2005 Research results

1. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains

Author

M.Tominaga, YH.Lee, R.Hayashi, Y.Suzuki, O.Yamada, K.Sakamoto, K.Gotoh, O.Akita.

Abstract

To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40.

Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.

Source

Appl. Environ. Microbiol.. 72, 484-490(2006)

2. Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo Visualization of the FmanIBp-GFP Fusion Protein

Author: T.Akao, M.Yamaguchi, A.Yahara, K.Yoshiuchi, H.Fujita, O.Yamada, O.Akita, T.Ohmachi, Y.Asada, T.Yoshida.
Abstract: 1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.
Source: Biosci. Biotechnol. Biochem., 70(2), 471-479(2006)

3. Structural and Retrogradation Properties of Rice Endosperm Starch Affect Enzyme Digestibility of Steamed Milled-Rice Grains Used in Sake Production

Author: M.Okuda, I.Aramaki, T.Koseki, N.Inouchi, and K.Hashizume
Abstract: Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2 - 20.2%, number-average degree of polymerization (DP(n)) of amylose was 900 - 1,400, and the ratio of short-to-long chain amylopectin was 2.7 - 3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4℃ after gelatinization was 20 - 31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It was determined that the structure and retrogradation properties of endosperm starch in Japanese rice cultivars affect the decreasing rate of enzyme digestibility of the steamed, milled rice grains.
Source: Cereal Chemistry 83(2):143-151 (2006)

4. Difference in the Physical Properties of White-Core and Non-White-Core Kernels of the Rice Varieties for Sake Brewing is Unrelated to Starch Properties

Author: M.Tamaki, S.Kurita, M.Toyomaru, T.Itani, T.Tsuchiya, I.Aramaki and M.Okuda
Abstract: This study was designed to determine whether or not the difference in the physical properties between white-core and non-white-core kernels of the rice varieties for sake brewing is associated with their starch properties. We used two rice cultivars for sake brewing, Senbon-nishiki and Yamada-nishiki, from three different plots in Hiroshima prefecture. Hardness values of kernels were significantly higher in non-white-core than in white-core kernels in both varieties. Vickers hardness (VH) values were lowest at the center of the kernel in both types of kernels. VH values of white-core tissues of white-core kernels were significantly lower than those of corresponding tissues of non-white-core kernels. No significant differences were observed between the two types of kernels in VH values of the surrounding translucent tissues and in the starch properties (amylose content, pasting properties analyzed using a rapid viscoanalyzer and gelatinization properties analyzed using a differential scanning calorimetry). These results suggest that the difference in physical properties between the two types of kernels of the rice varieties for sake brewing are associated with the difference in structure of endosperm cells and not in starch properties.
Source: Plant Prod.Sci.9(1): 78-82 (2006)

5. Effects of temperature on anthocyanin biosynthesis in grape berry skins

Author: T.Yamane, S.T.Jeong, N.Goto-Yamamoto, Y.Koshita, and S.Kobayashi
Abstract: Although coloration of grape berry skins is influenced by temperature, the details of its effects have not been reported. To find temperature sensitive stages for coloration and to clarify the mechanisms that underlie the effect of temperature on anthocyanin accumulation, two-week treatments at temperatures of 20℃ and 30℃ were carried out at four different stages of development and ripening using each of three potted vines of Aki Queen (Vitis labrusca x V. vinifera). Anthocyanin accumulation in the skins was significantly higher at 20℃ than at 30℃ after the temperature treatment, and the most sensitive stage for the temperature treatment was from one to three weeks after coloring began (stage III). Furthermore, at harvest, the grapes treated at 20℃ in stage III contained the highest concentration of anthocyanin. After temperature treatment in stage III, the concentration of abscisic acid (ABA), a plant hormone related to anthocyanin accumulation, in the berry skins was 1.6 times higher at 20℃ than at 30℃. The copy numbers of accumulated mRNA of anthocyanin biosynthetic enzyme genes and a myb-related regulate gene, VvmybA1, were also higher at 20℃ than at 30℃. These results and previous reports indicate that the high and low temperatures during ripening, especially in stage III, likely affect the production and/ or degradation of ABA in berry skins and that the endogenous ABA level affects the expression of VvmybA1; the product of VvmybA1 then controls the expression of the anthocyanin biosynthetic enzyme genes.
Source: Am. J. Enol. Vitic., 57 (1) 54-59 (2006)

6. Development of Grape Microsatellite Markers and Microsatellite Analysis Including Oriental Cultivars

Author: N.Goto-Yamamoto, H.Mouri, M.Azumi, and K.J.Edwards
Abstract: Nine newly developed microsatellite markers for grapes were characterized. Using these markers and eight reported markers, seven Occidental and eight Oriental cultivars were analyzed, including Japanese and Chinese cultivars of Vitis vinifera, two cultivars of V. labrusca, and one sample each of V. riparia and V. rotundifolia. Calculated phenetic distances (1 - proportion of shared alleles) agreed well with the classification of grapes. A dendrogram based on the phenetic distances showed a clear separation of species of Vitis, as well as Oriental and Occidental cultivars. Results suggested the importance of Oriental cultivars as genetic resources of grapes.
Source: Am. J. Enol.Vitic., 57 (1), 105-108 (2006)

7. Rice Bran Fractions Improve Blood Pressure, Lipid Profile, and Glucose Metabolism in Stroke-Prone Spontaneously Hypertensive Rats

