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ABSTRACT: D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives.
Applied Microbiology and Biotechnology 05/2013; · 3.42 Impact Factor
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ABSTRACT: Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD(+), the other for NADP(+) and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81 %), but was not high in the region recognizing the 2' position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties.
Extremophiles 03/2013; · 2.94 Impact Factor
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ABSTRACT: Pediococcus lolii NGRI 0510Q(T) was isolated from ryegrass silage produced on Ishigaki Island, Okinawa Prefecture, Japan. Here we present a draft genome sequence for this strain, consisting of 103 contigs for a total of 2,047,078 bp, 2,154 predicted coding sequences, and a G+C content of 42.1%.
Genome announcements. 01/2013; 1(1).
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ABSTRACT: The effect of alkali treatment on the isomerization of amino acids was investigated. The 100×D/(D+L) values of amino acids from peptide increased with increase in the number of constituent amino acid residues. Furthermore, the N-terminal amino acid of a dipeptide was isomerized to a greater extent than the C-terminal residue.
Journal of Bioscience and Bioengineering 07/2012; 114(4):457-9. · 1.79 Impact Factor
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ABSTRACT: A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.
Biotechnology Letters 05/2012; 34(9):1693-9. · 1.68 Impact Factor
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ABSTRACT: To elucidate the mechanism of silica biodeposition in hot spring water, which is induced by Al(3+) ions bound to the surface of microbes, a chelate resin (Chelex 100) was used as a model compound of the surface of microbes. No silicic acid was adsorbed on the Na type Chelex 100, whereas silicic acids were significantly adsorbed to the Al type Chelex 100. In the Al type Chelex 100, the Al(3+) ions were present as 1:1 tridentate complex with iminodiacetate (IDA) group. After adsorption of silicic acid to Al type Chelex 100, a IDA-Al-O-Si-(OH)(3) site formed. The site acted as a template for the successive adsorption of silicic acids to form silica sheets around Al type Chelex 100 particles. In conclusion, Al(3+) ions bound to the surface of microbes play a key role as a trigger for the biodeposition of silica in hot spring water.
Colloids and surfaces. B, Biointerfaces 03/2012; 95:208-13. · 2.60 Impact Factor
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ABSTRACT: We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The Km values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible.
AMB Express. 11/2011; 1(1):43.
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ABSTRACT: We report that the lees in salmon fish sauce consist of Tyr and Phe. The concentration of free l-Tyr (2.0mM) was almost same as the saturated concentration (2.4mM) in water at 20°C. This result shows that lees are formed by Tyr precipitation due to its saturation in the sauce.
Journal of Bioscience and Bioengineering 06/2011; 112(3):256-8. · 1.79 Impact Factor
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ABSTRACT: Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.
Analytical Biochemistry 10/2010; 409(1):1-6. · 3.00 Impact Factor
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ABSTRACT: The activity of a dye-linked L-proline dehydrogenase (dye-L: -proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5-9.0 for 30 min at 50 °C. The enzyme catalyzed L-proline dehydrogenation to Δ(1)-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for L-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-L-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.
Applied Microbiology and Biotechnology 10/2010; 89(4):1075-82. · 3.42 Impact Factor
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ABSTRACT: Thermus thermophilus cells formed siliceous deposits in the presence of supersaturated silicic acid (600 p.p.m SiO(2)). The supersaturated silicic acid promoted interaction between cells and the inside walls of glass culture bottles, leading to the development of cell aggregates or biofilms. Electron probe microanalysis showed that within the aggregates most of the cell surfaces were covered with silica. Under these conditions, there was remarkable production of silica-induced protein (Sip), a solute-binding component of the Fe(3+)-binding ABC transporter. Furthermore, supersaturated silica enhanced resistance to the peptide antibiotics bacitracin, colistin and polymyxin B, which all act on the cell envelope. By contrast, supersaturated silica did not induce resistance to ampicillin, chloramphenicol, kanamycin and tetracycline, which inhibit peptide synthesis. Although strong expression of Sip was detected in liquid cultures of T. thermophilus in the presence of supersaturated silica and colistin, upregulated transcription of putative efflux pump and multidrug resistance ABC transporter genes were not detected by quantitative real-time PCR analysis. These findings suggest Sip promotes silica deposition on the surfaces of cells, after which the silicified outer membrane may serve as a 'suit-of-armor,' conferring resistance to peptide antibiotics.
