2007 Research results

1. Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

Author: T. Koseki, Y. Mese, S. Fushinobu, K. Masaki, T. Fujii, K. Ito, Y. Shiono, T. Murayama, H. Iefuji
Abstract: The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl -D-glucoside, p-nitrophenyl -D-cellobioside, and p-nitrophenyl -D-cellotrioside.
Source: Appl Microbiol Biotechnol. 77(6), 1279-1285 (2008)

2 . Effects of experience, eating habits, and way of drinking on sake preference

Author: Yasuko Manabe, Hitoshi Utsunomiya, Hanae Izu, Kuniaki Kiso, and Tohru Fushiki
Abstract: Palatability of food is affected by not only taste, but also physiological alteration after ingestion. To understand whether the palatability of sake in the oral sensation is also affected by its ingestion, we examined the relation between the preference order based on the preference test without swallowing sake and that based on the intake volume for 1hr in experienced sake tasters and sake beginners. We also investigated whether the palatability of sake in humans is similar to that in rats, whose preference is mostly attributed to the instinctive and physiological appetite. In the experienced sake tasters under fasting condition, the preference rank based on the intake volume was not affected by the ingestion of sake, which did not correspond to the rat preferences. The preference rank of the sake beginners under fasting differed between the experiments with or without swallowing sake, while their preference rank after eating was independent of the way of drinking. The preference rank of the sake beginners under fasting is affected by the way of drinking, but that of the experienced sake tasters remained unchanged.
Source: J. Brew. Soc. Japan, 102(12), 897-906 (2007)

3. Analysis of Asbestos and Microbes in Alcohol Beverages and Sake Cake

Author: Kuniyasu GOTO
Source: Report of the Research Institute of Brewing, 179, 44-48 (2007)

4. Elevated expression of genes under the control of stress response element (STRE) and Msn2p in an ethanol tolerance sake yeast Kyokai no.11

Author: Mamoru Watanabe, Kenichi Tamura, Jose Paolo Magbanua, Kaname Takano, Katsuhiko Kitamoto, Hiroshi Kitagaki, Takeshi Akao and Hitoshi Shimoi,
Abstract: The sake yeast strain Kyokai no. 11 (K11) is an ethanol tolerant mutant of strain Kyokai no. 7 (K7), which shows higher viability in an ethanol solution than strain K7. To clarify the mechanism underlying the ethanol tolerance of this strain, the gene expression profiles of K7 and K11 were analyzed using DNA microarrays. The results indicate that many genes induced by stresses were highly expressed in strain K11 not expected to stresses. Analysis of HSP12, one of the most highly expressed genes in strain K11 compared with strain K7, revealed that a trans-acting factor of strain K11 was involved in the elevated expression of HSP12. Many of the highly expressed genes in strain K11 including HSP12 were under the control of a cis-acting factor called the stress response element (STRE). The addition of STRE sequences to a promoter region of a reporter gene resulted in constitutive high-level expression in strain K11. It was reported that transcription factors Msn2p and Msn4p bind to STRE sequences. DNA sequence analyses of MSN2 and MSN4 of strains K7 and K11 revealed that only Msn2p was functional in these strains. When two copies of MSN2 in strain K11 were disrupted, the expression level of the reporter gene under the control of STRE decreased to the level of strain K7, indicating that Msn2p is required for the elevated expression of the STRE-controlled genes in K11.
Source: Journal of Bioscience and Bioengineering、104、163-170、(2007)

5. Application of Pesticide Captan in Viticulture and Its Inhibitory Effect on Fermentation

Author: Nami GOTO-YAMAMOTO, Hiroyuki YAMASHITA, Tomokazu HASHIGUCHI, Mineyo NUMATA
Abstract: The pesticide Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) has been reported to inhibit alcohol fermentation during wine making. However, further information on this effect is not available in Japan. Therefore, we investigated the inhibitory effect of Captan by adding it to a small-scale vinification process. In the making of red wine, Captan at 5 mg/L delayed the onset of fermentation by 1 to 2 days. In white or blush wine making, Captan at 1 mg/L delayed the onset by 1 day, and that at 3 mg/L delayed the onset by 2 to several days. Thus, even if the residual concentration of Captan is lower than that specified by Japanese regulations (5 mg/kg of grape berries, without stem), the concentration must be considered in wine making. Notably, when applied to Merlot grapes at 30 days before harvest, Captan did not influence the fermentation profile or the sensory evaluation of the wine produced. As Captan was not detected in the wine after fermentation, it is surmised that Captan gradually degraded under acidic conditions, as has previously been reported.
Source: J. ASEV Jpn., 18, 79-84 (2007)

