2004 Research results

1. Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence

Author: T.Koseki, K.Takahashi, S.Fushinobu, H.Iefuji, K.Iwano, K.Hashizume, and H.Matsuzawa
Abstract: We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei(RmLIP) and Humicola lanuginose(HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward α-naphthylbutyrate (C4) and α-naphthylcaprylate (C8), respectively, was due to a lower K_m value. The higher catalytic efficiency of D77N toward C4 substrate was due to combination of decreased K_m and considerably increased k_cat. The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.
Source: Biochim. Biophys. Acta 1722, 200-208(2005)

2. Characteristics of Long-term Stored Rice for Sake Brewing

Author: M.Okuda, S.Sakurao, M.Numata, T.Koseki, F.Hata, T.Hasuo, O.Shyojima, I.Aramaki and K.Hashizume
Abstract: Sixteen rice samples stored under 15℃ for 5 to 7 years after harvest were examined for their characteristics for sake brewing compared with fresh rice, Nipponbare, harvested in 2003. Some of long-term stored rice samples showed high loss and high ratio of broken grains in the polishing tests both by a grain testing mill and by a 60kg scale mill. The properties in the polishing test widely differed among the samples tested. The water absorbance of long-term stored rice samples was lower than that of the fresh rice sample, while their digestibility was about thesame as the fresh rice sample. The long-term stored rice samples contained potassium and hexanal at highers level than the fresh rice sample, but we could not detect hexanal in steamed and then cooled long-term stored rice samples. The moromi mash of the long-term stored rice showed a slightly faster fermentation rate, and the yield of the sake from the mash was slightly higher than that of the fresh rice sample. Athough some panels pointed out bitterness or zatsumi to the sake brewed from long-term stored rice, the general quality of the sake did not show significant differences with the sake from fresh rice. The level of dimethylsulfide in the all sake samples brewed on a small scale was less than 1 ppb, which is far below the stimated threshold value in sake (10ppb).
Source: J. Brew. Soc. Japan., 100,189-198(2005)

3. Cloning and Heterologous Expression of the Antibiotic Peptide (ABP) Genes from Rhizopus oligosporus NBRC 8631

Author: O.Yamada, K.Sakamoto, M.Tominaga, T.Nakayama, T.Koseki, A.Fujita and O.Akita
Abstract: We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the α-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.
Source: Biosci. Biotechnol. Biochem., 69(3),477-482,2005

4. A Novel Amine Oxidase-Encoding Gene from Aspergillus oryzae

Author: K.Matsumura, H.Hisada, H.Obata, Y.Hata, A.Kawato, Y.Abe and O.Akita
Abstract: We cloned a novel gene (aoxA) encoding amine oxidase (AOX) from Aspergillus oryzae. One cDNA clone showing extreme homology to the AOX-encoding genes was found in an expressed sequence tag (EST) library of A. oryzae. Molecular analysis revealed that the aoxA carried four exons interrupted by three introns and had an open reading frame encoding 672 amino acid residues. The deduced amino acid sequence showed about 83.5% identity to the Aspergillus niger AO-I. The strictly conserved residues for co-factor and copper binding in copper/quinine-containing AOXs were also preserved at Tyr 405, His 456, His 458 and His 617 in the cDNA sequence. When the aoxA was overexpressed in the homologous hyperexpression system of A. oryzae, AOX activity in the transformant was enhanced 75-fold. An apparent molecular weight of 159,000 by gel filtration and a subunit molecular weight of 75,000 by SDS-PAGE of the purified enzyme were estimated, suggesting that the enzyme molecule is a homo-dimer similar to other copper/quinine-containing AOXs. The A. oryzae AOXA preferentially oxidized aliphatic monoamines of C2-C6 rather than aromatic amines or diamines. From these results, the aoxA gene product obtained by homologous hyperexpression system of A. oryzae is undoubtedly a functional AOX.
Source: Journal of Bioscience and Bioengineering, 98,359-365 (2004)

5. Effects of strains of Saccharomyces cerevisiae and incubation conditions on the in vitro degradability of yeast and roughage