The ISME Journal 03/2010; 4(6):809-16. · 7.38 Impact Factor
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ABSTRACT: The adhesion of Escherichia coli onto quartz, hematite and corundum was experimentally investigated. A strain of E. coli was used that had the genes for expressing protein for silica precipitation. The maximum cell adhesion was observed at pH <4.3 for quartz and at pH 4.5-8.5 for corundum. For hematite, cell adhesion remained low at all pH values. The microbe-mineral adhesion was assessed by the extended DLVO theory approach. The essential parameters for calculation of microbe-mineral interaction energy (Hamaker constants and acid-base components) were experimentally determined. The extended DLVO approach could be used to explain the results of the adhesion experiments. The effect of E. coli on the floatability of three oxide minerals was determined and the results showed that E. coli can act as a selective collector for quartz at acidic pH values, with 90% of the quartz floated at 1.5 x 10(9)cells/ml. However, only 9% hematite and 30% corundum could be floated under similar conditions. By using E. coli and no reagents, it was possible to separate quartz from a hematite-quartz mixture with Newton's efficiency of 0.70. Removal of quartz from the corundum mixture was achieved by E. coli with Newton's efficiency of 0.62.
Colloids and surfaces. B, Biointerfaces 08/2009; 74(1):140-9. · 2.60 Impact Factor
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ABSTRACT: A Gram-positive, coccus-shaped, lactic acid bacterium, strain NGRI 0510Q(T), was isolated from ryegrass silage produced in Okinawa Prefecture, Japan. The cell is non-spore-forming, non-motile, and occurs in pairs or tetrads. The strain is homofermentative and produces d- and l-lactic acid from glucose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NGRI 0510Q(T) belongs to the genus Pediococcus and clusters within the Pediococcus acidilactici and Pediococcus pentosaceus group, with 98.2 and 96.9 % sequence identity, respectively. DNA-DNA relatedness between strain NGRI 0510Q(T) and P. acidilactici JCM 8797(T) and P. pentosaceus JCM 5890(T) was 19.3 and 17.3 %, respectively. Based on its phenotypic characteristics, phylogenetic relationship and DNA-DNA relatedness, NGRI 0510Q(T) (=JCM 15055(T)=DSM 19927(T)) represents the type strain of a novel species, for which the name Pediococcus lolii sp. nov. is proposed.
International journal of systematic and evolutionary microbiology 06/2009; 59(Pt 5):1007-10. · 2.27 Impact Factor
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ABSTRACT: The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75 degrees C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO(2)), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe(3+) ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe(3+)-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.
Applied and environmental microbiology 03/2009; 75(8):2406-13. · 3.69 Impact Factor
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ABSTRACT: An alcohol dehydrogenase (ADH) gene, ST0053, from Sulfolobus tokodaii was expressed in Escherichia coli. The purified recombinant enzyme was an NAD-dependent medium-chain ADH with high thermostability and tolerance of a wide range of pHs. This is the first step in creating an experimental functionality library of 10 genes annotated as ADHs in the S. tokodaii genome.
Applied and environmental microbiology 02/2009; 75(6):1758-63. · 3.69 Impact Factor
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ABSTRACT: A gene (ST1218) encoding a D-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35kDa and was highly thermostable. It preferably acted as an NAD-dependent D-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.
Archives of Biochemistry and Biophysics 03/2008; 470(2):120-8. · 2.93 Impact Factor
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ABSTRACT: Numerous rod-shaped bacterial cells were present in amorphous silica which had formed on the copper plate placed in geothermal hot water, under conditions of pH 7.2 and 85±2°C. Bulk genomic DNA in the siliceous deposit was extracted, using lysozymes and the freeze-thaw method. The volume of siliceous deposit formed on one copper plate and the amount of genomic DNA extracted from siliceous deposits exponentially increased with the time of incubation. The phylogenetic diversity in these DNA extracts was investigated by cloning and sequencing of partial 16S rRNA genes obtained by PCR. The bacterial community was composed mainly of three phylogenetic types in domain Bacteria. Cluster I (8 clones) was affiliated with the Aquificales and cluster II (15 clones) was closely related to the genus Thermus. Cluster III (2 clones), the sequences of which were homologous with Gram-positive anaerobic thermophilic bacteria, was also detected. These extremely thermophilic bacteria may possibly contribute to the rapid aggregation of amorphous silica.
FEMS Microbiology Ecology 01/2006; 24(1):41 - 48. · 3.41 Impact Factor
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ABSTRACT: Streptomycetes and phages were isolated from soil in different locations of Thailand. The isolated phages, distinguished by host range determination, plaque morphology and phage morphology under transmission electron microscope, were partially purified by single plaque isolation. From host range determination in a variety of phages, phage Roi-1 was selected to serve as an antigen for immunization in rabbits. High titers of phage suspension were prepared by removal of host cells passing through 0.45 µm membrane filter. Five milliliters of the pure phage suspension was injected to three rabbits twice a week for 1 month. Sera from rabbits were subsequently collected and tested for cross reactivation against another 8 phages. The results revealed that phage Nsaw-30 gave a high degree of relatedness to phage Roi-1.
The Natural History Journal of Chulalongkorn University. 11/2003; 3:1-6.
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ABSTRACT: Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).
Bioscience Biotechnology and Biochemistry 05/2003; 67(4):797-802. · 1.28 Impact Factor
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ABSTRACT: To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.
Journal of Bioscience and Bioengineering 02/2003; 95(5):518-25. · 1.79 Impact Factor
