Osamu Kurita

Kinetics of fatty acid binding ability of glycated human serum albumin.

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.

Improvement on the freeze–thaw stability of corn starch gel by the polysaccharide from leaves of Corchorus olitorius L.

Effect of the polysaccharide from leaves of Corchorus olitorius L. (PLC) on the freeze-thaw (FT) stability of corn starch gel was studied. PLC was incorporated into the starch gel at 0.7% and total solid was adjusted to 6.0%. The syneresis was measured by the centrifugal-filtration method and, as a result, addition of PLC reduced effectively the syneresis of the starch gel even after 5 FT cycles, which was less than one third that of the normal starch gel. The rheological changes of the starch/PLC gel during the FT treatments were evaluated while the gel remained on the rheometer plate. The starch/PLC gel had less significant changes in the rheological parameters during the FT cycles than starch/guar gum or xanthan gum gel systems. SEM images showed that PLC stabilized the gel matrix surrounding pores, which would contribute to both a lower syneresis production and a higher stability in the rheological behavior at FT.

Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939.

A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized. IFO 10939 had the ability of maintaining intracellular pH. The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0. Alkalification of growth medium exhibited a significant increase in acetate and propionate production. The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH.

Extraction and Characterization of the Pectic Substances from Japanese Pepper (Zanthoxylum piperitum DC.) Fruit

Pectic substances were extracted from Japanese pepper (Zanthoxylum piperitum DC) fruit with hot diluted HCl (90°C, pH 2.2). The yield of the pectic substances was 8.4% (w/w) based on dry material. The contents of total carbohydrate, anhydrogalacturonic acid, ash, protein, and moisture of the pectic substances were 84.1, 66.9, 3.6, 2.7, and 9.6% (w/w), respectively. The degree of methylation was estimated to be 80.3% by using alcohol oxidase test. This indicated that the pectic substances are high methoxyl pectins and have an ability to form sugar gels. The jelly grade of the pectic substances was estimated to be 130 (USA-SAG) at standard condition. Viscosity-average molecular weight was relatively low (30.3 kDa). When the molecular weight distribution was checked by gel-permeation chromatography on Sepharose CL-4B, the elution profile of the pectic substances was highly polydispersed extending from 40 kDa (or less) to 2000 kDa.

Isolation and characterization of a high-acetate-producing sake yeast Saccharomyces cerevisiae

The result of sensory evaluation of sake showed that acetic acid imparted desirable acidity when the proportion of acetic acid to lactic acid was about 1/3, even if the concentration of acetic acid was 0.75 g/l. Glycerol balanced the acidity and brought about a harmony between sweetness and acidity in sake. A high-acetate producing sake yeast (MHA-3) was isolated from mutants having low NADH dehydrogenase (NDE) activity. MHA-3 produced 15 times more acetate and 5 times more lactate than the parental strain Kyokai no. 901 (K-901) in a small-scale sake brewing test using 10 kg of rice. In addition, the concentrations of glycerol in sake brewed with MHA-3 were approximately 1.5-fold higher than in that brewed with K-901. The proportion of acetic acid to lactic acid was about 1/3 in sake fermented with MHA-3 and it exhibited a good balance between sweetness and acidity. The activities of glycerol-3-phosphate dehydrogenase (GPD) and aldehyde dehydrogenase (ALD) in MHA-3 were 1.4-fold and 3.1-fold, respectively, higher than those in K-901 while the activity of NDE was 40% that of K-901. MHA-3 accumulated higher amounts of acetate and glycerol than K-901 in static YNB10 medium. The concentrations of acetic acid produced, depending on the quantity of yeast cells added, increased in conjunction with increases in glycerol produced. We suggest that NDE might be linked with GPD and that the nde mutants, which can be used in sake brewing, produced higher amounts of acetate and glycerol.

Overexpression of Peroxisomal Malate Dehydrogenase MDH3 Gene Enhances Cell Death on H2O2 Stress in the ald5 Mutant of Saccharomyces cerevisiae

Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration. With use of the mutant, we examined the detoxication of H2O2 generation by fatty acid -oxidation in peroxisome. The ald5 mutant (AKD321), as well as the 746 0 mutant, was more resistant to H2O2 stress than the wild type. However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid -oxidation caused a decrease in cell viability of AKD321 to H2O2 stress, while the 746 0 mutant had no such effect. Intracellular H2O2 concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321. The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321. And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 0 strain, decreasing by 14% to that of 746 0 mutant. The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains. Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress. These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.

Feasible protein aggregation of phosphorylated poly-γ-glutamic acid derivative from Bacillus subtilis ( natto )

Poly-γ-glutamic acid (PGA) was modified with phosphorylating agents such as sodium metaphosphate and potassium metaphosphate in the culture medium of Bacillus subtilis (natto). The highly phosphorylated PGA derivatives were prepared and investigated for their chemical and physicochemical properties. The PGA derivatives had approximately 7% (W/W) inorganic phosphorus and characteristic absorbance PO2- bands at 1082cm-1 and 1260cm-1 by Fourier Transform Infrared Spectroscopy. The derivative modified by sodium metaphosphate (J-5) was easily hydrated in water and had extremely low viscosity. The shear rate-induced transition leading to the decrease of viscosity was not observed in J-5 whereas the derivative modified by potassium metaphosphate (J-6) as well as unmodified PGA (J-1) showed the typical decrease of viscosity. In circular dichroism (CD) measurement of J-5, there was a significant loss of the negative chirality CD signal, implying that protein aggregation occured at decreasing pH from 6.2 to 4.4. The thioflavin T fluorescence intensity of the aqueous solution in the J-5 was extremely high despite the absence of heat-treatment. The results indicate that the J-5 is the likeliest type of aggregation by β-sheet cross-linking which is relevant to protein diseases like Alzheimers disease.