Author: S.Ando, Y.Nishiguchi, K.Hayasaka, Y.Yoshihara, J.Takahashi, H.Iefuji
Abstract: The in vitro degradability of yeast and the effect of yeast on the in vitro degradability of forage may differ in terms of the specific yeast strains or their incubation conditions. Thus in experiment I, two strains of sake yeast (strainK7 and strainK9) and one strain of bakers' yeast (KY5649) were incubated in an aerobic condition. In experiment 2, aerobically or anaerobically incubated K7 was used for investigating the in vitro degradability of yeast, the effect of yeast on the in vitro degradability of forage, and the degradability of yeast by pepsin and pronase treatment. The in vitro degradability of bakers' yeast was significantly (p<0.05) higher than those of sake yeasts. The in vitro degradability of anaerobically incubated yeast was significantly (p<0.01) higher than that of aerobically incubated yeast. The degradability of bakers' yeast by pepsin treatment was significantly (p<0.01) higher than that of the sake yeasts. The degradability of bakers' yeast by pronase treatment was slightly higher than that of the two sake yeasts, while the degradability of anaerobically incubated yeast by both enzymes, respectively, was significantly (p<0.01) higher than that of aerobically incubated yeast. The degradability of forages was increased significantly (p<0.05) by the addition of yeasts. The degradability of roughage by sake yeast tended to be higher than that by the bakers' yeast. The degradability of roughage was significantly (p<0.05) higher by anaerobically incubated yeast than by aerobically incubated yeast. Given the above results, it seems that in vitro degradability of yeast and the magnitude of the increment of roughage degradation differ among the yeast strains and their incubation conditions.
Source: Asia-Aust.J.Anim.Sci., 354-357 (2005)

6. Inhibitory Effects of Extracts from Shochu Distillation Remnants on the Growth of Human Stomach Tumor Cells

Author

Y.Kadota, S.Hirose, H.Iefuji, T.Matsushita, Y.Matsumoto, R.Ueoka

Abstract

Development of a new processing method for the effective use of rice-Shochu distillation remnants is an important subject for the reduction of environmental pollution. In this study, inhibitory effects on the growth of human stomach tumor cells along with the induction of apoptosis were obtained for powder treated with ethanol from freeze-dried supernatant of rice-Shochu distillation remnants in vitro. Furthermore, the powder obtained from rice-Shochu distillation remnants showed no toxicity to normal rats in vivo experiments.

These results suggest that rice-Shochu distillation remnants might be effectively used not only as medicine but also as food supplements for health.

Source

Journal of Chemical Engineering of Japan 38(2),154-157(2005)

7. The Use of a 2-Thiobarbituric Acid Reaction in the Measurement of Sake Staling(Part3)

Author: Y.Nomura, A.Mizuno, K.KisoM
Abstract: In our previous papers, we reported that the reaction mixture of sake stored at high temperatures with a TBA reagent produced a red pigment, which was similar to the product of the TBA reaction with malonaldehyde, and that the TBA reaction of sake was caused not by oxidized fat but partially by 3-Deoxyglucoson(3-DG). In this paper, we report the development of a measurement method for 3-DG concentration, which was not affected by pyruvic acid or other carbonyl compounds in sake. We confirmed that the 3-DG concentrations in commercial sake have a correlation with the degree of staleness from sensory tests. As a result of a multivariate analysis of the value of the TBA reaction, 3-DG concentration and sake coloring of commercial sake, and an irradiation experiment with sunlight, it was suggested that not only the 3-DG concentration but also melanoidin had an effect on the TBA reaction.
Source: J. Brew. Soc. Japan., 100, 141-145(2005)

8. Cloning and Enhanced Expression of the Cytochrome P450nor Gene (nicA; CYP55A5) Encoding Nitric Oxide Reductase from Aspergillus oryzae

Author: M.Kaya, K.Matsumura, K.Higashida, Y.Hata, A.Kawato, Y.Abe, O.Akita, N.Takaya and H.Shoun
Abstract: We cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.
Source: Biosci. Biotechnol. Biochem., 68(10),2040-2049,2004

9. Measurement of thresholds for reference compounds for the sensory profiling of Sake (Part2)

Author

H.Utsunomiya, A.Isogai and H.Iwata

Abstract

The sensory profiling of sake is very important for research and product analysis. However, a systematic flavor terminology for sake has not been defined yet. Objective reference standards are a prerequisite for improved sensory profiling. Thus, we selected 20 compounds as potential reference standards for sake aroma characteristics. In these studies reported here, a panel from our institute determined detection and recognition thresholds for 10 compounds -- ethyl acetate, isoamyl acetate, ethyl hexanoate, isoamyl alcohol, 2-phenylethanol, acetic acid, butyric acid, isovaleric acid, hexanoic acid and octanoic acid -- dissolved in sake using a forced - choice ascending concentration series method of limits (ASTM, E679-91). Recognition thresholds of 90% were calculated for evaluation of the perception of reference compounds.

In comparison with beer, detection threshold values of isoamyl acetate, 2-phenylethanol, isovaleraldehyde, methyl sulfide, 2,4,6-trichloroanisole, acetic acid, hexanoic acid and octanoic acid were lower than beer's. Threshold values of the rest 10 of the compounds (except sotolon, furfural) were not different from beer's.

Source

J. Brew. Soc. Japan., 99, 729-734（2004）

10. Amakara categories for type designation

Author

H.Utsunomiya, A.Isogai and H.Iwata

Abstract

We determined the amakara (Dryness) categories of sake for type designation. Multiple regression analysis was applied to the sensory evaluation score by trained panel and chemical data (R2=0.797). We defined the following discriminant model of amakara by simplification of the regression equation.

