Shigeru Morimura

Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales

Tests were conducted to evaluate the inhibitory effects of collagen peptides in the hydrolysate of sea bream scales on the activity of angiotensin I converting enzyme (ACE, EC3.4.15.1). The scales were hydrolyzed using an alkaline protease treatment by which 92% of the peptides were degraded to form hydrolysate. The 50% inhibitory concentration of the peptides was as high as 0.57 mg ml−1. In addition, using spontaneously hypertensive rats, oral administration of 300 mg of the peptides (kg of body weight)−1 d−1 was shown to decrease blood pressure significantly (P<0.05). Four peptides that demonstrated high ACE inhibitory activities were isolated from the hydrolysate of the scales using chromatographic methods. The ACE inhibitory activities of the isolated peptides were 5–20 times higher than that of the unpurified hydrolysate. The amino acid sequences of inhibitory peptides were determined to be Gly–Tyr, Val–Tyr, Gly–Phe and Val–Ile–Tyr.

PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra‐ or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat‐generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5‐fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome. Copyright

Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis

The requirement of Ni2+ and Co2+ addition on methanogenic activity and the coenzymes involved in methanogenesis were investigated in anaerobic continuous cultivation with synthetic wastewater using acetate as the sole carbon source. Addition of Ni2+ and Co2+ to the synthetic wastewater drastically increased the maximum dilution rate of the cultivation. The concentrations of coenzymes F430 and corrinoids in the biomass increased to 0.62 micromol-Ni/g-VSS and 0.67 micromol-Co/g-VSS, respectively with the increase of the dilution rate. Methanogenic activity of the culture broth also increased with an increase of dilution rate. However, without addition of Ni2+ and Co2+, F430 and corrinoids were not detected in the biomass and methanogenic activity was only a trace level at a dilution rate of 0.025 d(-1). When the amounts of Ni2+ and Co2+ added at a dilution rate of 0.6 d(-1) were lowered in steps, the concentrations of F430 and corrinoids in the biomass and methanogenic activity decreased with decreasing amounts of Ni2+ and Co2+ added. These results suggest that Ni2+ and Co2+ were required for the methane-producing reactions via increases of coenzymes F430 and corrinoids.

Antioxidative and antimutagenic activities of 4-vinyl-2,6-dimethoxyphenol (canolol) isolated from canola oil

A potent antioxidative compound in crude canola oil, canolol, was recently identified, and reported herein are studies of its scavenging capacity against the endogenous mutagen peroxynitrite (ONOO(-)). ONOO(-) is generated by the reaction between superoxide anion radical and nitric oxide, both of which are produced by inflammatory leukocytes. Among various antioxidative substances of natural or synthetic origin, canolol was one of the most potent antimutagenic compounds when Salmonella typhimurium TA102 was used in the modified Ames test. Its potency was higher than that of flavonoids (e.g., rutin) and alpha-tocopherol and was equivalent to that of ebselen. Canolol suppressed ONOO(-)-induced bactericidal action. It also reduced intracellular oxidative stress and apoptosis in human cancer SW480 cells when used at a concentration below 20 microM under H(2)O(2)-induced oxidative stress. In addition, canolol suppressed plasmid DNA (pUC19) strand breakage induced by ONOO(-), as revealed by agarose gel electrophoresis.

Effect of dilution rate on metabolic pathway shift between aceticlastic and nonaceticlastic methanogenesis in chemostat cultivation

ABSTRACT Acetate conversion pathways of methanogenic consortia in acetate-fed chemostats at dilution rates of 0.025 and 0.6 day−1 were investigated by using 13C-labeled acetates, followed by gas chromatography-mass spectrometry (GC-MS) analysis of the CH4 and CO2 produced. Nonaceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens was suggested to occupy a primary pathway (approximately 62 to 90%) in total methanogenesis at the low dilution rate. In contrast, aceticlastic cleavage of acetate by aceticlastic methanogens was suggested to occupy a primary pathway (approximately 95 to 99%) in total methanogenesis at the high dilution rate. Phylogenetic analyses of transcripts of the methyl coenzyme M reductase gene (mcrA) confirmed that a significant number of transcripts of the genera Methanoculleus (hydrogenotrophic methanogens) and Methanosarcina (aceticlastic methanogens) were present in the chemostats at the low and high dilution rates, respectively. The mcrA transcripts of the genus Methanosaeta (aceticlastic methanogens), which dominated the population in a previous study (T. Shigematsu, Y. Tang, H. Kawaguchi, K. Ninomiya, J. Kijima, T. Kobayashi, S. Morimura, and K. Kida, J. Biosci. Bioeng. 96:547-558, 2003), were poorly detected at both dilution rates due to the limited coverage of the primers used. These results demonstrated that the dilution rate could cause a shift in the primary pathway of acetate conversion to methane in acetate-fed chemostats.

