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Byung-Gee Kim

Publications and source records attributed to Byung-Gee Kim.

At least 19 recordsLinked to original sources
PubMed2006

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor.

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.

Calorimetry↗
PubMed2006

Simultaneous profiling of N-glycans and proteins from human serum using a parallel-column system directly coupled to mass spectrometry.

A method for the rapid identification of proteins and their N-glycans was developed through the use of two parallel columns directly connected to a mass spectrometer. Both porous graphitic carbon (PGC) and C18 capillary columns were connected in parallel with two switching valves for the simultaneous analysis of glycans and peptides, respectively. To verify the efficiency of the analytical system, profiling of N-glycans and proteins from human serum was demonstrated. This method is suitable for high-throughput analysis and automation, is contamination-free for the identification of N-glycans and proteins in a complex biological sample, and can be applied to glycomics and proteomics.

Amino Acid Sequence↗
PubMed2006

Engineering aromatic L-amino acid transaminase for the asymmetric synthesis of constrained analogs of L-phenylalanine.

An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-diphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobacter sp. BK2K-1. To rationally redesign the enzyme, structural model was constructed by the homology modeling. The structural model was experimentally validated by the site-directed mutagenesis of the predicted pyridoxal-5'-phosphate (PLP) binding site and the substrate-recognition region, and the cell-free protein synthesis of mutated enzymes. It was suggested that Arg281 and Arg375 were the key residues to recognize the distal carboxylate and alpha-carboxylate group of the substrates, respectively. The model also predicted that Tyr66 forms hydrogen bond with the phosphate moiety of PLP and interacts with the side chain attached to beta-carbon of the amino acid substrate. Among the various site-directed mutants, Y66L variant was able to synthesize L-diphenylalanine with 23% conversion yield for 10 h, whereas the wild-type AroATEs was inactive for the transamination between diphenylpyruvate and L-phenylalanine as amino acceptor and amino donor, respectively.

Amino Acids, Aromatic↗
PubMed2006

Pro-antibiotic substrates for the identification of enantioselective hydrolases.

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2-phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.

Anti-Bacterial Agents↗
PubMed2006

A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip.

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.

Biosensing Techniques↗
PubMed2006

Synthesis of enantiomerically pure trans-(1R,2R)- and cis-(1S,2R)-1-amino-2-indanol by lipase and omega-transaminase.

Syntheses of trans-(1R,2R) and cis-(1S,2R)-1-amino-2-indanol (AI) were accomplished by a series of enantioselective enzymatic reactions using lipase and transaminase (TA). Lipase catalysed enantioselective hydrolysis of 2-acetoxyindanone was employed to prepare (R)-2-hydroxy indanone (HI). trans-AI (5 mM) (de > 98%) was produced from 20 mM (R)-2- HI using omega-TA and 50 mM (S)-1-aminoindan as an amino donor in water-saturated ethyl acetate. For the production of cis-AI, the diastereomeric (2R)-AI was synthesized from (R)-2-HI using reductive amination, and the kinetic resolution was performed with omega-TA. The enantioselectivity of omega-TA for (2R)-AI was increased to 22.1 in the presence of 5% gamma-cyclodextrin. cis-AI (15.4 mM) (96% de) was obtained from 40 mM (2R)-AI using 30 mM pyruvate and omega-TA (25 mg) in 10 mL of 100 mM phosphate buffer (pH 7.0).

Indans↗
PubMed2006

Preparative synthesis of dTDP-L-rhamnose through combined enzymatic pathways.

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.

Bacterial Proteins↗
PubMed2006

The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney.

The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.

Animals↗
PubMed2006

Selection of peptides for lipopolysaccharide binding on to epoxy beads and selective detection of Gram-negative bacteria.

Lipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 10(6) cells ml(-1).

Bacterial Typing Techniques↗
PubMed2006

Protective effect of Rosa laevigata against amyloid beta peptide-induced oxidative stress.

The amyloid beta (Abeta) peptide is known to increase free radical production in nerve cells, leading to cell death. To investigate the effect of Rosa laevigata against Abeta-induced oxidative damage, in vitro assays and in vivo behavioral tests were performed. R. laevigata showed cell protective effects against oxidative stress-induced cytotoxicity. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay exhibited significant increase in cell viability when rat pheochromocytoma (PC 12) cells were treated with R. laevigata extracts. Administration of R. laevigata extracts to mice significantly reversed the Abeta-induced learning and memory impairment in in vivo behavioral tests. These results suggest that R. laevigata extracts can reduce the cytotoxicity of Abeta in PC 12 cells, possibly by the reduction of oxidative stress, and these extracts may be useful in the prevention of Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed2006

Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples.

An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment size of approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18S rDNA groups under standard polymerase chain reaction conditions.

DNA, Ribosomal↗
PubMed2005

A novel electrochemical detection method for aptamer biosensors.

A beacon aptamer-based biosensor for the detection of thrombin was developed using electrochemical transduction method. Gold surface was modified with a beacon aptamer covalently linked at 5'-terminus with a linker containing a primary aliphatic amine. Methylene blue (MB) was intercalated into the beacon sequence, and used as an electrochemical marker. When the beacon aptamer immobilized on gold surface encounters thrombin, the hairpin forming beacon aptamer is conformationally changed to release the intercalated MB, resulting a decrease in electrical current intensity in voltamogram. The peak signal of the MB is clearly decreased by the binding of thrombin onto the beacon aptamer. The linear range of the signal was observed between 0 and 50.8 nM of thrombin with 0.999 correlation factor. This method was able to linearly and selectively detect thrombin with a detection limit of 11 nM.

Aptamers, Nucleotide↗
PubMed2005

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal.

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.

Aptamers, Nucleotide↗
PubMed2005

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis.

In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its beta-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher beta-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.

Bacillus subtilis↗
PubMed2005

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis.

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA-CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA-CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction.

Bacterial Proteins↗
PubMed2005

The ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis.

A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.

Acetylation↗