AV = G - A

AV : Amakara Value, G : Glucose (g/dl), A : Total acidity (ml)

Karakuch (DRY): AV<=0.2,

Yaya_karakuchi (Medium Dry): 0.3<=AV<=1.0, Yaya_amakuchi (Medium Sweet): 1.1<=AV<=1.8, Amakuchi (Sweet) : AV>= 1.9

To verify this model, the calculate amakara values were compared with sensory intensities of amakara by two type panels, one is the panel who has much experience of drinking sake (NRIB trainee) and the other is the panel who has little experience of it (University students). Amakara values correlated well to sensory intensities of amakara by both panels (R2=0.712, R2=0.757).

Source

J. Brew. Soc. Japan., 99, 882-889（2004）

11. Identification of Unknown Grape Cultivars Using Simple Sequence Repeat(SSR) Analisys

Author: N.Goto-Yamamoto, Guanghua WAN,M.Numata, Isao Aramaki and K.Hashizume
Abstract: Simple sequence repeat (SSR) analysis is known to be a powerful tool to detect DNA polymorphism, and more reproducible than RAPD analysis. Thus, SSR analysis is regarded as a suitable method for cultivar identification of grapes. However, the length of SSR fragments of the same cultivar with the same marker reported by different groups are sometimes different, which is probably caused by the difference of the detection methods of SSR fragments. For identification of an unknown cultivar, we selected candidate cultivars from reported SSR data, which showed higher correlation coefficients of SSR alleles with the unknown cutivar. Then, the identities of the SSR alleles of the candidates and the unknown cultivar were confirmed after correction of the difference of SSR data of control cultivars that were analyzed both in the report and in our laboratory. Thus, we identified one of two unknown cultivars, which were originally planted as Pinot blanc, as Sauvignon blanc, and the other was found to be very similar to Melon.
Source: J.ASEV Jpn., 15：52-57 (2004)

12. Crystal structure of a family 54 α-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose

Author: A.Miyanaga, T.Koseki, H.Matsuzawa, T.Wakagi, H.Shoun and S.Fushinobu
Abstract: As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 α-L-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a β-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a β-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecule is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cystein residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu221 and Asp297, respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
Source: J. Biol. Chem., 279, 44907-44914 (2004)

13. A hap1 Mutation in a Laboratory Strain of Saccharomyces cerevisiae Results in Decreased Expression of Ergosterol-Related Genes and Cellular Ergosterol Content Compared to Sake Yeast

Author: K.Tamura, Y.Gu, Q.Wang, T.Yamada, K.Ito and H.Shimoi
Abstract: DNA microarray and Northern blot analysis revealed that a sake yeast strain Kyokai no. 7 (K7) showed higher expression of genes encoding proteins involved in ergosterol biosynthesis than a laboratory yeast strain X2180. We hypothesized that these differences in expression levels were caused by a defect of a transcriptional factor Hap1, because strain X2180 contained a Ty1 insertion mutation in the HAP1 gene. To confirm this, we constructed a strain X2180 derivative (strain HX) that contained the wild-type HAP1 genes originating from strain K7. The expression levels of ergosterol-related genes and cellular ergosterol content in strain HX were higher than those in strain X2180 and were almost comparable to those in strain K7. These results suggest that the differences in the expression levels of ergosterol-related genes and ergosterol content between strains K7 and X2180 were largely caused by the hap1 mutation in strain X2180. Involvement of the mutated Hap1 in the differential gene expression between strain K7 and strain X2180 was further confirmed by a lacZ reporter assay of HMG1, one of the Hap1-regulated genes. We also revealed that the HMG1 promoter region between -500 and -376 was important in the transcriptional activation by Hap1.
Source: Journal of Bioscience and Bioengineering, 98, 159- 166(2004)

14. A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

Author: H.Kitagaki, K.Ito and H.Shimoi
Abstract: Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a △dcw1 △dfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3△dfg5). When DC61 cells were incubated at 37℃, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37℃.At 37℃, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37℃, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37℃, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (△dcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.
Source: Eukaryotic Cell, 3, 1297- 1306(2004)

15. High Gravity Brewing Utilizing α-glucosidase (Part 3) Control of Degree of Fermentation Using Immobilized α-glucosidase