6. Change in Monoterpene Concentration of Grapes during Berry Development and Influence of Vinification Conditions on Monoterpene Concentration in Muller-Thurgau Wine

Author: Noriko SOMA, Nami GOTO-YAMAMOTO , Atsuko ISOGAI, Jyunzo FURUKAWA
Abstract: Monoterpene alcohols are known to contribute to the aroma of Muller-Thurgau wine. In this study, we examined the change in monoterpene concentration of Muller-Thurgau grapes during maturation. Also, the effects of grape maturity and enological pectinase preparations on the monoterpene concentration in Muller-Thurgau wine were investigated. Linalool was the major monoterpene in Muller-Thurgau grapes, existing in both free and bound forms, and the total concentration of monoterpenes increased during maturation. Muller-Thurgau wine made from grapes collected 14 weeks after flowering (overripe) contained higher levels of monoterpenes and received higher organoleptic evaluation than wine made from 10-week-old grapes. Treatment with enological pectinase preparations possessing glycosidase activity increased the monoterpene concentration in Muller-Thurgau wine and improved the results of organoleptic evaluation. However, the increased monoterpene concentrations were not organoleptically detectable in ethanol solution. Thus, the enzyme treatment possibly increases the concentrations of unknown aroma compounds as well as monoterpenes.
Source: J. ASEV Jpn., 18, 85-93 (2007)

7. Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.) fruit at ripening stages

Author: Adebanjo A. Badejo, Seok T. Jeong, Nami Goto-Yamamoto, Muneharu Esaka
Abstract: Acerola (Malpighia glabra L.) is one of the richest natural sources of L-ascorbic acid (AsA; vitamin C). GDP-D-mannose pyrophosphorylase (GMP; EC 2.7.7.13) was found to play a major role in the proposed AsA biosynthetic pathway in plants, considering that Arabidopsis vtc1-1 mutant with point mutation in this gene has a highly reduced AsA content. GMP cDNA was isolated from acerola fruits, designated MgGMP, using rapid amplification of cDNA ends (RACE), and its expression was monitored during fruit ripening. The full-length cDNA was found to have an ORF of 1083 bp encoding a polypeptide of 361 amino acids. In silico analysis of the predicted amino acid sequence showed a pI of 6.45 and molecular mass of 39.7 kD. MgGMP showed over 80% amino acid sequence identity with other plant GMP homologues. The phylogenetic tree shows the close relation of MgGMP to the GMP of other plants as against those from parasite, yeasts and mammals. Southern analysis indicated that M. glabra contains not less than two copies of GMP genes. Northern blot analysis showed the transcript abundance of MgGMP in all the organs of acerola examined, with the fruit having the highest expression. The relative transcript abundance of MgGMP mRNA levels in the fruits changes as the ripening process progresses, with the unripe green fruits having the highest relative mRNA level, and the lowest was found in the fruits at advanced ripening stage. A strong correlation was also observed between the relative MgGMP mRNA levels and the AsA contents of acerola during fruit ripening.
Source: Plant Physiology and Biochemistry 45、665-672 (2007)

8. Protein expression of Saccharomyces cerevisiae in response to uranium exposure

Author: F.Sakamoto, T.Nankawa, N.Kozai, T.Fujii, H.Iefuji, A.J.Francis, T.Ohnuki
Abstract: Protein expression of Saccharomyces cerevisiae grown in the medium containing ²³⁸U(VI) and ²³³U(VI) was examined by two-dimensional gel electrophoresis. Saccharomyces cerevisiae of BY4743 was grown in yeast nitrogen base medium containing glucose and glycerol 2-phosphate and ²³⁸U of 0, 2.0, and 5.0 × 10^-4 M or ²³³U of 2.5 × 10^-6 M (radioactivity was higher by 350 times than 2.0 × 10^-4 M ²³⁸U) and 5.0 × 10^-6 M for 112 h at 30 ℃. The growth of Saccharomyces cerevisiae was monitored by measuring OD600 at 112 h after the inoculation. Uranium concentrations in the media also were measured by radiometry using a liquid scintillation counter. The growths of the yeast grown in the above media were in the following order: control > 2.5 × 10^-6 M ²³³U > 2.0 × 10^-4 M ²³⁸U > 5.0 × 10^-6 M ²³³U > 5.0 × 10^-4 M ²³⁸U. This result indicated that not only radiological but also chemical effect of U reduced the growth of the yeast. The concentrations of U in the medium containing ²³⁸U or ²³³U decreased, suggesting U accumulation by the yeast cells. The 2-D gel electrophoresis analysis showed the appearance of several spots after exposure to ²³⁸U or to ²³³U but not in the control containing no uranium. These results show that the yeast cells exposed to U express several specific proteins.
Source: Journal of Nuclear and Radiochemical Sciences, 8(2), 99-102 (2007)

