a reaction medium. At the optimum conditions for enzymatic glycosylation of acrylic acid and vinyl methacrylate, we attained up to 80% conversion for glyco-acrylate from acrylic acid and 90% conversion for glyco-methacrylate from vinyl methacrylate. The polymerizable glyco-acrylates and methacrylate have biomedical application as hydrophilic monomers and hydration modifiers to be use for hydrogel contact lens formulation.
Batch and Fed-batch Fermentation for the Lovastatin Production by Cerulenin-resistant Aspergillus terreus Mutant
KSBB Journal, volume 16, issue 1, 2001, Pages 87~94
The biosynthesis of Lovastatin, a cholesterol lowering agent formed by the filamentous fungus, cerulenin-resistant Aspergillus terreus mutant was studied in shake flasks and bioreactors. The lovastatin production could be improved by fed-batch under the limited condition of carbon source. The relationship between the fungal morphology and the lovastatin production was also examined during the fed-batch cultures. The fed-batch studies in shake flasks were carried out to find the optimum glucose feeding method, and the pulsed feeding of glucose from 3 days onward at 24 hours intervals was found to be optimal to increase the lovastatin production and reduce the average pellet size. When the pH was controlled at around 5.8 during the whole fermentation period, the lovastatin concentration reached 384 mg/L, which is much higher than the values obtained pH-uncontrolled and pH 7.4. The optimal glucose feeding strategies was found that 30 g/L of glucose was added initially in batch mode, and then fed-batch was conducted by continuous addition of glucose solution(180 g/L) from 72 to 240 hr at a rate of 1.2 mL/hr at
, pH 5.8, 400 rpm, and 1.0 vvm. The lovastatin concentration of 547 mg/L was obtained in 168 hr. It was about 1.5 times higher than the value of the batch fermentation.
Ethanol Modified Supercritical
Extraction of Daidzein from Soybean
KSBB Journal, volume 16, issue 1, 2001, Pages 95~98
Various factors affecting the supercritical carbon dioxide extraction of daidzein from soybean were studied. Daidzein was not extracted with pure supercritical carbon dioxide. The ethanol was an efficient modifier for supercritical carbon dioxide to extract daidzein. The extraction efficiency increased as the pressure increased up to 300 bar. At
and 300 bar, 93% of daidzein was extracted with supercritical carbon dioxide modified with 15% of ethanol.