Taketo Ohmori

Amino acid components of lees in salmon fish sauce are tyrosine and phenylalanine

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.

Distribution of D-amino acids in vinegars and involvement of lactic acid bacteria in the production of D-amino acids

Levels of free D-amino acids were compared in 11 vinegars produced from different sources or through different manufacturing processes. To analyze the D- and L-amino acids, the enantiomers were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine. This was followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using ultra-performance liquid chromatography. The analyses showed that the total D-amino acid level in lactic fermented tomato vinegar was very high. Furthermore, analysis of the amino acids in tomato juice samples collected after alcoholic, lactic and acetic fermentation during the production of lactic fermented tomato vinegar showed clearly that lactic fermentation is responsible for the D-amino acids production; marked increases in D-amino acids were seen during lactic fermentation, but not during alcoholic or acetic fermentation. This suggests lactic acid bacteria have a greater ability to produce D-amino acids than yeast or acetic acid bacteria.

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

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.

Increased tyrosine in the brain and serum of mice by orally administering dipeptide SY

We examined the effect of orally administering L-Ser-L-Tyr (SY) dipeptide on the brain of a serine deficiency disease model mouse to attain the efficient delivery of L-Tyr and L-Ser into the mouse brain. Oral SY administration increased the L-Tyr level more efficiently than L-Tyr administration with the same intake dose, but did not significantly affect the L-Ser level when compared with L-Ser administration.

Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by d-amino acids

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of d-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using 14C-labeled d-Trp showed that d-Trp in the medium was used as a protein component in the cells, suggesting the presence of d-amino acid metabolic enzymes. Pyridoxal 5′-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with d-amino acids, especially d-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of d-amino acids for growth.

Soy Peptide Ingestion Increases Neuroactive Amino Acids in the Adult Brain of Wild-Type and Genetically Engineered Serine-Deficient Mice

The aim of this study was to assess the neurochemical effects produced by short-term dietary soy peptide ingestion in C57BL/6 wild-type mice and in serine-deficient mice that were created as a genetic serine-deficiency disease model. D, L-Amino acid analysis demonstrated that overnight oral ingestion of a 35% (w/v) soy peptide solution significantly increased the hippocampal tissue content of certain neuroactive amino acids in both genotype groups of mice. These amino acids included the neurotransmitter l-glutamate, its precursor L-glutamine, the neuromodulator d-aspartate, and branched-chain amino acids l-valine, l-leucine, and l-isoleucine. Soy peptide ingestion caused similar increases in contents of L-glutamine and branched chain amino acids in the cerebral cortex. Oral ingestion of a 150 mM L-serine solution did not alter contents of these amino acids in the hippocampus and the cerebral cortex in both genotype groups. The present findings indicate that the short-term oral intake of soy peptide positively modulates the levels of certain neuroactive amino acids in the adult brain.

Effects of alkali or acid treatment on the isomerization of amino acids

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.

Draft Genome Sequence of d-Branched-Chain Amino Acid Producer Lactobacillus otakiensis JCM 15040T, Isolated from a Traditional Japanese Pickle

ABSTRACT Lactobacillus otakiensis strain JCM 15040T was isolated from an unsalted pickling solution used in the production of sunki, a traditional Japanese pickle. Here, we prepared a draft genome sequence for this strain consisting of 40 contigs containing a total of 2,347,132 bp, 2,310 predicted coding sequences, and a G+C content of 42.4%.

A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima

ABSTRACT The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T). Graphical Abstract The bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) from Thermotoga maritima is a novel type of the L-threonine-sensitive AK-HseDH.