Geun-Bae Kim - Academia.edu (original) (raw)
Papers by Geun-Bae Kim
Journal of Dairy Science, 2004
Bile salt hydrolases were purified to electrophoretic homogeneity from Bifidobacterium bifidum AT... more Bile salt hydrolases were purified to electrophoretic homogeneity from Bifidobacterium bifidum ATCC 11863, Bifidobacterium infantis KL412, Bifidobacterium longum ATCC 15708, Bifidobacterium longum KL507, and Bifidobacterium longum KL515. Three different types (A, B, and C) of bile salt hydrolase (BSH) were revealed during the purification study, exhibiting the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was around 35 kDa, and the native molecular mass in all five Bifidobacterium strains was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, an isoelectric point value of 4.45 was obtained with BSH (type B) from B. bifidum ATCC 11863 and the other BSH (types A and C) showed the similar pI values around 4.65. N-Terminal amino acid sequencing for the proteins of types A and C revealed that 6 out of 20 amino acid residues were different, and highly conserved residues were identified in both N-terminal sequences of types A and C. All BSH enzymes from five strains hydrolyzed six major human bile salts, and they showed a better deconjugation rate on glycine-conjugated bile salts than on taurine-conjugated forms. (Key words: bile salt hydrolase, purification, Bifidobacterium)
Journal of Dairy Science, 2006
Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane)... more Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane) were studied for the gas chromatographic analysis of conjugated linoleic acids (CLA) from probiotic bacteria grown in de Man, Rogosa, and Sharpe medium. As compared with chloroform/methanol and hexane/isopropanol, hexane showed comparable extraction efficiency for CLA from unspent de Man, Rogosa, and Sharpe medium, but showed minimal extraction of oleic acid originated from the emulsifier in broth. The extraction efficiency of CLA by hexane was influenced by the broth pH, showing the optimal pH of 7.0. Repeated extraction with hexane increased the yield. Extraction with hexane showed excellent recovery of spiked CLA from the spent broth with up to 97.2% (standard deviation of 1.74%). This represents the highest recovery of CLA from culture broth ever reported. The sample size was also successfully reduced to 0.5 mL to analyze CLA from the broth without impairment of analytical data. This smaller sample size in the 1.5-mL microcentrifuge tube using a small bench-top centrifuge reduced analytical time significantly.
Bioconjugate Chemistry, 2007
Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived f... more Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3 γ recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.
Biotechnology Letters, 2005
A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expr... more A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P bsh ) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.
Applied and Environmental Microbiology, 2004
Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifid... more Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.
International Dairy Journal, 2003
Deconjugation of bile salts by Lactobacillus acidophilus strains isolated from men, fermented mil... more Deconjugation of bile salts by Lactobacillus acidophilus strains isolated from men, fermented milk, and pigs resulted in precipitate halo, opaque granular white colonies, shiny precipitate halo or clear zone around the microbial colonies on bile salt-MRS agar plates depending on the type of bile salts added. None of the L. acidophilus cultures tested exhibited 7a-dehydroxylase activity that transforms cholate into deoxycholate. L. acidophilus SNUL020 and SNUL01 deconjugated both taurocholate (TCA) and glycocholate (GCA) at similar rates, while L. acidophilus FM01 deconjugated GCA more rapidly than TCA. Most L. acidophilus strains tested precipitated more soluble cholesterol in the media containing taurochenodeoxycholate (TCDCA) and taurodeoxycholate (TDCA) than with TCA and GCA. In the medium containing TDCA, L. acidophilus SNUL020, SNUL01, and FM01 precipitated more than 50% of soluble cholesterol. All L. acidophilus strains tested were more resistant to taurine-than to glycine-conjugated bile salts. Fecal excretions of cholesterol and deoxycholate increased slightly with acidophilus milk intake containing live cells of L. acidophilus SNUL01.
Journal of Food Science, 2002
ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactob... more ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactobacillus acidophilus NCFM plus 1 strain each of Bifidobacterium longum and Lactobacillus casei, were used to make yogurt-like products. The lactobacilli and bifidobacteria were tested for growth in the products and subsequent viability during refrigerated storage. During fermentation, L. casei Com-5 actually declined in numbers, while L. casei E5 and E10 increased about 2 fold. Numbers of B. longum S9 increased about 3 fold while B. longum Com-4 did not increase. During storage, L. acidophilus NCFM appeared stable in all mixtures and both strains of bifidobacteria decreased. Lactobacillus casei E5 and E10 were more stable than was L. casei Com-5.
