• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.294-301
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• 2001

The effects of including glycine and isonicotinic acid hydrazide (INH) in the growth medium (Luria broth, LBG) on the subsequent lysozyme-imduced protoplast formation and transformation efficiency of Corynebacterium glutamicum were studied. The transformation efficiency of C. glutamicum AS019 increased up to 100-fold as the ocncentrationof glycine in the media increased from 0% to 5% (w/v), relative to cells grown in the absence of glycine. The presence of 5 mg/ml INH in the growth medium led to a further 10-fold increase in transformation efficiency. In addition, this transformation protocol was successfully applied to other strains of C. glutamicum. Both chemicals affected the mycolic acid attachment to the cell surface of C. glutamicum, when INH, the relative percentage of fatty acids of AS019 to the total lipids (mycolic acid plus fatty acids) decreased from 76.9% (in LBG) to 72.9% (in LBG-2% glycine) and 66.4% (in LBG-8 mg InG/ml), thereby suggeting that these chemicals also inhibit fatty acid synthesis.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.678-682
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• 2005

The promoter region of a carbon starvation gene isolated from Pseudomonas putida was cloned and analyzed for its potential use for in situ bioremediation and bioprocessing. We constructed a recombinant plasmid pMKD101 by cloning the 0.65 kb promoter region of the gene into the promoter proving vector, pMK301, which contains the lacZ for ${\beta}$-galactosidase activity as a reporter gene. pMKD101 was transformed into the wild-type P. putida MK1, resulting in P. putida RPD101, and analyzed for ${\beta}$-galactosidase activity under different culture conditions. When RPD101 was grown on the minimal medium plus $0.1\%$ glucose as a sole carbon source in batch cultures, ${\beta}$-galactosidase activity was found to be 3.2-fold higher during the stationary phase than during the exponential phase. In chemostat cultures, ${\beta}$-galactosidase activity was found to be 3.1-fold higher at the minimal growth rate (dilution rate=$0.05\;h^{-1}$) than at the maximal growth rate (dilution rate=$0.173;h^{-1}$). The results suggest that a carbon starvation promoter can be utilized to maximize the expression of a desired gene under nutrient limitation.

• KSBB Journal
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• v.20 no.5 s.94
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• pp.338-340
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• 2005

Covalent modification of a protein with polyethylene glycol (PEG) has become one of the most widely used and well established drug enhancement strategies in the biopharmaceutical industry. The general benefits enjoyed by PEGylation, such as prolonged serum half-lives or reduced immunogenicity in vivo, are well known. By now the PEGylation process has been performed with purified proteins, and it is required to recover the desired PEGylate by a multi-step purification process. The ultimate aim of our research is to develop an integrated process of PEGylation and in vitro refolding starting with inclusion body material. For this, we investigated the feasibility that a protein could be PEGylated under a denaturing condition and also the PEGylated proteins could be refolded correctly. Using lipase as a model protein, we found that it was PEGylated in the presence of 8M urea and that the PEG molecules covalently attached to lipase did not appear to hinder its refolding.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.65-72
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• 2006

Recent technical advances in the biorecognition engineering and the microparticle fabrication may enable us to develop the single step purification using magnetic particle, because of its simplicity, efficacy, ease of automation, and process economics. In this study, we used commercial magnetic particles from Seradyn, Inc. (Indianapolis, USA). It was ca. 2.8 micron in diameter, consisted of polystyrene core and magnetite coating, and its surface had carboxyl groups. The model, capture protein was IgG and anti-IgG was used as the ligand molecule. We studied the different surfaces ('nude', ester-activated, and anti-IgG coated) for their biorecognition of IgG. At a high pH condition, we could reduce non-specific binding. Also anti-IgG immobilized magnetic particle could capture IgG more selectively. We attempted 'oriented immobilization' of anti-IgG, in which the polysaccharides moiety near the C-terminus was selectively oxidized and linked to the hydrazine-coated MP, to improve the efficacy of biorecognitive binding. Using this method, the IgG capturing ability was improved by ca. 2 fold. From the binary mixture of the IgG-insulin, IgG could be more selectively captured. In summary, the oriented immobilization of oxidized anti-IgG proved to be as effective as the streptavidin-biotin system and yet simpler and cost-effective. This immobilization method can find its applications in protein biochips and biotargeting.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.187-201
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• 2005