Author: A.Mizuno, N.Shinoda and Y.Nomura
Abstract: We studied the application of immobilized α-glucosidase as a means of controlling the degree of fermentation. Immobilized α-glucosidase was sterilized by 70% aqueous ethanol in a reactor for 10 minutes, after which 94% activity remained. Following sterilization, fermenting wort was circulated through the reactor by pumps at varying stages of fermentation. The use of the reactor containing immobilized α-glucosidase increased or decreased the real degree of fermentation of 20%Plato worts depending on the stage of fermentation at which α-glucosidase reaction was undertaken. When the wort was circulated through the reactor for 48 hours from the beginning of fermentation, the glycosyl-transfer reaction of α-glucosidase formed non-fermentable sugars such as isomaltose, panose and isomaltotriose, which resulted in an 11% reduction in the degree of fermentation of the beer compared with the control. When the reactor was used from the 7th day of fermentation or later, the α-glucosidase hydrolyzed polysaccharides to produce glucose resulting in a 10% increase in the degree of fermentation of the beer. In both cases, production of acetic acid decreased. The use of immobilized α-glucosidase was not only effective in controlling the degree of fermentation, but also overcame common problems of high gravity brewing such as insufficient sugar attenuation and the increased production of acetic acid.
Source: J. Brew. Soc. Japan., 99, 809-815（2004）

16. High Gravity Brewing Utilizing α-glucosidase (Part 4) Application of Parallel Fermentation Utilizing α-glucosidase

Author

A.Mizuno, N.Shinoda and Y.Nomura

Abstract

High gravity brewing has come to be widely used for increased brewery efficiency. However, it is not easy to maintain good yeast performance during fermentation because of the stress on yeast caused by high sugar and/or ethanol concentrations. Therefore, sugar attenuation tends to be insufficient and the finished beer contains high concentrations of acetic acid and esters.

We previously reported on the development of a new fermentation method by which several difficulties in high gravity brewing could be overcome. In this method, α-glucosidase added at the beginning of fermentation enables the conversion of wort sugars by glycosyl-transfer and hydrolytic reactions to proceed in parallel with ethanol formation by yeast. By using α-glucosidase, we succeeded in high gravity brewing with a top fermentation brewer's yeast at a constant fermentation temperature of 15℃. However, bottom fermenting brewer's yeast is more widely used for commercial brewing around the world. In this paper we report results of using the method with bottom fermenting brewer's yeast. We confirmed that the method could be successfully applied to high gravity wort fermentation with bottom fermenting yeast at low temperature. When α-glucosidase was added to 20%Plato wort at a concentration of 800 mg/l, the wort was successfully fermented at 10℃ with a bottom fermenting brewer's yeast, W34/70. The real degree of fermentation was 77%, which was 16% higher than that of the control fermentation, and the concentration of acetic acid was 200 mg/l, which was 85 mg/l lower than that of the control. The addition of α-glucosidase was also effective for increasing the degree of fermentation of dextrinous wort. When α-glucosidase was added at the concentration of 400 mg/l to 20%Plato wort containing 6.2% dextrin, the content of dextrin decreased to 3.7% by the end of fermentation with a top fermenting brewer's yeast, NCYC1245; the real degree of fermentation was 75%, which was 19% higher than that of the control. It is suggested that the use of α-glucosidase is applicable for brewing good beers from wort that have a low proportion of fermentable sugars, meaning that malt with lower diastatic power could be utilized. The use of enzymes in fermentation would open several new possibilities to add to existing enzyme methods, such as the use of acetolactate decarboxylase to shorten the conditioning period or glucoamylase for dry beer brewing.

Source

J. Brew. Soc. Japan., 99, 873-877（2004）

17. Increase in Spontaneous Locomotor Activity in Rats Fed Diets Containing Sake Lees or Sake Yeasts

Author: Y.Manabe, M.Shobayashi, T.Kurosu, S.Sakata, T.Fushiki, H.Iefuji
Abstract: The process of sake making generates a great deal of secondary product called sake lees. Part of the sake lees is disposed of as waste, so finding ways to recycle it are a matter of concern. Because sake lees contains many nutritious substances derived from both rice and sake yeast, we studied the physiological effects of these materials on rats. Sprague-Dawley rats were fed one of three experimental diets for two weeks. Rats fed diets containing either 20% sake lees or 2% sake yeast (Kyokai 9 yeast) both had higher spontaneous locomotive activity than rats fed a control casein diet. Body weight and food consumption were not different among the three groups. These results suggest that sake lees and sake yeast have a reinforcing effect on spontaneous locomotive activity.
Source: Food Sci. Technol. Res., 10, 6-9, (2004)

18. Measurement of thresholds for reference compounds for the sensory profiling of Sake (Part1)

Author: H.Utsunomiya, A.Isogai and H.Iwata
Abstract: The sensory profiling of Sake is very important for research and product analysis. However, a systematic flavor terminology for sake has not been defined yet. Objective reference standards are a prerequisite for improved sensory profiling. Thus, we selected 20 compounds as potential reference standards for sake aroma characteristics. In these studies reported here, a panel from our institute determined detection and recognition thresholds for 10 compounds -- acetaldehyde, isovaleraldehyde, benzaldehyde, diacetyl, furfural, sotolon, ethyl mercaptan, methyl sulfide, methyl trisulfide and 2,4,6-trichloroanisole -- dissolved in sake using a forced - choice ascending concentration series method of limits (ASTM, E679-91). Recognition thresholds of 90% were calculated for evaluation of the perception of reference compounds.Detection threshold values of isovaleraldehyde and sotolon were over 10 - fold lower than these reported values.
Source: J. Brew. Soc. Japan., 99, 652- 658 (2004)