Journal of Dairy Science, 2004
Bile salt hydrolases were purified to electrophoretic homogeneity from Bifidobacterium bifidum AT... more Bile salt hydrolases were purified to electrophoretic homogeneity from Bifidobacterium bifidum ATCC 11863, Bifidobacterium infantis KL412, Bifidobacterium longum ATCC 15708, Bifidobacterium longum KL507, and Bifidobacterium longum KL515. Three different types (A, B, and C) of bile salt hydrolase (BSH) were revealed during the purification study, exhibiting the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was around 35 kDa, and the native molecular mass in all five Bifidobacterium strains was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, an isoelectric point value of 4.45 was obtained with BSH (type B) from B. bifidum ATCC 11863 and the other BSH (types A and C) showed the similar pI values around 4.65. N-Terminal amino acid sequencing for the proteins of types A and C revealed that 6 out of 20 amino acid residues were different, and highly conserved residues were identified in both N-terminal sequences of types A and C. All BSH enzymes from five strains hydrolyzed six major human bile salts, and they showed a better deconjugation rate on glycine-conjugated bile salts than on taurine-conjugated forms. (Key words: bile salt hydrolase, purification, Bifidobacterium)
Journal of Dairy Science, 2006
Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane)... more Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane) were studied for the gas chromatographic analysis of conjugated linoleic acids (CLA) from probiotic bacteria grown in de Man, Rogosa, and Sharpe medium. As compared with chloroform/methanol and hexane/isopropanol, hexane showed comparable extraction efficiency for CLA from unspent de Man, Rogosa, and Sharpe medium, but showed minimal extraction of oleic acid originated from the emulsifier in broth. The extraction efficiency of CLA by hexane was influenced by the broth pH, showing the optimal pH of 7.0. Repeated extraction with hexane increased the yield. Extraction with hexane showed excellent recovery of spiked CLA from the spent broth with up to 97.2% (standard deviation of 1.74%). This represents the highest recovery of CLA from culture broth ever reported. The sample size was also successfully reduced to 0.5 mL to analyze CLA from the broth without impairment of analytical data. This smaller sample size in the 1.5-mL microcentrifuge tube using a small bench-top centrifuge reduced analytical time significantly.
Bioconjugate Chemistry, 2007
Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived f... more Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3 γ recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.
Biotechnology Letters, 2005
A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expr... more A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P bsh ) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.
Applied and Environmental Microbiology, 2004
Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifid... more Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.
International Dairy Journal, 2003
Deconjugation of bile salts by Lactobacillus acidophilus strains isolated from men, fermented mil... more Deconjugation of bile salts by Lactobacillus acidophilus strains isolated from men, fermented milk, and pigs resulted in precipitate halo, opaque granular white colonies, shiny precipitate halo or clear zone around the microbial colonies on bile salt-MRS agar plates depending on the type of bile salts added. None of the L. acidophilus cultures tested exhibited 7a-dehydroxylase activity that transforms cholate into deoxycholate. L. acidophilus SNUL020 and SNUL01 deconjugated both taurocholate (TCA) and glycocholate (GCA) at similar rates, while L. acidophilus FM01 deconjugated GCA more rapidly than TCA. Most L. acidophilus strains tested precipitated more soluble cholesterol in the media containing taurochenodeoxycholate (TCDCA) and taurodeoxycholate (TDCA) than with TCA and GCA. In the medium containing TDCA, L. acidophilus SNUL020, SNUL01, and FM01 precipitated more than 50% of soluble cholesterol. All L. acidophilus strains tested were more resistant to taurine-than to glycine-conjugated bile salts. Fecal excretions of cholesterol and deoxycholate increased slightly with acidophilus milk intake containing live cells of L. acidophilus SNUL01.
Journal of Food Science, 2002
ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactob... more ABSTRACT: Multiple species cultures, including 2 strains of Streptococcus thermophilus and Lactobacillus acidophilus NCFM plus 1 strain each of Bifidobacterium longum and Lactobacillus casei, were used to make yogurt-like products. The lactobacilli and bifidobacteria were tested for growth in the products and subsequent viability during refrigerated storage. During fermentation, L. casei Com-5 actually declined in numbers, while L. casei E5 and E10 increased about 2 fold. Numbers of B. longum S9 increased about 3 fold while B. longum Com-4 did not increase. During storage, L. acidophilus NCFM appeared stable in all mixtures and both strains of bifidobacteria decreased. Lactobacillus casei E5 and E10 were more stable than was L. casei Com-5.