Isolation, Identification, and Structure of a Potent Alkyl-Peroxyl Radical Scavenger in Crude Canola Oil, Canolol *

Alkylhydroperoxides in oxidized oil are undesirable components because they become alkylperoxyl radicals (ROO•) in the presence of heme, hemoglobin, or myoglobin in red meat. ROO• are biochemically reactive and damage nucleic acids and proteins, thereby harming living cells. We isolated a component, a highly potent ROO• scavenger, from crude canola oil (rapeseed), which we designated canolol, and identified its chemical structure, 4-vinyl-2,6-dimethoxyphenol. The canolol content of crude canola oil greatly increased after the seed was roasted as compared with that from unroasted seed, but it decreased in highly purified oil. This anti-ROO• activity was highest in crude oil, deceased after each refining step, and was lowest in highly purified refined oil. Canolol was thus generated during roasting. As shown previously, canolol is one of the most potent anti-ROO• components in crude canola oil and its potency is much greater than that of well-known antioxidants, including α-tocopherol, vitamin C, β-carotene, rutin, and quercetin.

Effect of Dilution Rate on Structure of a Mesophilic Acetate-Degrading Methanogenic Community during Continuous Cultivation

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d(-1) were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea, the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria. Within the domain Archaea, all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria, the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.

Synthesis of chitosan-caffeic acid derivatives and evaluation of their antioxidant activities

In this study, the antioxidant activities of different molecular weights (M(w)) and grafting ratios of chitosan-caffeic acid derivatives were investigated. The grafting process was achieved using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) as covalent connector under different conditions such as molecular-weight of chitosan, molar ratio of chitosan and caffeic acid, reaction temperature, pH, and reaction time. The half-inhibition concentrations (IC₅₀) of products were calculated by reduction of the 1,1-diphenyl picryl hydrazyl in the radical-scavenging assay and reduction of the Fe³+/ferricyanide complex to the ferrous form in reducing power assay. The EDAC showed maximum activity at 3-h, pH 5.0 and room temperature conditions, except high-molecular-weight chitosan in pH 2.0. The products were water-soluble in all pH and showed lower viscosity than native chitosan. The highest grafting ratio of caffeic acid was observed at 15% in low-molecular-weight chitosan. After 5% grafting of caffeic acid into chitosan, the grafting efficiency was increased by decreasing molecular-weight of chitosan at the same conditions. Caffeic acid has main role in the antioxidant activity of products. The maximum IC₅₀ of radical-scavenging activity (0.064 mg/ml) was observed at the highest caffeic acid containing derivative. Water-soluble chitosan and caffeic acid derivatives were obtained by this study without activity loss.

Ethanol production by repeated-batch fermentation at high temperature in a molasses medium containing a high concentration of total sugar by a thermotolerant flocculating yeast with improved salt-tolerance

An attempt was made to improve the salt tolerance of the thermotolerant flocculating yeast Saccharomyces cerevisiae strain KF-7 (KF-7) by maintaining a high concentration of KCl in the medium. Among selected strains, K211 had the highest cell viability and ethanol productivity in a molasses medium containing 25% (w/v) total sugar at 35°C. Strain K211 accumulated higher concentrations of glycogen and trehalose than did KF-7, and also remained alive in the stationary phase during batch fermentation in a jar fermentor. As a result of repeated-batch fermentation tests with K211, stable ethanol production was achieved with an ethanol concentration of 92 g/l and a productivity of 3.5 g/l·h at 33°C in 22% molasses medium. With KF-7, stable ethanol production could not be attained under these conditions. In addition, even at the higher temperature of 35°C, strain K211 gave stable ethanol production with an ethanol concentration of 91 g/l and a productivity of 2.7 g/l·h.

Repeated-batch fermentation process using a thermotolerant flocculating yeast constructed by protoplast fusion

Abstract A thermotolerant flocculating yeast, Saccharomyces cerevisiae KF-7 (KF-7), was constructed by protoplast fusion of the flocculating yeast S. cerevisiae IR-2 (IR-2) and the thermotolerant yeast S. cerevisiae EP-1 (EP-1). In repeated-batch fermentation with KF-7 at 30°C in a molasses medium containing 20% (w/v) total sugar, stable fermentations could be performed continuously and an ethanol productivity of 5 g/l·h was obtained. Even at 35°C a productivity of 3.6 g/l·h was achieved, which was 1.7 times higher than that achieved with IR-2. In a flask-cultivation test, the maximum growth rate and the decay constant of KF-7 were not influenced by an increase in temperature, unlike the results for IR-2. Thus, the fusant KF-7 had acquired thermotolerance. However, KF-7 may be inferior to IR-2 in terms of osmotic tolerance.