Yukimichi Koizumi

Structure of green pigment formed by the reaction of caffeic acid esters (or chlorogenic acid) with a primary amino compound.

A marked greening observed in some foods such as sweet potato, burdock, and others during food processing was shown to be due to green pigment formation by the condensation reaction of two molecules of chlorogenic acid or caffeic acid ester with one molecule of a primary amino compound under aeration in alkaline solution. Reduction of the green pigment by ascorbic acid or NaBH4 gave a yellow product, which readily turn green and then blue in air. The reduced and acetylated product of the green pigment was identified to be a novel trihydroxy benzacridine derivative, and the yellowish ethanol solution of this product immediately turned green upon addition of butyl amine or diluted alkali. Therefore, the green pigment was assumed to be an oxidized quinone type product of trihydroxy benzacridine. This identification of the structure was supported by the correspondence of the measured absorption spectra with those calculated by the molecular orbital method. A possible charge transfer complex between products of different oxidation steps in green solution was proposed.

Cloning and characterization of groESL operon in Acetobacter aceti.

Abstract The groESL operon of Acetobacter aceti was cloned and sequenced. We observed that GroES and GroEL of A. aceti had high amino acid sequence homologies to GroES and GroEL of Escherichia coli and Bacillus subtilis . The upstream region of the groESL operon contained the heatshock promoter, which was previously reported in α-purple proteobacteria, and the highly conserved inverted repeat sequence. Phylogenetic analysis revealed that the A. aceti GroES and GroEL are very closely related to those of other α-purple proteobacteria. Transcription of this operon in A. aceti was induced by heat shock as well as by exposure to ethanol and acetic acid, which are present during fermentation of acetic acid. A. aceti that overexpressed the groESL was more resistant than the control strain to Stressors such as heat, ethanol, or acetic acid, indicating that GroES and GroEL are closely associated with the characteristic nature of Acetobacter and play an important role in acetic acid fermentation.

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase from Acetobacter aceti and Some Properties of its Disruptant

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Cloning and Characterization of the dnaKJ Operon in Acetobacter aceti

The dnaKJ operon of Acetobacter aceti was cloned and sequenced. The profile of the gene configuration was similar to that of other alpha-proteobacteria. In the DnaK and DnaJ proteins of A. aceti, the characteristic domains/motifs reported in other organisms were well conserved. This operon was transcribed in response to a temperature shift and exposure to ethanol/acetic acid. The overexpression of this operon in A. aceti resulted in improved growth compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against stressors present during fermentation, although the overexpression did not increase the resistance to acetic acid.

Purification and characterization of intracellular esterases related to ethylacetate formation in Acetobacter pasteurianus

Acetobacter sp. produce ethylacetate during acetic acid fermentation. We found that high ethylacetate-producing strains contained more intracellular esterase than low ethylacetate-producing strains. Acetobacter pasteurianus N-23, a high ethylacetate-producing strain, formed two major intracellular esterases (esterase-1 and esterase-2) that were regulated by ethanol. These two esterases were purified to homogeneity by means of hydrophobic interaction, ion exchange, gel filtration, and hydroxyapatite chromatographies. The two esterases hydrolyzed various esters, but only esterase-1 showed ethylacetate synthesis activity at pH 3. The molecular weights of esterase-1 and esterase-2 were 72 kDa and 44 kDa, respectively, as determined by gel filtration, and 38 kDa and 42 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Esterase-1 was inhibited by cysteine enzyme inhibitors such as diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These esterases may play some role in the production of ethylacetate by Acetobacter sp. during vinegar fermentation.

Alkalibacterium gilvum sp. nov., slightly halophilic and alkaliphilic lactic acid bacterium isolated from soft and semi-hard cheeses.