Bioprocessing technologies utilizing 'biorecognition' between a solid matrix and a protein is being widely experimented as a means to replacing the conventional, solution-based technology. Frequently the matrices are chromatographic resins with specific functional groups exposed outside. Since the reactions of and interactions with the proteins occur as they are attached to the solid matrix, this 'solid-phase' processing has distinct advantages over the solution-phase technology. Solid-phase refolding of inclusion body proteins uses ion exchange resins to adsorb denaturant-dissolved inclusion body. As the denaturant is slowly removed from the micromoiety around the protein, it is refolded into a native, three-dimensional structure. Once the refolding is complete, the folded protein can be eluted by a conventional elution technique such as the salt-gradient. This concept was successfully extended to 'EBA (expanded bed adsorption)-mediated refolding,' in which the denaturant-dissolved inclusion body in whole cell homogenate is adsorbed to a Streamline resin while cell debris and other impurity proteins are removed by the EBA action. The adsorbed protein follows the same refolding steps. This solid-phase refolding process shows the potential to improve the refolding yield, reduce the number of processing steps and the processing volume and time, and thus improve the overall process economics significantly. In this paper, the experimental results of the solid-phase refolding technology applied to several biopharmaceutical proteins of various types are presented.

• Natural Product Sciences
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• v.4 no.1
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• pp.9-14
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• 1998

Jaceidin, Jaceidin-7-O-methylether and quercetin were isolated from-Juniperus phoenicea L. alcoholic extract, however, Sequoiaflavone was isolated from Cupressus semperiverns L. In addition, the alcoholic extracts of both plants were found to contain also kaempferol-3-O-rhamnoside, quercetrin, myricitrin, cupressuflavone. The chemical identities of the isolated compounds were established using UV, IR, $^1H-and\;^{13}C-NMR$ spectroscopy.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.309-314
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• 2005

We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface plasmon resonance biosensor. We immobilized anti­HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to the dextran layer on a CM5 chip surface that had previously been activated by N-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately $17.6 ng/mm^2$. The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up to ca. $40{\mu}g$/mL. This linearity was much higher than that of the ELISA method. It appeared the anti­gen-antibody binding increased as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method as a rapid, simple and multi­sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg quantification for replacing the ELISA.

• Journal of the Korea Organic Resources Recycling Association
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• v.11 no.3
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• pp.67-74
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• 2003

Starvation promoters can be utilized for in situ bioremediation and for the efficient bioprocessing. Previously we have cloned and characterized strong starvation promoters from envrionmentally relevant bacteria, Pseudomonas putida strains (Y. Kim, and A. Matin, J. Bacteriol. 177:1850-1859, 1995). Here we report the construction of the plasmid pYKS101 using one of the starvation promoters from P. putida MK1. The pYKS101 was found to be useful for a novel starvation promoter-driven gene expression system. Under this system, the target reporter gene, lacZ, was stably integrated into the chromosomal DNA of P. putida MK1. ${\beta}$-galactosidase activity was found to be four-fold higher upon carbon starvation than during exponential growth. The resultant recombinant strain is indigenous to the environment contaminated with various toxic materials, hence can be a good candidate for in situ bioremediation.

• KSBB Journal
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• v.17 no.2
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• pp.176-181
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• 2002

Astaxanthin (3,3＇-dihydroxy-${\beta}$, ${\beta}$-carotene-4-4＇-dione), a natural pigment of pink to red color, is widely distributed in nature particularly in the skin layer of salmonoids and the crust of shrimp, lobster, etc. Recently, it was produced from the yeast culture of Phaffia rhodozyma. Because of its high thermal stability and antioxidant functionality, its applications can be extended into food, cosmetics, and pharmaceutical ingredient beyond the traditional feed additive. Because of its very high lipophilicity, astaxanthin has been extracted traditionally by strong organic solvents such as chloroform, petroleum ether, acetone, etc. In this study, we developed a surfactant-based solubillization system for astaxanthin, and used it to extract astaxanthin from disrupted yeast cells. Among Tween 20, Triton X-100 and SDS, Tween 20 was identified as the most suitable surfactant in terms of extraction capacity and safety. The ethylene oxide group of Tween 20 was identified as the most significant factor to increase the HLB value that determined the extraction capacity. The effects of micelle formation condition, such as the molar ratio of astaxanthin and Tween 20, pH, and ionic strength were also investigated. pH and ionic strength showed no significant effects. The optimal molar ratio between astaxanthin and Tween 20 was 1 : 12. Antioxidant activity of astaxanthin was higher than ${\beta}$-carotene and ${\alpha}$-tocopherol. Astaxanthin in the crude extract from the yeast cell was more resistant to air and/or light degradation than pure astaxanthin, probably because of the presence of other carotenoids and lipids.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.3
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• pp.138-149
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• 2002

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stage etc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.