Author: Ardiansyah,* H.Shirakawa, T.Koseki, K.Ohinata, K.Hashizume, and M.Komai
Abstract: Effect of dietary supplementation of two types of rice bran fraction on blood pressure (BP), lipid profile, and glucose metabolism in stroke-prone spontaneously hypertensive rats was studied. Male 4-week-old rats were divided into one group fed the AIN-93M-based control (C) diet and two groups fed diet supplemented with 60 g/kg of Driselase and ethanol fractions (DF and EF, respectively) of rice bran. After 8 weeks feeding, the BP decreased in the DF and EF groups in comparison with the C group (p < 0.01). Plasma ACE inhibitory activity, BUN, BUN/creatinine ratio, albumin, triglyceride, and glucose levels were lower in the DF and EF groups than in the C group (p < 0.01). Plasma nitric oxide and urinary 8-hydroxy-2'-deoxyguanosine levels were lower in the DF and EF groups than in the C group (p < 0.01). Rice bran fractions appear to have a beneficial dietary component that improves hypertension, hyperlipidemia, and hyperglycemia.
Source: J. Agric. Food Chem., 54 (5), 1914 -1920, 2006.

8. An Aspergillus oryzae acetyl xylan esterase: Molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris

Author: T.Koseki, Y.Miwa, T.Akao, O.Akita, K.Hashizume
Abstract: We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused α-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.
Source: Journal of Biotechnology 121 (2006) 381-389

9. Characterization of Low-Acetic-Acid-Producing Yeast Isolated from 2-Deoxyglucose-Resistant Mutants and Its Application to High-Gravity Brewing

Author: A.Mizuno, H.Tabei, and M.Iwahuti
Abstract: We isolated a mutant with low acetic acid and high ethanol productivities from 2-deoxyglucose-resistant mutants of brewer's yeast NCYC1245 (Saccharomyces cerevisiae). To determine the mechanism for these properties in the mutant (2DGR19) during fermentation, gene expression and enzyme activity related to acetic acid and ethanol production were investigated. DNA microarray analysis revealed that the transcriptional levels of many genes involved in glycolysis were higher in 2DGR19 than in NCYC1245. Among these transcriptional levels of 2DGR19 relative to NCYC1245, the expression level of ADH4 encoding alcohol dehydrogenase (ADH) was highest, which corresponded to the high ADH activity in 2DGR19. Quantitative PCR analysis also revealed that the transcriptional level of ADH4 was the highest among ADH1 to ADH4. Although no significant differences in the transcriptional levels of ALD2 to ALD6 encoding acetaldehyde dehydrogenase (ALD) between 2DGR19 and NCYC1245 were observed, ALD activity in 2DGR19 was lower. Using quantitative PCR analysis, ALD6 was found to be the most highly expressed among the ALD2 to ALD6 genes. These results indicate that ALD6 contributes to a low ALD activity, depending on post-transcriptional regulation. A high ADH activity appeared to be the major reason for the high ethanol productivity of 2DGR19. A low ALD activity was considered to be principally responsible for a low acetic acid productivity, although a high ADH activity also might have played a role. Beer brewed using 2DGR19 in pilot-scale high-gravity brewing contained about half as much acetic acid and 1.1% more ethanol compared with that brewed using NCYC1245. The use of 2DGR19 may overcome difficulties associated with high-gravity brewing.
Source: J. Biosci. Bioeng., 101, 31-37 (2006)

10. GPI-anchored cell suurface protein of white koji mold

Author

Y.Nakamura, H.Shimoi and K.Ito

Abstract

A candidate of the GPI-anchored cell surface protein (CwpA) was obtained from Aspergillus kawachii. This protein possessed hydrophobic regions in both the N-terminus and C-terminus, a feature indicating the characteristics of a GPI-anchored protein.

CwpA existed in the membrane fraction of the cell, and it was extracted into the detergent phase (Triton X114) when not treated with PI-PLC, but it remained in the aqueous phase when treated with PI-PLC. CwpA, which was deleted from the GPI anchor signal by the insertion of an artificial stop codon just before the C-terminal hydrophobic region, was secreted. These results suggest that CwpA is a GPI-protein.

Fluorescence was observed at the cell surface in the transformant of the GFP-CwpA fusion gene, indicating the fact that CwpA is anchored to the cell membrane via GPI.

Source

J. Brew. Soc. Japan, Vol.101, 53-60 (2006)

11. Aroma compounds responsible for “hineka” in commercial sake

Author: A.Isogai, H.Utsunomiya, R.Kanda, H.Iwata and S.Nakano
Abstract: To examine aroma compounds responsible for “hineka” in commercial sake, we carried out a quantitative analysis of aroma compounds and a sensory analysis for commercial sake in which “hineka” was pointed out in the national survey of commercial sake. We also analyzed commercial sake without “hineka” and commercial aged sake (stored for more than 3 years) for comparison. Volatile aldehydes, ethyl esters, polysulfides (DMDS and DMTS), furfural and sotolon increased in the order of : commercial sake without “hineka”, commercial sake with “hineka”, commercial aged sake. Result of principal component analysis suggested that commercial sake with “hineka” was characterized by relatively high levels of polysulfides, while commercial aged sake stored for long periods was characterized by high levels of sotolon, volatile aldehydes, furfural and diethyl succinate. A sensory analysis of the commercial sake showed that the logarithm of DMTS concentration correlated with “hineka” score, while that of sotolon did not. These results suggest that DMTS contributes greatly to “hineka” in commercial sake
Source: J. Brew. Soc. Japan、Vol.101, 125-131 (2006)

12. A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

Author

M.Shobayashi, N.Mukai, K.Iwashita, Y.Hiraga, H.Iefuji

Abstract

S-adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study.

In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolate by this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.

