Showing papers by "Byung-Gee Kim published in 2010"
TL;DR: Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli.
Abstract: Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.
46 citations
TL;DR: This first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA is reported.
42 citations
TL;DR: The basic properties of BCAT en and BCAT es such as substrate specificity, enantioselectivity, and kinetic parameters are characterized and coupling reactions were carried out with l -glutamate dehydrogenase (GDH)/formate dehydrogen enzyme (FDH) and AspAT.
Abstract: l -Tert-leucine (Tle) and l -3-hydroxyadamantylglycine of (HAG) are important intermediates for a variety of pharmaceutical classes. They were asymmetrically produced from corresponding keto acids using branched-chain aminotransferase (BCAT) with l -glutamate (Glu) as an amino donor. For the production of l -Tle and l -HAG, BCAT from Enterobacter sp. TL3 (BCATen) and BCAT from Escherichia coli K12 (ilvE, newly named as BCATes) were used, respectively. In our current study, we characterized the basic properties of BCATen and BCATes such as substrate specificity, enantioselectivity, and kinetic parameters. The activities of BCATen and BCATes were inhibited severely by α-ketoglutarate which is a deaminated product of l -Glu. In the presence of 10 mM α-ketoglutarate, both enzymes activities were reduced up to 80%. In order to overcome product inhibition by α-ketoglutarate and the problem of equilibrium of the transamination reaction, coupling reactions were carried out with l -glutamate dehydrogenase (GDH)/formate dehydrogenase (FDH) and AspAT. The coupling reaction dramatically increased the yields of both target compounds. 135 mM of l -Tle (>99% ee) was produced from 150 mM corresponding keto acid in BCATen/GDH/FDH coupling reaction with 90% conversion. In addition, 90.5 mM l -HAG (>99% ee) was produced from 100 mM corresponding keto acid in BCATes/AspAT coupling reaction using recombinant whole-cells.
36 citations
TL;DR: Recombinant Pichia pastoris expressing ω-transaminase (TA) was used as a whole-cell biocatalyst to kinetically resolve α-methylbenzylamine (MBA) and the reaction condition of endogenous oxidoreductases was optimized.
Abstract: Recombinant Pichia pastoris expressing ω-transaminase (TA) was used as a whole-cell biocatalyst to kinetically resolve α-methylbenzylamine (MBA). To overcome product inhibition of ω-TA by acetophenone (deaminated product of α-MBA), the reaction condition of endogenous oxidoreductases, which can catalyze the reduction of acetophenone into non-inhibitory 1-phenylethanol, was optimized. When the whole-cell reaction was carried out using recombintat P. pastoris in 100 mM Tris/HCl buffer (pH 9.0) containing 2.5% glucose and 1% methanol, 100 mM α-MBA was successfully resolved to (R)-α-MBA (> 99% ee) at a conversion of 52.2%.
25 citations
TL;DR: Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours and it is expected that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.
Abstract: Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.
20 citations
TL;DR: Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase were utilized for the production of UDP-galactose (UDP-Gal), and the reaction was optimized by increasing the concentration of AK.
Abstract: Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.
20 citations
TL;DR: An easy preparation method of multilayer fluorescence optically encoded beads for protein detection is presented and the ligands are identified easily by their color codes.
19 citations
TL;DR: Homology modeling and docking simulation found that Thr247 is responsible for the 3′-position hydroxylation reaction by forming a hydrogen bond with the 4′-hydroxyl group of daidzein that forces the proton at the 3″-position to face the heme center.
16 citations
TL;DR: ManB isolated from Streptomyces coelicolor was shown to have both phosphomannomutase and phosphoglucomutase activities, and this result provides useful information for the design of strategies that enhance antibiotic production through the control of carbon flux.
Abstract: Phosphomannomutase (ManB), whose main function is the conversion of mannose-6-phosphate to mannose-1-phosphate, is involved in biosynthesis of GDP-mannose for numerous processes such as synthesis of structural carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins in prokaryotes and eukaryotes. ManB isolated from Streptomyces coelicolor was shown to have both phosphomannomutase and phosphoglucomutase activities. Deletion of manB in S. coelicolor caused a dramatic increase in actinorhodin (ACT) production in the low-glucose Difco nutrient (DN) medium, whereas the wild-type strain did not produce ACT on this medium. Experiments involving complementation of the manB deletion showed that increased ACT production in DN media was due to blockage of phosphomannomutase activity rather than phosphoglucomutase activity. This result therefore provides useful information for the design of strategies that enhance antibiotic production through the control of carbon flux.
12 citations
TL;DR: π-TA from Vibrio fluvialis JS17 (ω-TAVf) is a novel amine:pyruvate transaminase that is capable of stereoselective transamination of aryl chiral amines and was overexpressed in Escherichia coli with engineered C-terminal His tags.
