2011 Research results

1. Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry

Author: Kei Takahashi, Masafumi Tokuoka, Hiromi Kohno, Nobuko Sawamura, Yuka Myoken, and Akihiro Mizuno
Abstract: Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, the comprehensive analysis of dipeptides in alcoholic beverages was performed by a high separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 μM in sake. The analysis was validated by dipeptide recovery of 64.0%-107.2% (2.5 μM of standard) with a relative standard deviation of ＜ 33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 40 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.
Source: J. Chromatogr. A, 1242, 17-25 (2012)

2. Lignin Is Linked to Ethyl-Carbamate Formation in Ume (Prunus mume) Liqueur

Author: Tomokazu HASHIGUCHI, Hanae IZU and Shigetoshi SUDO
Abstract: Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.
Source: Biosci. Biotechnol. Biochem., 76, 148-152 (2012)

3. Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

Author: Chiemi Noguchi, Daisuke Watanabe, Yan Zhou, Takeshi Akao, and Hitoshi Shimoi
Abstract: Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a △ppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.
Source: Applied and Environmental Microbiology, 78, 385-392 (2012)

4. Positional Differences in Swelling Degree, Gelatinization Temperature, and Protein Content of Rice Flour from Large and Small Rice Grains

Author: Jiro Koura, Masahiko Tamaki, Isao Aramaki and Tomio Itani
Abstract: The swelling degree, gelatinization temperature, and protein content of rice flour prepared from the ventral and dorsal parts of rice grains used for sake brewing (larage grains) and staple food (small grains) were examined. The swelling degree of rice flour was higher in the order: ventral part of large grains >> ventral part of small grains > dorsal part of large grains ≒ dorsal part of small grains. The rice flour obtained from the ventral part of large grains also exhibited the highest swelling power, lowest gelatinization temperature, and lowest protein content. The white precipitate remaining after the measurement of swelling power was subjected to iodine and Berlin blue reactions and observed microscopically. From these findings, we speculate that rice flour containing large amounts of protein may be difficult to gelatinize because protein firmly adheres to starch, resulting in reduced water absorption and consequently, reduced gelatinization.
Source: Journal of the Japanese Society of Agricultural Technology Management, 18, 87-94, 2011

5. Yeast Genes Involved in Uranium Tolerance and Uranium Accumulation: A Functional Screening Using the Nonessential Gene Deletion Collection

Author: Fuminori Sakamoto, Takuya Nankawa, Toshihiko Ohnukia, Tsutomu Fujii and Haruyuki Iefuji
Abstract: We screened 4908 non-essential gene deletion mutant yeast strains for uranium sensitivity and low accumulation by growth in agar medium containing uranium. All mutant strains grew successfully on agar media containing 0 or 0.5 mM uranium for one week at 30℃. Thirteen strains with single gene deletions showed reduced growth in the agar medium containing 0.5 mM uranium and were identified as uranium-sensitive mutant strains. The phosphate transporter genes of PHO86, PHO84, PHO2, and PHO87 were among the deleted genes in the uranium-sensitive mutant strains, suggesting that genes concerned with phosphate transport contribute to uranium tolerance. Seventeen single-deletion strains showed lower uranium accumulation than the wild-type after exposure to agar medium containing 0.5 mM uranium, and were identified as mutant strains with low uranium accumulation. Among the deleted genes in these strains were cell membrane proteins, phospholipid-binding proteins, and cell wall proteins, suggesting that cell surface proteins contribute to uranium accumulation.
Source: Geomicrobiology Journal 29, 470-476 (2012)

6. Characterization and Isolation of Mutants Producing Increased Amounts of Isoamyl Acetate Derived from Hygromycin B-Resistant Sake Yeast

Author: Inoue T, Iefuji H, Katsumata H.
Abstract: Hygromycin B is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. Twenty-four hygromycin B-resistants mutants were isolated from sake yeast, and were divided into three different degrees of strength according to hygromycin B resistance. Three of four hygromycin B strongly resistant mutants produced increased amounts of isoamyl acetate in sake brewing test, although isoamyl alcohol levels remained unchanged. Many hygromycin B-resistants mutants showed higher E/A ratios than K-701 in culture with koji extract medium. Strain HMR-18 produced the largest amount of isoamyl acetate, and its alcohol acetyltransferase (AATFase) activity was 1.3-fold that of K-701. DNA microarray analysis showed that many genes overexpressed in HMR-18 were involved in stress responses (heat shock, low pH, and so on) but HMR-18 showed thermo- and acid-sensitivity. It was strongly resistant to hygromycin B and another aminoglycoside antibiotic, G418.
Source: Biosci Biotechnol Biochem. 76, 60-66 (2012)

7. Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2, and enhancement of the production of a cutinase-like enzyme.

