Yuta Mutaguchi

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.

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.

Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family.

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%.

Production of d-Branched-Chain Amino Acids by Lactic Acid Bacteria Carrying Homologs to Isoleucine 2-Epimerase of Lactobacillus buchneri

Isoleucine 2-epimerase (ILEP) is a novel branched-chain amino acid racemase isolated from Lactobacillus buchneri. In this study, we examined production of free d-branched-chain amino acids such as d-valine, d-leucine, and d-allo-isoleucine, using lactic acid bacteria carrying homologs to ILEP. Twelve selected strains of lactic acid bacteria were grown at optimal growth temperatures and accumulation of d-branched-chain amino acids in the medium was monitored in exponential, early stationary, and stationary phases. To analyze the d-branched-chain amino acids, enantiomers in the medium were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-isobutyryl-l-cysteine. The resultant fluorescent isoindole derivatives were analyzed on an octadecylsilyl stationary phase using ultra-high performance liquid chromatography. The analyses revealed that the seven following lactic acid bacteria carrying homologs showing 53–60% amino acid sequence identity to the L. buchneri ILEP accumulate d-branched-chain amino acids: Lactobacillus fermentum and Weissella paramesenteroides produce d-valine, d-leucine, and d-allo-isoleucine; Lactobacillus reuteri, Leuconostoc mesenteroides subsp. mesenteroides, and Leuconostoc gelidum subsp. gasicomitatum accumulate d-leucine and d-allo-isoleucine; and Lactobacillus vaginalis and Leuconostoc pseudomesenteroides produce d-allo-isoleucine. These results suggest that d-branched-chain amino acids are produced by a variety of lactic acid bacteria species, particularly those carrying homologs to the ILEP.

d-Amino Acids in Fermentative Foods

In recent years, d-amino acids have been shown to be present, in a free or bound state, in a wide variety of foods and beverages, and to exhibit properties that differ from those of l-isomers. It is anticipated that as more is learned about the properties of d-amino acids in foods, they will be applied for the improvement of food characteristics. In this section, we will summarize what is known about the distribution and metabolism of d-amino acids in foods and beverages, especially fermented types, as well as what is known about the enzymes involved in the metabolism of d-amino acids. In addition, applications in which d-amino acids are being used to improve the characteristics of food are described.