9. Bitter-tasting Sake Peptides derived from the N-Terminus of the Rice Glutelin Acidic Subunit

Author: Katsumi HASHIZUME, Masaki OKUDA, Mineyo NUMATA, Kazuhiro IWASHITA
Abstract: Five bitter-tasting peptides were isolated from charcoal-untreated sake, following a Sepabeads resin separation, an initial reverse-phase chromatography (RP-HPLC), a gel permeation-chromatography, and a second RP-HPLC. The isolated peptides consisted of six to thirteen amino acid residues. The N-termini were uniformly pyroglutamate residues. Based on the rice protein database, the peptides were derived from two different N-termini of the rice glutelin acidic subunit. One of them was reported as a prolyl endopeptidase inhibitor. The thirteen-amino acid peptides in charcoal-untreated ginjyo-type sakes were lower than that in charcoal-untreated jyunmai-type sakes. The thirteen-amino acid peptides were not detected in the commercial ordinary-type sake analyzed. The concentration of analyzed peptides of nine to thirteen amino acid residues in charcoal-untreated sake exceeded their preliminary estimated sensory threshold values, suggesting that they contribute to the sensory quality of charcoal-untreated sake.
Source: Food Sci. Technol. Res., 13(3),270-274,2007

10. Characterization of Peptides Generated in Proteolytic Digest of Steamed Rice Grains by Sake Koji Enzymes

Author: Katsumi Hashizume, Masaki Okuda, Mineyo Numata, Yan Zhou, and Takuya Koseki
Abstract: High-molecular-weight peptides (approximately 10-30 kDa) generated in a digest of steamed rice grains by sake koji enzymes were characterized. Among thirteen major spots resolved by 2-D gel electrophoresis, twelve contained peptides having N-termini of rice glutelin as determined by mass fingerprinting analysis and/or MS/MS. The source of these peptides was presumed to be the acidic subunit of rice glutelin. An addition of up to 25% glucose in the digestion of an isolated rice protein body induced the accumulation of these peptides. The level of accumulation of these peptides in the digest of 70% polished rice samples correlated well with the crude protein content of the rice grains. The degree of accumulation of these peptides in Yamadanishiki and low-polish-rate rice was low, whereas that observed in 90% polished rice samples was extremely low.
Source: J. Biosci. Bioeng. 104,251-256,2007

11. Isc1 regulates sphingolipid metabolism in yeast mitochondria

Author: Hiroshi KITAGAKI, L. Ashley COWART, Nabil MATMATI, Silvia VAENA DE AVALOS, Sergei A. NOVGORODOV, Youssef H. ZEIDAN, Jacek BIELAWSKI, Lina M. OBEID, Yusuf A. HANNUN
Abstract: The Saccharomyces cerevisiae inositolsphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in -hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1⊿ in the post-diauxic phase showed drastic reduction in the levels of -hydroxylated phytoceramide (93.1 % loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1⊿ showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1⊿ exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.
Source: Biochem et Biophys Acta Biomembranes 1768, 2849-2861 (2007)

12. Mitochondrial dynamics of yeast during sake brewing

Author: Hiroshi KITAGAKI and Hitoshi SHIMOI
Abstract: The presence of mitochondria during alcohol fermentation has not been studied. Here, we examined the yeast mitochondrial structure during sake brewing using the green fluorescent protein. Mitochondrial structures were observed throughout brewing and they fragmented as brewing proceeded. This study is the first to show direct evidence of the presence of mitochondria during alcohol fermentation.
Source: J. Biosci. Bioeng., 104, 227-230 (2007)

13. Analysis of Sake Component Presented to the Sake Contest in 2005

Author: S. Nakano, A. Fujita, H. Utsunomiya, A. Isogai, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 179, 1-17 (2007)