Source

Appl. Microbiol. Biotechnol. 69(6), 704-710 (2006)

13. Homocysteine accumulation causes a defect in adenine biosynthesis: further characterization of Schizosaccharomyces pombe methionine auxotrophs

Author

Y.Fujita, E.Ukena, H.Iefuji, Y.Giga-Hama, K.Takegawa.

Abstract

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis i.e.,

methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, thus the gene was identified as a methionine synthase and designated met26+. We investigated additional phenotypes of the met26 mutant and found that it exhibited a remarkable growth defect in the absence of adenine even in media supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae that has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from S. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into S. pombe met26D cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in met26D cells was higher than in other methionine auxotrophs and that introduction of the S. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in adenine biosynthesis in the met26 mutant.

Source

Microbiology, 152, 398-404 (2006)

14. Comparison of the chiral recognition of prochiral substrates in the acetylation reaction by a novel lipase (CSL) from the yeast, Cryptococcus spp. S-2 with immobilized PPL Enzyme-catalyzed desymmetrization and asymmetrization of prochiral 2-substituted 1,3-propanediols by CSL and immobilized PPL

Author: C.Lin, Y.Hiraga, K.Masaki, H.Iefuji and K.Ohkata
Abstract: In order to elucidate the nature of a novel lipase (CSL), isolated from the yeast Cryptococcus spp. S-2, in chiral recognition by comparison with that of immobilized PPL, the desymmetrization and asymmetrization of prochiral 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c), 2-benzyl-2-methyl-1,3-propanediol (1d), 2-ethyl-2-phenyl-1,3-propanediol (1e), and 2-benzyl-2-ethyl-1,3-propanediol (1f) by acetylation was investigated. Acetylation of 1a with excess vinyl acetate by the CSL-enzyme catalyst gave the corresponding monoacetate 2a with high enantioselectivity (80% ee) in 46% yield. Very high levels of desymmetrization were observed in the tertiary systems of 1c-f, giving the corresponding monoacetates 2c-f, respectively, in >97%. In the desymmetrization of diols 1a, 1c, 1d, and 1f, the sense of chiral differentiation of CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).
Source: J. Molecular Catalysis B: Enzymatic 38 1-10 (2006)

15. Organ-Specific Transcription of Putative Flavonol Synthase Genes of Grapevine and Effects of Plant Hormones and Shading on Flavonol Biosynthesis in Grape Berry Skins

Author: A.Fujita, N.Goto-Yamamoto, I.Aramaki, and K.Hashizume
Abstract: In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis.
Source: Biosci. Biotechnol. Biochem., 70 (3), 632-638 (2006)

16. Properties of Starch and Protein of "Hattan-Type Varieties" of Rice Suitable for Brewing Original Hiroshima Sake

Author: M.Tamaki, R.Kihara, M.Okuda, I.Aramaki, Z.Katsuba and T.Tsuchiya
Abstract: By successive crossing using Hattan-type varieties originating from Hattanso" as a parent, "Hattan-type varieties" of rice suitable for brewing the original Hiroshima sake have been bred. In this study, the difference in the properties of starch and protein among the Hattan-type varieties was examined. Six Hattan-type varieties, Hattanso, Hattan No.10, Hattan No.35, Hattan No.40, Hattan-nishiki No.1 and Hattan-nishiki No.2, were used. As the properties of starch, amylose content, pasting properties and gelatinization properties were examined. The pasting and gelatinization properties were examined using a rapid viscoanalyzer (RVA) and a differential scanning calorimetry (DSC), respectively. As the properties of protein, the compositional ratio of two types of protein bodies (PB-II/PB-I) was analyzed. However, no significant differences in the above properties were observed among these Hattan-type varieties. The above properties of starch and protein in Hattanso seem to be retained in all of these varieties. In these varieties, breeding might not have been aimed at improvement of the properties of starch and protein.
Source: Plant Prod.Sci. 8(5):586- 591 (2005)

17. Expression of the flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes and flavonoid composition in grape (Vitis vinifera)

Author: S.T.Jeong, N.Goto-Yamamoto, K.Hashizume, and M.Esaka
Abstract: Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are involved in the biosynthesis pathway of cyanidin- and delphinidin-based anthocyanins, as well as quercetin and myricetin (flavonols), and procyanidin and prodelphinidin (flavan-3-ols). Grape (Vitis vinifera) accumulates these three classes of flavonoids and is presumed to have both F3'h and F3'5'h. Thus, we obtained the genomic sequences of F3'h and F3'5'h from grape and determined their mRNA levels as well as the flavonoid composition of several grape organs to investigate whether the transcription of these genes controls the flavonoid composition. The flower, stem, tendril, and seed, which accumulated a higher level of mRNA of F3'h than F3'5'h, showed reasonable compositions of flavonols and/or flavan-3-ols. The organs that accumulated a high level of F3'5'h mRNA contained a high level of delphinidin-based anthocyanins (berry skin at the harvest stage) or prodelphinidin (small leaf), consistent with the mRNA levels. However, not all the classes of flavonoid showed a similar composition according to the mRNA levels of F3'h and F3'5'h. The compositions of anthocyanins and flavonols in the small leaf were inconsistent with the mRNA levels. Consequently, other mechanisms in addition to gene transcription are also expected to control the composition of each flavonoid class.
Source: Plant Sci. 170: 61-69 (2006)

