Biomedical subjects

Yu-Ryang Pyun

Publications and source records attributed to Yu-Ryang Pyun.

At least 19 recordsLinked to original sources

PubMed2007

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

Arabinose↗

PubMed2006

Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov.

A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.

Amino Acid Sequence↗

PubMed2006

Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh.

We have isolated a bacterium (TP-6) from the Indonesian fermented soybean, Tempeh, which produces a strong fibrinolytic protease and was identified as Bacillus subtilis. The protease (TPase) was purified to homogeneity by ammonium sulfate fractionation and octyl sepharose and SP sepharose chromatography. The N-terminal amino acid sequence of the 27.5 kDa enzyme was determined, and the encoding gene was cloned and sequenced. The result demonstrates that TPase is a serine protease of the subtilisin family consisting of 275 amino acid residues in its mature form. Its apparent K (m) and V (max) for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 259 microM and 145 micromol mg(-1) min(-1), respectively. The fibrinogen degradation pattern generated by TPase as a function of time was similar to that obtained with plasmin. In addition, N-terminal amino acid sequence analysis of the fibrinogen degradation products demonstrated that TPase cleaves Glu (or Asp) near hydrophobic acids as a P1 site in the alpha- and beta-chains of fibrinogen to generate fragments D', E', and D' similar to those generated by plasmin. On plasminogen-rich fibrin plates, TPase did not seem to activate fibrin clot lysis. Moreover, the enzyme converted the active plasminogen activator inhibitor-1 to the latent form.

Amino Acid Sequence↗

PubMed2006

Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1.

Pediocin PA-1 is a representative class IIa bacteriocin which is small and heat-stable and has a consensus motif, -YGNGV-. The plasmid pQE40PED, encoding pediocin PA-1 fused with His-tagged mouse dihydrofolate reductase (DHFR), was constructed and introduced into Escherichia coli M15 strain. The fusion protein was overexpressed in the strain after induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active pediocin PA-1, the purified fusion protein was cleaved by Factor Xa protease and the liberated pediocin PA-1 was finally purified by ultrafiltration with a 75% yield. The molecular mass of the purified recombinant pediocin PA-1 was the same as that of native pediocin PA-1 on an electrophoresis gel.

Amino Acid Sequence↗

PubMed2006

Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from raw chicken meat and human stools in Korea.

Prevalence of Campylobacter in raw chicken meat and human stools and subsequent antibiotic resistance profiles of the pathogenic isolates obtained from 2000 through 2002 were investigated. Campylobacter jejuni and Campylobacter coli were isolated from 570 of the 923 raw chicken meat samples collected from traditional markets, large retail stores, or department stores in Korea, resulting in the isolation rate of 61.8%. A total of 579 Campylobacter isolates were obtained from raw chicken (36.3% for C. jejuni and 26.4% for C. coli) with the average population of 335.6 CFU/g. From 513 human stool samples, 15 isolates of Campylobacter were detected. Seasonal variation in the quantification of C. coli was not noticeable throughout the year, while the isolation rate of C. jejuni was the highest in September through October (840 CFU/g) followed by that of July through August and May through June in decreasing order, showing a significant seasonal effect (P < 0.05). Contamination of Campylobacter was more severe in raw chicken meat sold in traditional markets than in those sold in large retail stores and department stores. Prevalence of Campylobacter in raw chicken sold in traditional markets was significantly influenced by seasonal changes (P < 0.05), whereas the samples obtained from other places was less affected by the seasonal changes. Susceptibilities of the 594 chicken isolates to ciprofloxaxin, chloramphenicol, erythromycin, kanamycin, nalidixic acid, and tetracycline were determined by an E-test. Campylobacter isolates were the most resistant to nalidixic acid (91.4%) followed by ciprofloxaxin (87.9%), tetracycline (87.2%), kanamycin (30.6%), erythromycin (19.4%), and chloramphenicol (1.3%). Human isolates showed a similar resistance to the six antibiotics tested. The proportion of Campylobacter isolates with multidrug resistance to four or more antimicrobials obtained from 2000 through 2002 ranged from 28 to 43.5%, indicating that it could be a serious health-threatening factor. This study suggests that it is prudent to establish an effective National Monitoring Program in Korea for the prevention and control of Campylobacter spp.

Animals↗

PubMed2005

Analysis of kimchi microflora using denaturing gradient gel electrophoresis.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 degrees C or 20 degrees C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.

Brassica↗

PubMed2005

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus.

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.

Aldose-Ketose Isomerases↗

PubMed2005

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures.

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.

Aldose-Ketose Isomerases↗

PubMed2005

Enhanced nisin production by increasing genes involved in nisin Z biosynthesis in Lactococcus lactis subsp. lactis A164.

Nisin Z production in Lactococcus lactis subsp. lactis A164 was improved by introducing multicopy genes, nisZ, nisRK, or nisFEG, involved in nisin biosynthesis into A164 strain. A similar growth profile was obtained from all strains tested. However, the cells expressing nisRK produced 25,000 AU nisin Z ml(-1) compared to 16,000 AU ml(-1) by the control strain. Northern blot analysis revealed that over-expression of nisRK promoted the transcription of the nisZ gene. The A164 strain expressing multicopy nisFEG also had an increased nisin Z production (25,000 AU ml (-1)) but produced the nisin more slowly than the cells expressing multicopy nisRK.