14. Results of Sensory Evaluation and Analysis of the Western Type Alcoholic Beverages Presented to the 44th Contests

Author: S. Mikami, N. Goto, K. Koyama and J. Hiramatsu
Source: Report of the Research Institute of Brewing, 179, 18-33 (2007)

15. Analysis of Traditional Shochu Presented to the 29th Sake Contest in 2006

Author: S. Mikami, H. Fukuda, Y. Satoh and J. Hiramatsu
Source: Report of the Research Institute of Brewing, 179, 34-43 (2007)

16. Comparison of the Anton Paar Alcolyzer Method and the Official GC-FID Method of the National Tax Administration Agency of Japan for the Evaluation of Alcohol Content in Beer, Happo-Shu, and Nonalcoholic Beer

Author

T. Kaneko, S. Furusho, R. Ganaha, T. Inui, A. Matsuyama, A. Mizuno, M. Morimoto, K. Takemoto

Abstract

he results were received from collaborators who analyzed a total of four sample pairs. The data were obtained using the Anton Paar Alcolyzer method and the GC-FID method, whichis based on the official method of the National Tax Administration Agency Japan. No outliers were identified using Dixon’s ratio test, and all results were used.
Based on the paired t test for the differences in the means of the alcohol content, no statistically significant difference was found between the Anton Paar Alcolyzer and GC-FID methods.
Reproducibility errors of the two methods were also measured using an F test, and there was a statistical difference between the two methods. The reproducibility error of the Anton Paar Alcolyzer method (pooled variance 0.00061) was smaller than that of the GC-FID method (pooled variance 0.01882).

Source

J. Am. Soc. Brew. Chem., 65, 246-247 (2007)

17. Stimulatory Effect of Ferulic Acid on the Production of Extracellular Xylanolytic Enzymes by Aspergillus kawachii

Author: Takuya Koseki, Naoko Mimasaka,Katsumi Hashizume, Yoshihito Shiono, and Tetsuya Murayama
Abstract: Production of extracellular β-1,4-xylanase, α-L- arabinofuranosidase, feruloyl esterase, and acetyl xylan esterase from Aspergillus kawachii was higher in a culture supplemented with ferulic acid than in a counterpart. Culture supernatant grown on oat spelt xylan supplemented with ferulic acid exhibited an increase in ferulic acid-releasing activity from insoluble arabinoxylan relative as compared to that from the ferulic acid-free culture.
Source: Biosci. Biotechnol. Biochem., 71, 1785 -1787, 2007

18. Intraspecies Diversity of the Industrial Yeast Strains Saccharomyces cerevisiae and Saccharomyces pastorianus Based on Analysis of the Sequences of the Internal Transcribed Spacer (ITS) Regions and the D1/D2 Region of 26S rDNA

Author: KAWAHATA Miho, FUJII Tsutomu, IEFUJI Haruyuki
Abstract: We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.
Source: Biosci Biotechnol Biochem、71、1616-1620、(2007)

19. Hepatoprotective Effects of Concentrate and Components of Sake against Galactosamine (GalN)-Induced Liver Injury in Mice

Author: Hanae IZU, Kazuhisa HIZUME, Kuniyasu GOTO, and Masato HIROTSUNE
Abstract: We investigated the hepatoprotective effects of concentrate of sake (CS) and its components against D-galactosamine (GalN)-induced liver injury by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mice. CS significantly suppressed GalN-induced elevations of ALT and AST activities. Each of four concentrated fractions extracted from sake (consisting mainly of basic amino acids, neutral and acidic amino acids, organic acids or sugars, respectively) suppressed GalN-induced elevations of ALT and AST activities. We focused on the sugar fraction containing glucose and ethyl a-D-glucoside (a-EG), which is a sake-specific sugar, as major components and demonstrated that only a-EG showed significant suppression of GalN-induced elevations of ALT and AST activities. We compared the effects of a-EG analogues, methyl a-D-glucoside and ethyl b-D-glucoside, on GalN-induced liver injury and confirmed that only a-EG significantly suppressed both ALT and AST activities. Moreover, CS and a-EG suppressed GalN-induced production of IL-6 and liver DNA fragmentation. Together these results show that CS and its component, a-EG, suppressed GalN-induced liver injury through inhibition of IL-6 production.
Source: Biosci. Biotechnol. Biochem.（2007 71(4):951-957）