19. Isolation and Characterization of a Novel Gene Encoding α-L-Arabinofuranosidase from Aspergillus oryzae

Author: K.Matsumura, H.Obata, Y.Hata, A.Kawato, Y.Abe and O.Akita
Abstract: We cloned and characterized a novel gene (abfA) encoding α-L-arabinofuranosidase (α-L-AFase) from Aspergillus oryzae. One clone homologous to the α-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the α-L-AFases from other organisms indicated that the α-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of α-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-α-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional α-L-AFase.
Source: Journal of Bioscience and Bioengineering, 98, 77- 84 (2004)

20. Effects of high temperature during grain-filling on characteristics of rice for sake brewing

Author: T.Koseki, M.Okuda, Y.Yonehara, K.Hachida, H.Iwata, I.Aramaki and K.Hashizume
Abstract: Characteristics of rice for sake brewing at high temperature during the grain-filling period were verified. The rices, Yamadanishiki and Nipponbare, that were cultivated at high temperature during grain-filling showed an increase in water absorption for 20 and 120 min and after steaming, but showed a decrease in digestibility (Brix). On the other hand, results suggest that the amounts of crude protein and potassium are affected by hours of sunlight as well as temperature conditions in the cultivation period of rice. The pasting and gelatinization properties of the rice, which was cultivated at different temperature during grain-filling, were analyzed by Rapid Visco Analyzer (RVA) and differencial scanning calorimetry (DSC), respectively. Pasting temperature, peak viscosity, and breakdown by RVA and onset and peak temperatures and transition heat by DSC were higher in the cultivars of increasing temperatures, confirming that the physicochemical properties of rice starches were affected by meteorological conditions. These observations suggest that meteorological conditions correlate to the characteristics of rice for sake brewing.
Source: J. Brew. Soc. Japan., 99, 591- 596 (2004)

21. Winemaking Using La France Pears and the Volatile Constituents of the Wine

Author: K.Hashizume, H.Ohura, N.Ohnuma and I.Aramaki
Abstract: A winemaking method reflecting the characteristic aroma of La France pears was developed and the volatile constituents of the wine produced were investigated. n-Butyl acetate and n-hexyl acetate were detected as major components that contribute to the characteristic aroma of La France pear wine through GC-sniffing analysis of the wine and also by comparative analysis of the aromas of raw fruit and wine. Fermentation of pear pulp provided more n-butyl and n-hexyl acetate and stronger aromatic characteristics than juice fermentation. Wine made from pulp by pulpy fermentation exhibited the highest levels of these esters and was rated the best among the wines tested. Wines made from skin and pulp had the highest phenolic levels and the darkest brown color and a low score of sensory evaluation, while wines made from core and pulp showed the brightest red color.
Source: J. Brew. Soc. Japan., 99, 597- 606 (2004)

22. The Use of 2-Thiobarbituric Acid Reaction in the Measurement of Sake Staling(Part2)

Author: Y.Nomura, A.Mizuno and K.Kiso
Abstract: In a previous paper, we reported that a red pigment, which was similar to a product of the TBA reaction with malonaldehyde, was formed in a TBA reaction of sake. However, the reactive substance of the TBA reaction of sake was not investigated. Essentially, oxidized fat caused the TBA reaction and red pigment formation. We analyzed concentrations of fatty acid component in sake, and concluded that the TBA reaction was not caused by oxidized fat. We thus investigated the TBA reactivity of many carbonyl compounds in sake. In these, only 3-Deoxyglucoson showed absorbance of the reactant mixture at a 530nm wave length. We also found that 3-Deoxyglucoson and TBA produced a red pigment which was similar to the product of the TBA reaction with malonaldehyde.
Source: J. Brew. Soc. Japan., 99, 521-525 (2004)

23. The role of riceα-glucosidase on kutikami-sake making

Author: H.Iwata, A.Isogai, H.Utsunomiya and N.Nishio
Abstract: To examine the role of rice-α-glucosidase on kutikami-sake brewing, a traditional sake making method, we made two test solutions with chewed uncooked rice and cooked rice. In the solution with chewed uncooked rice, 5% of glucose with no maltose was produced, whereas in the solution with chewed cooked rice 0.8% of glucose with 5% of maltose was produced after 24hours incubation. This difference was mainly due to the glucose formation from maltose derived from α-glucosidase contained in uncooked rice, the maltose being produced by α-amylase in saliva. The lactate produced by microorganisms caused pH decrease to around 4, an optimum pH forα-glucosidase, and aided in the extraction of α-glucosidase from unncooked rice. From the above results, it was determined that unncooked rice was used as a material in kutikami-sake brewing.
Source: J. Brew. Soc. Japan., 99, 534-538 (2004)

24. Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags

Author: H.Maeda, M.Sano, Y.Maruyama, T.Tanno, T.Akao, Y.Totsuka, M.Endo, R.Sakurada, Y.Yamagata, M.Machida, O.Akita, F.Hasegawa, K.Abe, K.Gomi, T.Nakajima, Y.Iguchi
Abstract: Aspergillus oryzae is a fungus used extensively in the fermentation industry. We constructed cDNA microarrays comprising 2,070 highly expressed cDNAs selected from the ~6,000 non-redundant expressed sequence tags (ESTs) in the A. oryzae EST database. Using the cDNA microarrays, we analyzed the gene expression profiles of A. oryzae cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions used during the construction of the EST database. The sets of genes identified by the cDNA microarray as highly expressed under each culture condition agreed well with the highly redundant ESTs obtained under the same conditions. In particular, transcription levels of most catabolic genes of the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle were higher under AC than AN conditions, suggesting that A. oryzae uses both EMP and TCA for glucose metabolism under AC conditions. We further studied the expression of genes encoding hydrolytic enzymes and enzymes involved in energy catabolism by using three industrial solid-phase biomass media, including wheat-bran. The wheat-bran culture gave the richest gene expression profile of hydrolytic enzymes and the lowest expression levels of catabolic genes (EMP,TCA) among the three media tested. The low expression levels of catabolic genes in the wheat-bran culture may release catabolite epression, consequently leading to the rich expression profiles of the hydrolytic enzymes.
Source: Appl Microbiol Biotechnol (2004) 65: 74-83

25. Relationship between the preference for sake Japanese rice wine) and the movements of metabolic parameters coinciding with sake intake.

Author: Y.Manabe, H.Utsunomiya, K.Gotoh, T.Kurosu, T.Fushiki
Abstract: Selection of foods by animals largely depends on their physiological condition. In this paper, we reported how physiological changes in rats after ingestion of sake (Japanese rice wine) affected their preference for different kinds of sake. Rats could discriminate among various kinds of sakes in a two-bottle choice test, even after adjustment of the glucose concentration or alcohol concentration of the sake, suggesting that the choice of a sake by animals was based on more than one ingredient. To identify effect of a rat's physiological condition on the selection of sake, we monitored the levels of blood glucose, nonesterified fatty acids (NEFA), and ketone bodies in serum in mice after forced intragastric ingestion of sake that had previously been offered to the rats in a two-bottle choice test. Blood glucose levels in mice were not different between the rats fed the palatable and unpalatable sakes, and the NEFA level and ketone level were high in rats fed the unpalatable sake. To further clarify the relationship between physiological condition and preference for sake, rats were offered eight kinds of Junmai-syu, which is made with only rice and no sub-ingredient. Rats could still discriminate among Junmai-syu. Furthermore, with two exceptions, the preference for a sake was significantly correlated with the ketone level. The order in the ratio of blood insulin level to blood glucagon level, which is an indicator of metabolism, was correlated with the order in preference to sake. These results suggest that some physiological factors besides oral stimulation also are important factors in the selection of a sake.
Source: Biosci Biotechnol Biochem., 2004 Apr;68(4):796-802.

26. Coloring of sake in cold storage by fluorescent light

Author: Y.Nomura, T.Naitoh, A.Ono, S.Mikami, T.Takahashi and K.Kiso
Abstract: Recently, sake has come to be frequently sold in transparent refrigerators fluorescently lighted. In such cases, the sake is sometimes colored highly. This seems due to the effect of fluorescent light. We researched the effects of fluorescent light sake coloring. As for the relations between sake coloring and the six types of fluorescent lights, a three band fluorescent light (natural white) colored sake most highly, second was a fluorescent light (natural white), and a high color rendering fluorescent light coated with UV absorption film (natural white) colored sake least. We researched the effects of four color-films on sake coloring. A red film was most effective for preventing sake coloring. A blue film was least ffective in preventing sake coloring. As a result of a multivariate analysis, light with a wavelength under 450nm produced an effect on sake coloring.
Source: J. Brew. Soc. Japan., 99, 289-294(2004)