Nine novel strains of halophilic and alkaliphilic lactic acid bacteria isolated from European soft and semi-hard cheeses by using a saline, alkaline medium (7 % NaCl, pH 9.5) were taxonomically characterized. The isolates were Gram-stain-positive, non-sporulating and non-motile. They lacked catalase and quinones. Under anaerobic cultivation conditions, lactate was produced from D-glucose with the production of formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. Under aerobic cultivation conditions, acetate and lactate were produced from D-glucose. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 2.0 % and 5.0 % (w/v), with a growth range of 0-1 % to 15-17.5 %. The optimum pH for growth ranged between 8.5 and 9.5, with a growth range of 7.0-7.5 to 9.5-10.0. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied a phylogenetic position within the genus Alkalibacterium, showing the highest sequence similarity (98.2 %) to Alkalibacterium kapii T22-1-2(T). The isolates constituted a single genomic species with DNA-DNA hybridization values of 79-100 % among the isolates and <29 % between the isolates and other members of the genus Alkalibacterium, from which the isolates were different in motility and flagellation, growth responses to NaCl concentrations and pH, and profiles of sugar fermentation. The DNA G+C contents were between 36.0 and 37.6 mol%. The cell-wall peptidoglycan was type A4β, Orn-D-Asp. The major components of cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9c. Based on the phenotypic characteristics and genetic distinctness, the isolates are classified as a novel species within the genus Alkalibacterium, for which the name Alkalibacterium gilvum sp. nov. is proposed. The type strain is 3AD-1(T) ( = DSM 25751(T) = JCM 18271(T)).

Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283.

The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.

Cloning and Characterization of clpB in Acetobacter pasteurianus NBRC 3283

The clpB gene in Acetobacter pasteurianus was cloned and characterized. Although the clpB gene was transcribed in response to a temperature shift and exposure to ethanol, the clpB disruption mutant was only affected by high temperature, suggesting that the ClpB protein is closely associated with heat resistance in A. pasteurianus.

Role of intracellular esterases in the production of esters by Acetobacter pasteurianus

Esters are the major flavor compounds produced by Acetobacter sp. during vinegar production. The two genes encoding the esterases in the bacteria were disrupted, and the effects of the disruptions studied. When cultured in the presence of ethanol, the est1 gene-disrupted mutant (DE1K) did not produce any ethyl acetate or isoamyl acetate. However, the disruption of est2 did not affect the ester production. Ethyl acetate production by N-23 (pME122P) and DE1K (pME122P), which contain est1, was 1.7-fold higher than that by the wild type, N-23. On analyzing the relationship between ethyl acetate production and the extracellular ethanol and acetic acid concentrations, we found that the highest amount of ethyl acetate was produced when the molar ratio of ethanol and acetic acid was 1:1. These results indicate that the ester production by Acetobacter sp. is mostly catalyzed by the intracellular esterase, esterase-1, with ethanol and acetic acid used as the substrates.

Marked Antioxidative Activity of Seed Oils Developed by Roasting of Oil Seeds Part I. Synergistic Action of the Antioxidative Components in Roasted Sesame Seed Oil.

1) 焙煎ゴマ油の濃褐色成分はアンバーライトXAD-7に吸着し,グリセリドを主成分とする非極性の区分と分離することができた.2) 濃褐色成分をさらにシリカゲルカラムクロマトグラフィーにより分画した.酢酸エチル,メタノール溶出区分は褐色,濃褐色を呈し,抗酸化性を示したが,これらの区分にはセサモールやトコフェロールはほとんど検出されなかったので,未知の抗酸化成分の存在が示唆された.3) 焙煎ゴマ油中の抗酸化物質であるセサモール,トコフェロール,弱い抗酸化性を示したセサミン,2)で分画した酢酸エチルおよびメタノール区分の抗酸化性および相乗作用について重量法で調べた.その結果,酢酸エチルおよびメタノール区分そのものの抗酸化性はそれほど高くなく,また,焙煎ゴマ油に含まれるどの抗酸化物質も単独ではゴマ油の高い抗酸化性には及ぼなかった.4) 焙煎ゴマ油中の4種の抗酸化成分(セサモール,トコフェロール,セサミン,メタノール溶出区分)をこの油中の含量に相当する量でリノール酸に添加して,相乗作用を検討した.2種ずつを組み合わせるとセサミン+メタノール溶出区分の組み合わせを除いて活性が高まり,特にトコフェロールとの組み合わせで著しく活性が高まった.3種,4種の混合でさらに高い活性が得られ,特に,4種の混合が最も高い抗酸化性を示した.