18. Anthocyanidin Reductase Gene Expression and Accumulation of Flavan-3-ols in Grape Berry

Author: A.Fujita, N.Soma, N.Goto-Yamamoto, H.Shindo, T.Kakuta, T.Koizumi, and K.Hashizume
Abstract: Proanthocyanidins, which are polymers and oligomers of flavan-3-ol units, are important components of grape for red winemaking and accumulate in the berry skins and seeds. Major flavan-3-ol units of grape proanthocyanidins are (-)-epicatechin and (-)-epigallocatechin. It was recently reported that (-)-epicatechin and (-)-epigallocatechin were biosynthesized from corresponding anthocyanidins by anthocyanidin reductase (ANR). In the current study, we obtained a genomic sequence of the ANR gene of Vitis vinifera cv. Cabernet Sauvignon (VvANR). The deduced amino acid sequence of VvANR conserved the characteristic sequence of ANR of other plants. Southern blot analysis showed that grape ANR is probably encoded by a single copy gene in its haploid genome. Real-time quantitative-PCR using berry skins and seeds showed that the mRNA of VvANR accumulates at the early stage of berry development and then decreases toward the ripening stage. Our analysis and other reports show that proanthocyanidins accumulate in the berry skins and seeds before veraison, especially during the early stage of development, and then decrease in concentration during ripening. Thus, the change of mRNA accumulation substantially coincides with the change of proanthocyanidin accumulation in the berry skins and seeds. These results indicate that at least a part of (-)-epicatechin and (-)-epigallocatechin biosynthesis is probably controlled by the transcription of VvANR.
Source: Am. J. Enol. Vitic., 56, 336-342 (2005)

19. Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics

Author: K.Masaki, N.R.Kamini, H.Ikeda, and H.Iefuji
Abstract: A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (e-caprolactone), and poly(3-hydroxybutyrate).
Source: Appl. Environ. Microbiol., 71 (11), 7548-7550 (2005)

20. Effects of the culture conditions on ergosterol biosynthesis by Saccharomyces cerevisiae

Author: M.Shobayashi, S.Mitsueda, M.Ago, T.Fujii, K.Iwashita, H.Iefuji
Abstract: Ergosterol is an essential component of yeast cell to maintain the integrity of membrane and investigated as an important factor of ethanol tolerance of yeast cells. We investigated the effects of brewing conditions on ergosterol contents of S. cerevisiae K-9, sake yeast, kinds of Saccharomyces cerevisiae, produce more than 20% of ethanol, and X2180-1A, laboratory yeast. K-9 has a higher total ergosterol contents under the all conditions which we examined than X2180-1A. Ethanol and hypoxia were found to have negative and synergistic effects on total ergosterol contents of both strains and significantly reduced the free ergosterol contents of X2180-1A but only slightly reduced the free ergosterol contents of K-9. The maintenance of free ergosterol contents under brewing conditions might be an important character of sake yeast strains. DNA microarray analysis also showed higher expression of ergosterol biosynthesis genes in K-9 than X2180-1A.
Source: Biosci.Biotechnol.Biochem.,69,2381-2388(2005)

21. Genome sequencing and analysis of Aspergillus oryzae

Author: M.Machida, K.Asai, M.Sano, T.Tanaka, T.Kumagai, G.Terai, K.Kusumoto, T.Arima, O.Akita, Y.Kashiwagi, K.Abe, K.Gomi, H.Horiuchi, K.Kitamoto, T.Kobayashi, M.Takeuchi, D.W.Denning, J.E.Galagan, W.C.Nierman, J.Yu, D.B.Archer, J.W.Bennett, D.Bhatnagar, T.E.Cleveland, N.D.Fedorova, O.Gotoh, H.Horikawa, A.Hosoyama, M.Ichinomiya, R.Igarashi, K.Iwashita, P.R.Juvvadi, M.Kato, Y.Kato, T.Kin, A.Kokubun, H.Maeda, N.Maeyama, J.Maruyama, H.Nagasaki, T.Nakajima, K.Oda, K.Okada, I.Paulsen, K.Sakamoto, T.Sawano, M.Takahashi, K.Takase, Y.Terabayashi, J.R.Wortman, O.Yamada, Y.Yamagata, H.Anazawa, Y.Hata, Y.Koide, T.Komori, Y.Koyama, T.Minetoki, S.Suharnan, A.Tanaka, K.Isono, S.Kuhara, N. Ogasawara and H. Kikuchi
Abstract: The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.
Source: Nature, 438(No.7071), 1157-1161 (2005)

22. Identification and Characterization of a Novel Biotin Biosynthesis Gene in Saccharomyces cerevisiae

Author: H.Wu, K.Ito, and H.Shimoi
Abstract: Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.
Source: Appl. Environ. Microbiol., 71, 6845-6855 (2005)

23. Analysis of ethyl carbamate in sake and shochu

Author: N.Mukai, K.Kiso
Abstract: The authors investigated the content of Ethyl Carbamate (EC), a suspected carcinogen, in 180 samples of alcoholic beverages including sake, synthetic-sake, and shochu. The commercial 157 sakes which we used for analysis was as follows: 125 normal products (without over three years of storage with a manufacturer), and 32 koshu (with over three years of storage with a manufacturer). The number of samples of synthetic-sake was 4. The commercial 19 shochu which we used for analysis were as follows: 6 sweet potato shochu, 5 wheat shochu, 4 rice shochu, one muscovado shochu, one soba shochu, one sesame shochu, and one awamori. The sake (normal products) contained 47 μg/kgof EC on the average, and 210 μg/kg of EC at the maximum. The sake (koshu) contained 183 μg/kgof EC on the average, and 1100 μg/kg of EC at the maximum. The content of EC in all of the synthetic-sakes was less than the limit of quantification (20 μg/kg). The content of EC in 15 of shochu was less than the limit of quantification, and the maxium level of EC in shochu was 37 μg/kg. A high regular correlation was present in koshu between ultraviolet absorption and EC content.
Source: J. Brew. Soc. Japan, 100(10), 705-714 (2005)