27. Retrotransposon-induced mutations in grape skin color

Author: S.Kobayashi, N.Goto-Yamamoto, H.Hirochika
Abstract: The transcript of a grape myb-related gene, VvmybA1, was detected in mature berry skins of red-skinned bud sports but not in those of their white-skinned progenitors. The white-skinned progenitors have only one gene (VvmybA1a) for VvmybA1, while the red-skinned sports contain two kinds of the gene (VvmybA1a and VvmybA1b). In VvmybA1a, a retrotransposon, Gret1, was inserted into a 5'-flanking region near the coding sequences of the gene, whereas Gret1 was missing in VvmybA1b, leaving behind its solo LTR. These results show that the bud mutation from white to red in berry skins of the grapes was caused by the deletion of Gret1.
Source: Science, 304, 982 (2004)

28. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins

Author: S.T.Jeong, N.Goto-Yamamoto, S.Kobayashi, M.Esaka
Abstract: Anthocyanins are important grape components for red wine making. Here we examined the effects of plant hormones, abscisic acid (ABA) and naphthaleneacetic acid (NAA), and shading treatments on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. The accumulation of anthocyanins was enhanced by ABA treatment and suppressed by NAA and shading. ABA enhanced the mRNA accumulation of VvmybA1, a putative regulatory gene of anthocyanin biosynthesis of grape, and all the tested enzyme genes of the anthocyanin biosynthetic pathway, while NAA and shading suppressed and retarded them. The change of the mRNA copy number of VvmybA1 coincided with that of all the tested anthocyanin biosynthetic enzyme genes and the accumulation of anthocyanins. We also showed that there were significant differences in the mRNA copy number among three Chss, between two Chis, and two F3hs during the coloration of grape berry skins.
Source: Plant Science, 167, 247-252 (2004)

29. Expression, purification, crystallization and preliminary X-ray analysis of α-L-arabinofuranosidase B from Aspergillus kawachii

Author: A.Miyanaga, T.Koseki, H.Matsuzawa, T.Wakagi, H.Shouna and S.Fushinobua
Abstract: α-L-Arabinofuranosidase (EC 3.2.1.55) is one of the hemicellulases that cleave the gylcosidic bonds between L-arabinofuranoside side chains and various oligosaccharides. In this study, the first crystallization and preliminary X-ray analysis ofα-L-arabinofuranosidase B from Aspergillus kawachii IFO4308 (AkAbfB), a family 54 glycoside hydrolase, is described. Recombinant AkAbfB was expressed in Escherichia coli and Pichia pastoris. The native crystals of recombinant AkAbfB produced by P.pastoris belong to the orthorhombic space group P212121 (unit-cell parameters a=39.5, b=98.2, c=144.0Å) and diffracted X-rays to a resolution of 1.82Å.
Source: Acta Cryst., D60, 1286-1288 (2004)

30. Variation of 2-Acetyl -1-Pyrroline Concentration in Aromatic Rice Grains Collected in the Same Region in Japan and Factors Affecting Its Concentration

Author: T.Itani, M.Tamaki, Y.Hayata, T.Fushimi and K.Hashizume
Abstract: Aromatic strength of aromatic rice varies with the genetic and environmental conditions. We determined the concentration of 2-acetyl-1-pyrroline (2AP), a key compound of the aroma of aromatic rice, in 62 samples of rice grains (brown rice) from 'Hieri' produced by 17-24 farmers in 3 years in the Kubokawa area of Kochi Prefecture, Japan. Many of them showed similar values and the standard deviations were 27-31%. However, a few samples showed extremely high (200%) or low (60%) 2AP concentrations compared to the individual year averages (100%). The influence of harvest time and temperature during ripening on the 2AP concentration in the brown rice was also examined using two cultivars. During grain development in an early-heading cultivar 'Miyakaori', the 2AP concentration in the brown rice reached a peak at 4 or 5 weeks after heading (WAH) and then decreased rapidly to 20% of the maximum at 7 or 8 WAH. In a late-heading cultivar 'Hieri', the 2AP concentration peaked at 4 WAH then gradually decreased to 40% of the maximum at 8 WAH. The 2AP concentration was higher in brown rice ripened at a low temperature (day:25℃/night:20℃) than that ripened at a high temperature (day:35℃/night:30℃) in both a short-grain cultivar 'Hieri' and a long-grain cultivar 'Sari Queen'.
Source: Plant Prod. Sci,,Vol 7(2), 178-183 (2004)