20. Change in the aroma of sake koji during koji-making

Author: Mie TAKAHASHI, Atsuko ISOGAI, Hitoshi UTSUNOMIYA, Shigeyoshi NAKANO, Takeo KOIZUMI, Akira TOTSUKA
Abstract: In order to clarify the changes in the aroma of sake koji during koji-making, we carried out sensory analyses and quantitative analyses of aroma compounds of sake koji. The concentration of phenylacetaldehyde reached a peak 40 hours after tokomomi. On the other hand, the concentrations of 1-octen-3-ol and 1-octen-3-one doubled in the 5 hours before de-koji (49 hours after tokomomi) and continued to increase after that. It was considered that the aroma of koji was sensed as kuri-ka (chestnut-like aroma) by the balance of the concentrations of those three compounds, and it was sensed as kinoko-ka (mushroom-like aroma) when the aroma of 1-octen-3-ol and 1-octen-3-one was strong, compared with other compounds. The formation mechanisms of these aroma compounds were also investigated. The concentration of linoleic acid, which is considered a precursor of 1-octen-3-ol, increased with the mycelial growth during koji-making. It was suggested that linoleic acid could be an index of the mycelial growth in koji-making. The crude enzymes were extracted from koji and added to a buffer solution containing linoleic acid or 1-octen-3-ol as substrate. It was confirmed that 1-octen-3-ol was generated from linoleic acid via 10-hydroperoxide (HOD) and 1-octen-3-one was generated by the enzymatic oxidation of 1-octen-3-ol. As the aroma of koji was the indicator of the growth of koji, it could also serve as an index for quality control in the koji-making process.
Source: J. Brew. Soc. Japan, 102, 403-411 (2007)

21. Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway.

Author: Hiroshi KITAGAKI, Yoshio ARAKI, Kouichi FUNATO and Hitoshi SHIMOI
Abstract: Cell death in yeast (Saccharomyces cerevisiae) involves several apoptotic processes. Here, we report the first evidence of the following processes, which are also characteristic of apoptosis, in ethanol-induced cell death in yeast: chromatin condensation and fragmentation, DNA cleavage, and a requirement for de novo protein synthesis. Mitochondrial fission protein, Fis1, appears to mediate ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation. However, mitochondrial fragmentation in response to elevated ethanol levels was not correlated with cell death. Further, in the presence of ethanol, generation of reactive oxygen species was elevated in mutant fis1D cells. Our characterization of ethanol-induced cell death in yeast as being Fis1-mediated apoptosis is likely to pave the way to overcoming limitations in large-scale fermentation processes, such as those employed in the production of alcoholic beverages and ethanol-based biofuels.
Source: FEBS Letters, 581, 2935-2942 (2007)

22. Influence of Maceration Temperature in Red Wine Vinification on Extraction of Phenolics from Berry Skins and Seeds of Grape (Vitis vinifera)

Author: Kazuya KOYAMA, Nami GOTO-YAMAMOTO, and Katsumi HASHIZUME
Abstract: The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions(cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, i.e., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, i.e., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, the modification of the temperature condition during maceration affected the progress in the concentration of phenolics, resulting in the alteration of their composition in the finished wine.
Source: Bioscience, Biotechnology, and Biochemistry、71、958-965、(2007)

23. Loss of anthocyanins in red-wine grape under high temperature

Author: Kentaro MORI, Nami GOTO-YAMAMOTO, Masahiko KITAYAMA, Katsumi HASHIZUME
Abstract: To determine the mechanism of inhibition of anthocyanin accumulation in the skin of grape berries due to high temperature, the effects of high temperature on anthocyanin composition and the responses in terms of gene transcript levels were examined using Vitis vinifera L. cv. Cabernet Sauvignon. High temperature (maximum 35 ℃) reduced the total anthocyanin content to less than half of that in the control berries (maximum 25 ℃). HPLC analysis showed that the concentrations of anthocyanins, with the exception of malvidin derivatives (3-glucoside, 3-acetylglucoside, and 3-p-coumaroylglucoside), decreased considerably in the berries grown under high temperature as compared with the control. However, Affymetrix Vitis GeneChip microarray analysis indicated that the anthocyanin biosynthetic genes were not strongly down regulated at high temperature. A quantitative real time PCR analysis confirmed this finding. To demonstrate the possibility that high temperature increases anthocyanin degradation in grape skin, stable isotope labelled tracer experiments were carried out. Softened green berries of Cabernet Sauvignon were cut and aseptically incubated on filter paper with 1 mM aqueous L- [1-¹³C]phenylalanine solution for 1 week. Thereafter, the changes in ¹³C-labelled anthocyanins were examined under different temperatures (15, 25, and 35 ℃). In the berries cultured at 35 ℃, the content of total ¹³C-labelled anthocyanins that were produced before exposure to high temperature was markedly reduced as compared with those cultured at 15 ℃ and 25 ℃. These data suggest that the decrease in anthocyanin accumulation under high temperature results from factors such as anthocyanin degradation as well as the inhibition of mRNA transcription of the anthocyanin biosynthetic genes.
Source: Journal of Experimental Botany, 58, 1935-1945, (2007)