24. Production of shochu fortified with 4-vinylguaiyacol

Author: N.Mukai, K.Kiso, H.Iefuji
Abstract: The authors attempted the production of shochu fortified with 4-vinylguaiacol (4-VG), which is thought to be the precursor of vanillin. In the small-scale fermentation tests of rice-shochu and awamori, the content of free ferulic acid in mash was increased by the addition of enzyme preparation hemicellulase, and consequently the content of 4-VG in distillate was also increased. The content of 4-VG in distillate increased more with the additional use of wine yeast, which has the ability to generate 4-VG from ferulic acid.
Source: J. Brew. Soc. Japan, 100(11), 832-835 (2005)

25. Effects of abscisic acid treatment and night temperatures on anthocyanin composition in Pinot noir grapes

Author: K.Mori, H.Saito, N.Goto-Yamamoto, M.Kitayama, S.Kobayashi, S.Sugaya, H.Gemma and K.Hashizume
Abstract: Potted Pinot noir grapevines were grown under continuous high temperature (30℃) or low night (15℃) and high day (30℃) temperatures after veraison. Half of the total number of clusters of each vine was sprayed with 250 ppm abscisic acid (ABA) at veraison. Anthocyanin accumulation in berry skins grown under high night temperatures was lower than that in berries grown under low night temperatures. HPLC analysis showed that the ratios of delphinidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside to the total anthocyanin content were greatly reduced under high night temperatures. ABA treatment enhanced anthocyanin accumulation under high night temperatures to almost the same level as under low night temperatures; the ratio of each anthocyanin to the total anthocyanin was not affected by ABA treatment.
Source: Vitis 44 (4), 161-165 (2005)

26. Mechanisms of uranium mineralization by the yeast Saccharomyces cerevisiae

Author: T.Ohnuki, T.Ozaki, T.Yoshida, F.Sakamoto, N.Kozai, E.Wakai, A.J.Francis, and H.Iefuji
Abstract: We determined the association of uranium in yeast cells S. cerevisiae grown in medium containing high (1g･L-1) or low (0.2g･L-1) concentrations of phosphate after exposure for 96 h to a 4 10-4mol･L-1 U(VI) solution at pH 3.2 or 4.7. The analysis was made using a field emission scanning electron microscope equipped with energy dispersive spectroscopy (FESEM-EDS), transmission electron microscopy (TEM), and visible diffuse reflectance spectrometry. Cells grown in the high-phosphate medium rapidly accumulated U(VI) from solution at pH 3.2 over the first 24 h, followed by a slow uptake until 96 h, whereas in cells grown in low-phosphate medium, U(VI) accumulation reached a steady state within 24 h. FESEM-EDS analyses revealed the formation of a U(VI)-bearing precipitate on the yeast cells grown in high-phosphate medium after only 48 h exposure; no precipitate was detected on cells grown in low-phosphate medium up to 96 h. These results suggest that sorption onto the cell surfaces was the dominant process initially. Analysis of the U(VI)-bearing precipitates by all three methods demonstrated the presence of H-autunite, HUO2PO4 ･ 4H2O. Thermodynamic calculations suggest that the chemical compositions of the solutions containing yeast grow in high-phosphate medium were undersaturated with respect to H-autunite, but were supersaturated with ten times more U(VI) and P than were actually observed. Apparently, the sorbed U(VI) on the cell surfaces reacts with P released from the yeast to form H-autunite by local saturation. The U(VI) uptake by yeast cells grown in high phosphate medium at pH 4.7, along with the thermodynamic calculation, indicated that more H-autunite is precipitated in neutral pH solution than in acid solution. Thus, U(VI)-phosphate mineralization on the cells of microorganisms should be taken into account for predicting U(VI) mobility in the environment.
Source: Geochimica et Cosmochimica Acta, 69(22), 5307-5316 (2005)

27. Effect of Uranium (VI) on the Growth of Yeast and Influence of Metabolism of Yeast on Adsorption of U (VI)

Author: F.Sakamoto, T.Ohnuki, N.Kozai, E.Wakai, T.Fujii, H.Iefuji, and A.J.Francise
Abstract: We have carried out the growth experiments of 3 strains of yeast in a medium containing uranium (VI) to elucidate the effect of U (VI) on the growth of microorganisms. Hansenula fabianii J640 grew in the liquid medium containing 0.1 mM U (VI) at lower rate than the control, but Saccharomyces cerevisiae did not grow under this condition. The H. fabianii J640 pre-cultured for 21 h in the liquid medium without U (VI) grew even after the exposure to 1 mM U (VI), but did not grow without pre-cultivation. For the pre-cultured H. fabianii J640, radioactivity of U in the medium was the same as the initial one for 110 h, and then gradually decreased. TEM-EDS analysis of H. fabianii J640 exposed to 1 mM U (VI) for 165 h showed accumulation of U (VI) on the cells. When H. fabianii J640 was not pre-cultured, radioactivity of U in the medium was lower than the initial one. These results indicated that U (VI) inhibits the growth of yeast, and that the adsorption of U (VI) by the cells depends on the metabolism of yeast.
Source: J. Nuclear and Radiochemical Sciences, Vol. 6, No.1, pp. 99-101, 2005

28. Amplified Fragment Length Polymorphism of the AWA1 Gene of Sake Yeasts for Identification of Sake Yeast Strains.

Author: M.Shimizu, K.Miyashita, H.Kitagaki, K.Ito, and H.Shimoi
Abstract: Sake yeasts are used for sake brewing and have a crucial role in the quality of sake, since they produce not only ethanol but also various compounds that provide sake flavors. Therefore, the appropriate selection and monitoring of a strain used in sake mash is important. However, the identification of specific sake yeast strains have been difficult, because sake yeasts have similar characteristics in taxonomic and physiological analyses. We found amplified fragment length polymorphisms (AFLPs) in the PCR products of the AWA1 gene of sake yeast strains. The AWA1 gene encodes a cell wall protein that is responsible for foam formation in sake mash. This polymorphism of the AWA1 gene can be used for the identification of sake yeast strains.
Source: J. Biosci. Bioeng., 100, 678-680 (2005)