31. Correlation between Structural Properties of Amylopectins and the Sake Brewing or Physicochemical Properties of Rice

Author: I.Aramaki, M.Yoshii, S.I.Wase, M.Ebe, Y.Sugano, Y.Kikunaga, M.Okuda, T.Koseki and K.Hashizume
Abstract: The structure of amylopectins in 70%-polished rice endosperm of fourteen cultivars, including nine varieties for sake brewing, was characterized by a high-performance anion-exchange chromatography system equipped with a pulsed amperometric detector (HPAEC-PAD) after hydrolysis with isoamylase. It was found that starch isoamylolyzates of the rice suitable for sake brewing were plentiful in shortish chains around DP 7-9, while the longish chains were poor in Yamadanishiki and Hattannishiki 1, and the middle chains were poor in Gohyakumangoku, Okuhomare, and Hattannishiki 1, compared with those of Koshihikari. Correlation analysis using all rice samples showed positive correlations between the shortish (DP: 6-9) chains and water absorption, or digestibility, whereas negative correlations existed between the longish (DP: 35-41) chains and digestibility. Also, there were positive correlations between the longish (DP: 43-51) chains and the DSC parameters (peak temperature, conclusion temperature, and heat of transition), but negative ones between the shortish (DP: 7-11) chains and these DSC parameters. The RVA parameters showed rather high correlations with the distribution ratio of some amylopectin side chains, but they were not much higher than those observed in the case of digestibility or DSC parameters.
Source: J. Brew. Soc. Japan., Vol99, 457-466 (2004)

32. Changes in the concentrationｓ of sotolon and furfural during the maturation of sake

Author: A.Isogai, H.Utsunomiya and H.Iwata
Abstract: The contribution of sotolon and furfural to the aroma of aged sake was investigated. These compounds were strongly detected in aged sake by GC-olfactometry. They were quantified by solvent extraction and GC-MS analysis. The coefficient of variation of sotolon decreased to less than 10% by using ¹³C₂-labeled sotolon instead of 3-octanol as an internal standard. Concentration of sotolon in aged sake was higher than its recognition threshold but furfural was not. The odor unit of sotolon in 33-year-old sake was 45. It was suggested that sotolon contributes greatly to the aroma of aged sake. Concentrationｓ of α-ketobutyric acid and acetaldehyde, which are considered to be precursors of sotolon, were investigated and a correlation with sotolon was observed. However, the possibility of another mechanism of sotolon formation was also suggested.
Source: J. Brew. Soc. Japan., 99, 374-380 (2004)

33. Effects of rice α-glucosidase on steeping and steaming processes of sake production

Author: H.Iwata, A.Isogai, H.Utsunomiya and N.Nishio
Abstract: We examined the effects of α-glucosidase in rice on steeping and steaming processes of sake production. The glucose formed in the rice steeping process increased with an increase in the steeping time and steeping temperature. Miglitol, a specific inhibitor of α-glucosidase, strongly inhibited the glucose formation during steeping. The activity of α-glucosidase showed a positive correlation with the glucose formed during the steeping except at 0 hour. On the digestibility test, absorbed water content and Brix value under miglitol addition were lower than those of the control.
These results suggest that α-glucosidase in rice exerts certain effects on rice during the steeping and steaming processes.
Source: J. Brew. Soc. Japan., 99, 467-473 (2004)

34. Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol.

Author: S.Kubota, I.Takeo, K.Kume, M.Kanai, A.Shitamukai, M.Mizunuma, T.Miyakawa, H.Shimoi, H.Iefuji, D.Hirata.
Abstract: The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. I ndeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.
Source: Biosci. Biotechnol. Biochem,, 2004 Apr;68(4):968-72.

35. Cloning of a Novel Tyrosinase-Encoding Gene(melB ) from Aspergillus oryzae and Its Overexpression in Solid-State Culture (Rice Koji)

Author: H.Obata. H.Ishida, A.Kawato, Y.Abe, T.Akao, O.Akita and E.Ichishima
Abstract: We have cloned a novel tyrosinase-encoding gene (melB) specifically expressed in solid-state culture of Aspergillus oryzae. A tyrosinase-encoding gene (melO) from A.oryzae was already cloned and the protein structures of its catalytic and copper binding domains were investigated. However, our recent results revealed that the melO gene was highly expressed in submerged culture but not in solid-state culture. Because tyrosinase activity was also detected in solid-state culture, we assumed that another tyrosinase gene other than melO is expressed in solid-state culture. Another tvrosinase gene was screened using the express ed sequence tag (EST) library. One redundant cDNA clone homologous with the tyrosinase gene was found in the collection of wheat bran culture. Northern blot analysis revealed that the gene corresponding to the cDNA clone was specifically expressed in solid-state culture (koji making), but not in submerged culture. Molecular cloning showed that the gene carried six exons interrupted by. five introns and had an open reading frame encoding 616 amino acid residues. This gene was designated as melB . The deduced amino acid sequence of the gene had weak homology (24%-33%) with MelO and other fungal tyrosinase but the sequences of the copper binding domains were highly conserved. When the melB gene was expressed under the control of the glaB promoter in solid-state culture, tyrosinase activity was markedly enhanced and the culture mass was browned with the melanization by MelB tyrosinase. These results indicated that the melB gene encodes a novel tyrosinase associated with melanization in solid-state culture.
Source: J. Biosci. Bioeng., 97,400-405(2004)