Blotting, Northern↗

PubMed2005

Nisin biosynthesis and its properties.

The antimicrobial peptide, nisin, produced by several strains of Lactococcus lactis, which belongs to the Class I bacteriocins called lantibiotics, is a small (3.4 kDa), 34-amino acid, cationic, hydrophobic peptide and has the five characteristic (beta-methyl)lanthionine rings formed by significant post-translational modification. A cluster of 11 genes has been involved in the biosynthesis of nisin and are proposed to be transcriptionally arranged as nisA(Z)BTCIP, nisRK, and nisFEG. The biosynthesis of nisin is regulated in a growth-phase-dependent manner including nisin-mediated induction which occurs via NisRK two-component regulatory system. This review outlines some of the more recent developments in the properties, regulation and applications of nisin biosynthesis.

Anti-Bacterial Agents↗

PubMed2005

Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum.

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

Aldose-Ketose Isomerases↗

PubMed2005

Secretion of recombinant pediocin PA-1 by Bifidobacterium longum, using the signal sequence for bifidobacterial alpha-amylase.

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial alpha-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.

Bacteriocins↗

PubMed2004

Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. lactis A164 using expanded bed ion exchange chromatography.

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.

Bacterial Proteins↗

PubMed2004

Transfer of Bacillus ehimensis and Bacillus chitinolyticus to the genus Paenibacillus with emended descriptions of Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb. nov.

The taxonomic status of Bacillus ehimensis and Bacillus chitinolyticus was examined, based on their 16S rDNA sequences, DNA-DNA hybridization and other taxonomic characteristics. A phylogenetic analysis using 16S rDNA sequences revealed that the two species belong to the genus Paenibacillus. In particular, B. ehimensis KCTC 3748(T) and B. chitinolyticus KCTC 3791(T) were found to be phylogenetically closely related to Paenibacillus koreensis YC300(T) (98.3 % sequence similarity) and Paenibacillus chinjuensis WN9(T) (95.2 % sequence similarity), respectively. DNA-DNA hybridization values between B. ehimensis KCTC 3748(T) and P. koreensis YC300(T) were less than 26 %. An experiment using Paenibacillus-specific PCR primers, PAEN515F and 1377R, revealed that B. ehimensis and B. chitinolyticus had the same amplified 16S rDNA fragment as members of the genus Paenibacillus. Accordingly, it is proposed that B. ehimensis and B. chitinolyticus be transferred to the genus Paenibacillus as Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb. nov., respectively.

Bacillus↗

PubMed2004

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima.

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.

Aldose-Ketose Isomerases↗

PubMed2003

The nontoxic mushroom Auricularia auricula contains a polysaccharide with anticoagulant activity mediated by antithrombin.

An acidic polysaccharide with anticoagulant activity was isolated from the edible mushroom Auricularia auricula using water, alkali or acid extracts. The alkali extract showed the highest anticoagulant activity and was thereby further purified using gel filtration chromatography. Specific anticoagulant activity of the purified polysaccharide was 2 IU/mg and its average mass was approximately 160 kDa. The polysaccharide from this species of mushroom contains mainly mannose, glucose, glucuronic acid and xylose but no sulfate esters. Its anticoagulant activity was due to catalysis of thrombin inhibition by antithrombin but not by heparin cofactor II. Inhibition of Factor Xa by antithrombin was not catalyzed by the polysaccharide. The glucuronic acid residues were essential for the anticoagulant action of the mushroom polysaccharide since the activity disappeared after reduction of its carboxyl groups. In ex vivo tests using rats orally fed with the polysaccharide, we observed an inhibitory effect on platelet aggregation as observed with aspirin, a well-known antiplatelet agent. The polysaccharides from these mushrooms may constitute a new source of compounds with action on coagulation, platelet aggregation and, perhaps, on thrombosis.

Animals↗

PubMed2003

Arthrobacter koreensis sp. nov., a novel alkalitolerant bacterium from soil.

Two Arthrobacter strains, CA15-8(T) and CA15-9, were isolated from soil, under alkali conditions, and characterized phenotypically, chemotaxonomically and genetically. These alkalitolerant organisms grew over a wide pH range (pH 7.0-12.0), with an optimum at pH 7.0-8.0. The mean G+C content of the DNA of these strains was 63+/-2 mol%. The strains contained MK-8(H(2)) and MK-9(H(2)) as the main respiratory quinones. The cell-wall peptidoglycan was Lys-Thr-Ala(2) and the whole-cell sugar was rhamnose. The major cellular fatty acids of the isolates were anteiso-C(15 : 0) and iso-C(15 : 0). A phylogenetic analysis, based on 16S rDNA sequence data, revealed that strains CA15-8(T) and CA15-9 formed an evolutionary lineage distinct from other Arthrobacter species. On the basis of morphological, physiological and chemotaxonomic characteristics, 16S rDNA sequence comparisons and DNA-DNA hybridization data, a novel species of Arthrobacter is proposed, namely Arthrobacter koreensis (type strain CA15-8(T)=KCTC 9922(T)=IFO 16787(T)).

Arthrobacter↗

PubMed2003

Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha.

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

DNA Replication↗