29. Cloning and Expression Analysis of Two Catalase Genes from Aspergillus oryzae

Author: H.Hisada, Y.Hata, A.Kawato, Y.Abe, O.Akita
Abstract: Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA). The catB gene predicts a 725-amino-acid polypeptide sharing 82% identity with A. fumigatus catalase (catB) and 75% with A.nidulans catalase (catB). However, the catA and catB genes share little homology (41%) with one another, suggesting that each gene belongs to a distinct gene family. Overexpression studies demonstrated that both genes encode a functional catalase. Promoter assays indicated that the catA gene is developmentally regulated as it was preferentially expressed in solid-state cultures undergoing sporulation. However, its expression was not affected by hydrogen peroxide treatment. Conversely, the catB gene was highly expressed under all culture conditions tested, and it was induced by hydrogen peroxide treatment. These results suggest that the catB gene may be mainly used for detoxification of oxidative stress while the catA gene may have another role such as chaperoning proteins in the spore.
Source: J. Biosci. Bioeng., 99, 562-568 (2005)

30. Changes in the Aroma Compounds of Sake during Aging

Author: A.Isogai, H.Utsunomiya, R.Kanda, and H.Iwata
Abstract: Changes in the aroma of sake during aging were investigated by aroma extract dilution analysis (AEDA) and quantitative analysis using the stirbar sorptive extraction (SBSE) method. In AEDA, more odor zones were detected in aged sake than in fresh. The dilution factors of aldehydes, polysulfides, and some esters were greater in the aged sake, and their increase during aging was confirmed through a quantitative analysis of sake stored for 0 to 35 years. Among these compounds, 3-methylbutanal, methional, and dimethyltrisulfide (DMTS) were present in aged sake at concentrations exceeding their odor thresholds, and the highest odor active-value was observed for DMTS. Sensory tests showed that supplementation with DMTS contributed to both the total odor intensity and the sulfury odor of aged sake aroma.
Source: J. Agric. Food Chem., 53, 4118-4123 (2005)

31. Identification of 2,4,6-Trichloroanisole (TCA) Causing a Musty/Muddy Off-Flavor in Sake and Its Production in rice koji and Moromi Mash

Author: A.Miki, A.Isogai, H.Utsunomiya, and H.Iwata
Abstract: 2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.
Source: J. Biosci. Bioeng., 100, 178-183 (2005)

32. Structural Characteristics, Properties, and In Vitro Digestibility of Rice

Author: M.Okuda, I.Aramaki, T.Koseki, H.Satoh, and K.Hashizume
Abstract: Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6 - 9.1%, amylose content was 4 - 27%, the DPn of purified amylose was 900 - 1,600, the amylopectin short/long chain ratio was 1.2 - 5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9-12.6 ^。Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounted for >80% of the observed variance in digestibility of steamed rice grains. Multiple regression revealed that the more digestible rice samples had starch with a lower amylose content and more shortchain amylopectin. Reassociation of amylose-lipid complex and recrystallization of amylopectin in the stored steamed rice grains was monitored by differential scanning calorimetry (DSC), and the observed retrogradation properties were related to the structural characteristics of starch and to the enzyme digestibility of steamed rice grains.
Source: Cereal Chem., 82(4):361 - 368

33. Varietal Difference of Polishing Characteristics and Suitability for Sake Brewing in "Hattan-Type Varieties" of Rice Suitable for Brewing Original Hiroshima Sake

Author: M.Tamaki, R.Kihara, T.Itani, Z.Katsuba, T.Tsuchiya, H.Matsumoto, K.Suenari and I.Aramaki
Abstract: By successive crossing using Hattan-type varieties originating from "Hattanso" as a parent "Hattan-type varieties" of rice suitable for brewing the original Hiroshima sake have been bred. The varieties were improved inheriting the flavor of Hattan-type sake, and Hattan-nishiki N0.1 and No.2 were bred in 1984. However, neither variety was suitable for brewing high-grade sake such as Ginjoshu and Daiginjoshu, which require high-degree polishing of rice grains. Therefore, their parent cultivar, Hattan No. 35, is attracting attention for the production of high-grade sake. We have been trying to breed a new variety, which retains the sake-brewing suitability of Hattan-type varieties but can endure high-degree polishing. In this study, to establish a guideline for further breeding, we examined the polishing characteristics and suitability for sake brewing of six Hattan-type varieties derived from Hattanso. In the process of breeding of "Hattan-type varieties" of rice, grain size, white-core size and % of white-core grains increased resulting in an increased suitability for sake brewing, such as water absorptivity and digestibility, in Hattan No.35, Hattan-nishiki N0.1 and Hattan-nishiki N0.2. However, Hattannishiki N0.1 and N0.2 had many ellipsoidal-white-cores with large white tissue, which cause the grains to be easily broken during high-degree polishing. On the other hand, Hattan N0.35, having grains with relatively many lined-white-cores with small white tissue, was superior for high-degree polishing. In the future, breeding of a new variety, which has the superior cultivation characteristics of Hattan-nishiki N0.1 and No.2, but with improved white-core characteristics, is expected.
Source: Plant Prod. Sci., 8(4), 468 - 474 (2005)