24. Effect of Shading on Proanthocyanidin Biosynthesis in the Grape Berry

Author: A. Fujita, N. Soma, N. Goto-Yamamoto, A. Mizuno, K. Kiso, and K. Hashizume
Abstract: Proanthocyanidins, which are oligomers and polymers of flavan-3-ol units (e.g., (+)-catechin and (-)-epicatechin), are important components of grapes for red winemaking. Flavan-3-ols are biosynthesized by the catalysis of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). In this study, we investigated the effect of shading on proanthocyanidin biosynthesis in berries of Vitis vinifera ‘Cabernet Sauvignon’. Shading of the berries reduced the accumulation of proanthocyanidins and the transcription of ANR and LAR genes in the skins during berry development, while no significant effect was observed in the seeds. Because the proanthocyanidins significantly decreased in the skins and seeds of the control berries during ripening, the levels of proanthocyanidins were similar in the shaded and control berries at the harvest stage.
Source: J. Japan. Soc. Hort. Sci., 76 (2), 112-119 (2007)

25. The Promoter Activity of Isovaleryl-CoA Dehydrogenase-Encoding Gene (ivdA) from Aspergillus oryzae is Strictly Repressed by Glutamic Acid

Author: Nobuo YAMASHITA, Kazutoshi SAKAMOTO Osamu YAMADA, Osamu AKITA and Akira NISHIMURA
Abstract: We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to β-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.
Source: Bioscience, Biotechnology, and Biochemistry Vol. 71 (2007), No. 6 pp.1561-1563

26. Analysis of Expressed Sequence Tags from the Fungus Aspergillus oryzae Cultured Under Different Conditions

Author: Takeshi AKAO, Motoaki SANO, Osamu YAMADA, Terumi AKENO, Kaoru FUJII, Kuniyasu GOTO, Sumiko OHASHI-KUNIHIRO, Kumiko TAKASE, Makoto YASUKAWA-WATANABE, Kanako YAMAGUCHI, Yoko KURIHARA, Jun-ichi MARUYAMA, Praveen Rao JUVVADI, Akimitsu TANAKA, Yoji HATA, Yasuji KOYAMA, Shotaro YAMAGUCHI, Noriyuki KITAMOTO, Katsuya GOMI, Keietsu ABE, Michio TAKEUCHI, Tetsuo KOBAYASHI, Hiroyuki HORIUCHI, Katsuhiko KITAMOTO, Yutaka KASHIWAGI, Masayuki MACHIDA, and Osamu AKITA
Abstract: We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.
Source: DNA Res., 14, 47-57 (2007)

27. Construction and analysis of Self-cloning Sake Yeasts that Accumulate Proline

Author: Hiroshi TAKAGI, Fumi MATSUI, Akari KAWAGYCHI, Hong WU, Hitoshi SHIMOI, Yoshito KUBO
Abstract: We constructed self-cloning diploid sake yeast strains that accumulate proline. The appropriate proline level is important for its protective effect against ethanol stress in yeast cells. Sake brewed with the proline accumulating strains contained two- to three fold more proline than the sake brewed with the parent strain. It was also suggested that intracellular proline does not affect overall fermentation profiles, but reduces fermentation time in terms of ethanol production rate.
Source: J. Biosci. Bioeng., 103, 377-380 (2007)

28. Effects of Accumulated S-Adenosylmethionine on Growth of Yeast Cells

Author: M. SHOBAYASHI, T. FUJII, H. IEFUJI
Abstract: S-adenosylmethionine (SAM) accumulated in cultured yeast cells and affected growth in two ways. High levels of intracellular SAM in yeast inhibited early growth, but increased growth in medium without sources of nitrogen and sulfur. Accumulated SAM in the yeast cells was recycled as a nutritional source depending on the sulfur and nitrogen contents of the medium.
Source: Biosci Biotechnol Biochem , 71(6), 1595-1597 (2007)