Abstract: Omega-transaminase (ω-TA) catalyzes the transfer of an amino group from a non-α-position amino acid or an amine compound with no carboxylic group to an amino acceptor. ω-TA from Vibrio fluvialis JS17 (ω-TAVf) is a novel amine:pyruvate transaminase that is capable of stereoselective transamination of aryl chiral amines. In this study, ω-TAVf was overexpressed in Escherichia coli with engineered C-terminal His tags. ω-TAVf was then purified to homogeneity and crystallized at 292 K. X-ray diffraction data were collected to a resolution of 2.5 A from a crystal belonging to the orthorhombic space group P212121, with unit-cell parameters a = 78.43, b = 95.95, c = 122.89 A.
12 citations
TL;DR: A member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor and could be a high-throughput method for the characterization of unknown regulators.
Abstract: To elucidate the function of an unknown regulator in Streptomyces, differences in phenotype and antibiotic production between a deletion mutant and a wild-type strain (WT) were compared. These differences are easily hidden by complex media. To determine the specific nutrient conditions that reveal such differences, we used a multiwell method containing different nutrients along with bromothymol blue. We found several nutrients that provide key information on characterization conditions. By comparing the growth of wild-type and mutant strains on screened nutrients, we were able to measure growth, organic acid production, and antibiotic production for the elucidation of regulator function. As a result of this method, a member of the MarR-like regulator family, SCO5405 (AbsC), was newly characterized to control pyruvate dehydrogenase in Streptomyces coelicolor. Deletion of SCO5405 increased the pH of the culture broth due to decreased production of organic acids such as pyruvate and alpha-ketoglutarate and increased extracellular actinorhodin (ACT) production in minimal medium containing glucose and alanine (MMGA). This method could therefore be a high-throughput method for the characterization of unknown regulators.
TL;DR: The expression levels of an ELP fusion protein were improved by almost 2-fold by adding specific amino acids, and it was determined that the amount of increase in expression levels depends on the length of the ELPs.
TL;DR: By selective addition of specific amino acids constituting ELP molecules, the expression level of the ELP fusion protein was improved by 1.3–1.8 times as high as positive control, implying that the expression amount of long repetitive polypeptides can be compensated to a degree by adding repeatedly consumed amino acids in vitro system.
TL;DR: It is shown how production of NDP-sugars could be enhanced by controlling mutase activity, using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, which was successfully synthesized.
Abstract: Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity.
TL;DR: This mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.
TL;DR: An integrative approach for discovering novel transcriptional regulators playing critical roles in antibiotic production and decoding their regulatory networks in Streptomyces species which contain many regulatory genes for synthesis of secondary metabolites and cell differentiation to spores is developed.
Abstract: In an age of burgeoning information on genomes and proteomes, determining the specific functions of a gene of interest is still a challenging task, especially genes whose functions cannot be predicted from their sequence information alone. To solve this problem, we have developed an integrative approach for discovering novel transcriptional regulators (TRs) playing critical roles in antibiotic production and decoding their regulatory networks in Streptomyces species which contain many regulatory genes for synthesis of secondary metabolites and cell differentiation to spores. The DNA affinity capture assay (DACA) cou- pled with clustering of DNA chip data was used to find new TRs controlling antibiotic biosynthetic gene clusters. Functions of these newly identified TRs were characterized using 96-well-based minimal media screening (antibiotic production mapping, APM), pH indica- tor method, comparative two-dimensional gel electrophoresis (2D-gel), reverse-transcription polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA), and scan- ning electron microscopy (SEM). Using these techniques, we were able to reconstruct a reg- ulatory network describing how these new TRs collectively regulate antibiotic production. This proposed approach providing additional key regulators and their interactions to an ex- isting incomplete regulatory network can also be applied in studying regulators in other bac- teria of interest.
Journal Article•
TL;DR: In this article, a rare isoflavone, 7, 8, 4'-trihydroxyisoflavones, was isolated from a 10-year-old fermented soybean food.
Abstract: A rare isoflavone, 7, 8, 4'-trihydroxyisoflavone, was isolated from a 10-year-old fermented soybean food. 7, 8, 4'-trihydroxyisoflavone was isolated for the first time from a Korean fermented soybean food. Evaluation tests of biological activity showed significantly inhibition activity for free radical scavenging on both non-enzymatic (DPPH system) and enzymatic method (xanthine oxidase system). DPPH radical scavenging effect of 7, 8, 4'-trihydroxyisoflavone was similar with vitamin C in a dose-dependent manner. In xanthine oxidase (XO) system 7, 8, 4'-trihydroxyisoflavone showed superoxide radical inhibition activity of 50 % at a concentration of . Also, the compound significantly suppressed cellular MMP-1 formation. These results suggest that 7, 8, 4'-trihydroxyisoflavone could be developed as a potential preventive or therapeutic agent against skin aging.