34. Analysis of the unique expression mode of acid-unstableα-amylase from Aspergillus kawachii

Author: T.Kato, K.Murashima, H.Shimoi and K.Ito
Abstract: In liquid cultures, the acid-unstable α-amylase (AU-amylase) of Aspergillus kawachii was produced at almost the same level as in a maltose medium when glycerol or glucose was used as a carbon source, while its production level was low compared with TAKA-amylase production by A.oryzae in a maltose medium. Although a sequence analysis showed an extensively high homology with TAKA-amylase gene, the AU-amylase gene depicted 64 base pairs of depletion in the promoter region. From the reporter gene analysis, it was suggested that the low expression level of AU-amylase was caused by deletion of the promoter region, while the unique expression mode on various carbon sources was due to the strain itself. In a manner similar to AU-amylase production, glucoamylase was also produced in A. kawachii (but not in A. oryzae) when glycerol or glucose was used as a carbon source.
Source: J. Brew. Soc. Japan, 100, 513-519 (2005)

35. Effects of low temperatures during grain-filling on characteristics of rice for sake brewing

Author: Y.Yonehara, T.Koseki, M.Okuda, I.Aramaki, K.Hashizume
Abstract: Characteristics of rice for sake brewing at low temperature during the grain-filling period were examined. The rices, Yamadanishiki and Nipponbare, cultivated at low temperatures during the grain-filling period, showed an increase in water absorption for 20 min and digestibility (Brix), but showed a decrease in potassium content. The pasting and gelatinization properties of the rice, which was cultivated at different temperatures during the grain-filling period, were analyzed by Rapid Visco Analyzer (RVA) and differential scanning calorimetry (DSC), respectively. RVA and DSC showed a significant decrease in pasting temperature in the rices cultivated at low temperatures during the grain-filling period. Both rices cultivated at low temperature showed an increase in amylose content or the ratio of short chain/long chain in amylopectin. These observations suggest that meteorological conditions affect the characteristics of rice for sake brewing.
Source: J. Brew. Soc. Japan, 100, 650-657 (2005)

36. Measurement of sake color by the Commission International de l'Eclairage (CIE) standard colorimetric system

Author: N.Mukai, K.Kiso
Abstract: The authors determined the color of sake by the Commission International de l'Eclairage (CIE) standard colorimetric system.Results showed b*, C*, Y, x and y to be highlycorrelated with the value of coloration degree (the absorbance of sake measured at a wavelength of exactly 430 nm) when measuring CIE color values of koshu (storaged over three years). CIE color values could be estimated from the value of coloration degree. The distribution of colors of koshu plotted on an x-y chromaticity diagram was similar to the distribution of colors of melanoidin solutions plotted on an x-y choromaticity diagram. As for the ruby-colored sake which was made by adenine-auxotrophic sake yeast or red-koji, it was suggested that the CIE color values could be appropriate for the attainment of a ruby color. Samples could be divided into three groups when performing a cluster analysis of koshu by means of L*a*b* values.
Source: J. Brew. Soc. Japan, 100(8), 588-595 (2005)

37. Association of VvmybA1 gene expression with anthocyanin production in grape (Vitis vinifera) skin-color mutants

Author: S.Kobayashi, N.Goto-Yamamoto, H. Hirochika
Abstract: By using white-skinned cultivars ('Italia' and 'Muscat of Alexandria') and putative red-skinned sports ('Ruby Okuyama' and 'Flame Muscat') from the white cultivars, we analyzed the expression and function of a myb-related gene, VvmybA1, involved in the regulation of anthocyanin biosynthesis in grapes. A simple sequence repeat (SSR) analysis showed that 'Ruby Okuyama' and 'Flame Muscat' are true derivatives by bud mutation from 'Italia' and 'Muscat of Alexandria', respectively. The VvmybA1 transcript was detected in berry skins of 'Ruby Okuyama' after coloring had begun; it was not detectable in those of 'Italia'. Three VvmybA cDNAs (VvmybA1, VvmybA2, and VvmybA3) from 'Ruby Okuyama' under the control of the 35S promoter were introduced into somatic embryos of 'Kyoho' by particle bombardment. Upon the introduction of VvmybA1, redo cells were induced in the embryos, whereas the introduction of VvmybA2 or VvmybA3 failed to do so. The VvmybA1 cDNA was also shown to have the ability to induce the anthocyanin-producing cells in the skin tissues of 'Muscat of Alexandria', The relationship between the skin-color mutations and the detailed structure of the VvmynA1 gene is also discussed.
Source: J. Japan. Soc. Hort. Sci. 74(3),196-203(2005)

38. Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

Author: T.Koseki, Y.Miwa, S.Fushinobu, K.Hashizume
Abstract: We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward α-naphthylacetate (C2), lower activity toward α-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser¹¹⁹, Ser¹⁴⁶, Asp¹⁶⁸ and Asp²⁰², were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser¹¹⁹ and Asp²⁰² are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased Km and decreased kcat. The catalytic efficiency of S146A was a 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.
Source: Biochim. Biophys. Acta, 1749, 7-13 (2005)

39. Decrease in the broken rice ratio at the milling of long-term stored rice grains

Author: K.Hashizume, M.Okuda, M.Numata, S.Sakurao, T.Koseki, F.Hata, T.Hasuo, and O.Shyojima
Abstract: The broken rice ratio at milling of long-term stored rice grains was closely related with the moisture content of the brown rice grains. Rice grain samples of low moisture content (11-13%) showed a low broken rice ratio in the milling process, while the broken rice ratio of the samples of high moisture content (over 15%) was very high. Drying treatment of the long-term stored rice grains was effective in preventing the breaking of rice grains in milling, and the availability of the treatment was confirmed using a 60 kg scale mill. Hardness of the long-term stored rice grains varied along with the moisture content of the rice grains. The relation between the hardness and the moisture content was not the same in the two examined samples, a finding which might affect the frequency of the breaking of different rice samples at the milling process. Drying treatment slightly raised the hexanal content of rice grains, and also raised the water absorption ratio of the polished rice grains.
Source: J. Brew. Soc. Japan,100, 362-369(2005)

40. Method for the Simultaneous Assay of Diacetyl and Acetoin in the Presence of α-Acetolactate: Application in Determining the Kinetic Parameters for the Decomposition of α-Acetolactate.

Author: K.Kobayashi, K.Kusaka, T. Takahashi, and K. Sato
Abstract: A simultaneous assay method for diacetyl and acetoin was developed to investigate the formation of diacetyl during the brewing of alcoholic beverages. A GC-MS analysis after the extraction from neutralized sample by ethyl acetate gave accurate assay results. The detection limit was below 0.1 mg/l and the assay was quantitative from 0.1 to 100 mg/l for both compounds. Unlike other methods, the assay results were unaffected by the presence of α-acetolactate (up to 26 mg/l), which easily decomposes to diacetyl or acetoin, because the extraction condition prevents the decomposition and extraction of this acidic compound. Since our assay is compatible with samples that contain α-acetolactate, the kinetic parameters for decomposition of α-acetolactate to diacetyl and acetoin were determined. The decomposition rate constants were affected by the ethanol concentration. Overall kinetics for the decomposition of α-acetolactate was formulated as a function of ethanol concentration, pH and temperature. The kinetics can be applied to alcoholic beverages such as sake.
Source: J. Biosci. Bioeng. 99, 502-511(2005)

41. Applied possibility to Teaching Materials about Biotechnology Production of Polygalacturonase protein by Escherichia coli expression system

Author: H.Fukuda, S.Nakatsu and S.Mikami
Abstract: The gene encoding polygalacturonase (PGU1) in Saccharomyces cerevisiae was subcloned into an expression vector (pQE80L), and was transformed into Escherichia coli Origami B strain. The polygalacturonase activity was detected in the E. coli Origami B strain under the induction condition. This enzyme was purified using chromatography on the market. Pgu1 protein (Pgu1p) was active in pH from 3.7 to 5.5, and the optimum temperature and pH of Pgu1p were 40℃ and 3.8, respectively. As it was easy to produce Pgu1p production in the E. coli, it was possible to be teaching materials as biotechnology related protein.
Source: Japan. J. Agric. Educ. 36(1):1-8 (2005)

42. KG1, a suppressor gene of synthetic lethality of kex2Δgas1Δ mutations, encodes a novel membrane protein that affects cell wall composition

Author: N.Tomishige, Y.Noda, H.Adachi, H.Shimoi and K.Yoda
Abstract: The fungal GAS1-related genes encode GPI-anchored beta-1,3-glucanosyltransferase, and their loss causes a defect in the assembly of the cell wall. The KEX2 gene encodes a processing protease in the late Golgi compartment and its loss also results in defects in the cell wall. Simultaneous mutations of these genes are lethal in Saccharomyces cerevisiae. To understand the basis of this synthetic lethality, we screened for multicopy suppressors and identified 13 SKG (suppressor of kex2 gas1 synthetic lethality) genes. SKG1 encodes a transmembrane protein that localizes on the inner surface of the plasma membrane at the bud and in the daughter cell. The multicopy SKG1 increases the sensitivity of cells to zymolyase, and the skg1Delta null mutation increases resistance to it. This zymolyase susceptibility corresponds to an increase of alkali-soluble beta-1,3-glucan and a decrease of chitin in the cell wall. Thus SKG1 encodes a novel protein that affects the cell wall polymer composition in the growing region of the cell.
Source: Yeast, 22, 141-155(2005)

43. High expression of unsaturated fatty acid synthesis gene in sake yeasts

Author: T. Yamada, H. Shimoi and K. Ito
Abstract: GeneFilters(r) and Northern blot analysis revealed that the sake yeaststrain Kyokai no. 7 (K7) showed a higher expression level of OLE1, whichencodes a Δ-9 fatty acid desaturase gene, compared with the laboratoryyeast strain X2180-1A. Other sake yeasts also showed a high expression levelof OLE1. Unsaturated fatty acid concentrations in strain K7 are higher thanthat in strain X2180-1A, suggesting that the higher expression level of OLE1in sake yeasts increases the unsaturated fatty acid content in the cellmembrane. Experiments using OLE1 promoter:lacZ fusion reporter genesrevealed that both the cis element of the OLE1 promoter and trans factorsare involved in the increased expression of OLE1 in sake yeasts.
Source: J. Biosci. Bioeng. 99, 512-516 (2005)

44. Trapping of enzymes in the fungal cell wall

Author: T.Kato, H.Shimoi and K.Ito
Abstract: Enzymes trapped in fungal cell wall and free enzymes produced by koji molds were measured separatelv. Little differences in a tendency for trapping were recognized between Aspergillus oryzae and A. kawachii. More enzymes were trapped in liquid cultures, while the ratios of the trapped enzymes decreased in solid cultures. More than 80% of α-glucosidase and more than 50% of glucoamylase were trapped in liquid cultures ; moreover, 10%~20% of the enzyme were trapped even in α-amylase. In solid culture, more than 20% of glucoamylase and more than 20% of α-glucosidase were still trapped, but the trapped α-amylase was negligible. The tendency of trapping was, a-amylase < glucoamylase < α-giucosidase, which was in order of molecular weight. 'I'he pH stabilitv of a trapped enzyme was greater than that of a free one.
Source: J. Brew. Soc. Japan,100, 355-361(2005)