Author: Masaki K., Tsuchioka H., Hirano T., Kato M., Ikeda H., Iefuji H.
Abstract: Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.
Source: Appl. Microbiol. Biotechnol., 93, 1627-1636 (2012)

8. Phylogenetic and biochemical characterization of the oil-producing yeast Lipomyces starkeyi.

Author: Oguri E., Masaki K., Naganuma T., Iefuji H.
Abstract: Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, D-glucose, D-xylose, L-arabinose, D-galactose, D-mannose, and D-cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted D-glucose, D-galactose, and D-mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted D-xylose, L-arabinose, D-galactose, and D-cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except d-cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from D-glucose, D-galactose, and D-mannose. The type of sugar converted depended on the subclade classification elucidated in this study.
Source: Antonie van Leeuwenhoek, 101, 359-368 (2012)

9. Purification, cloning and expression of an Aspergillus niger lipase for degradation of poly(lactic acid) and poly(ε-caprolactone)

Author: Nakajima-Kambe T., Edwinoliver N.G., Maeda H., Thirunavukarasu K., Gowthaman M.K., Masaki K., Mahalingam S., Kamini N.R.
Abstract: A lipase from Aspergillus niger MTCC 2594 was purified 53.8-fold to homogeneity by hydrophobic interaction chromatography using octyl sepharose and the enzyme showed two protein bands with apparent molecular mass of 35 and 37 kDa respectively. The lipase exhibited maximum activity at pH 7.0 and 37 ℃ and was stable between pH 4.0 and 10.0 and temperatures up to 50 ℃. The values of Km and Vmax were 3.83 mM and 32.21 μmol/min/mg respectively, using olive oil as substrate. Lipase encoding gene, lipA, coded for 297 amino acid residues with conserved pentapeptide sequence, G-H-S-L-G, was cloned and expressed in Pichia pastoris. Although lipA showed high homology with the known Aspergillus lipases, it exhibited differences in putative lid domain. Both native and recombinant lipases have potential for degradation of poly(lactic acid) and poly(ε-caprolactone), and the present study will serve as a baseline of initial studies for its exploitation in polymer degradation.
Source: Polym. Degrad. Stab., 97, 139-144 (2012)

10. Compatibility evaluation method for food and refined sake

Author: Shigetoshi SUDO, Akiko FUJITA, Michiko ENDO, Ryoko KANDA, Atsuko ISOGAI
Abstract: For refined sake, which is a liquor consumed during meals, compatibility with food is an important characteristic. Generally compatibility tends to be accompanied by ambiguity. Therefore, to obtain reliable compatibility, it is essential to develop an evaluatory method with accuracy and repeatability. We examined the factors affecting evaluation of compatibility with food and refined sake by using a food set A (black sesame tofu, tuna sashimi, saury-yakimono, diced steak, egg custard, vinegared flounder, cheesecake), and food set B (pudding, flounder sashimi, yose-hot pot, simmered fish, sirloin steak, lettuce, dry taste curry) and junmai-sake. From verification testing by 18 or 19 panelists, the following results were obtained. As for the eating and drinking intervals, less than 5 seconds were suitable. Compatibility with the sensitivity of drinking after eating was estimated to have about 3 times more effect on the evaluation of compatibility from that of eating after drinking. The effect of a placement of taste consciousness on the evaluation of compatibility was not clear. It was considered a problem during verification. The taste request from food to junmai-sake was suggested to affect the evaluation of compatibility. A preconception of compatibility affected the quality of junmai-sake which was directly linked to the evaluation. On the basis of the obtained results, we set up a method for evaluating compatibility.
Source: J. Brew. Soc. Japan, 107, 49-56 (2012)

11. Effects of Sake Components on Physiological Preference and Blood Glucose Level Coinciding with Sake Intake in Mice

Author: Gaku HITOMI, Hanae IZU, Yasuko MANABE, Kei TAKAHASHI, Tomokazu HASHIGUCHI, Sachie HORII2, Shigetoshi SUDO
Abstract: We previously reported that some physiological factors besides oral stimulation are important for Sake preference. In this paper, we show the effects of Sake components on Sake physiological preference and blood glucose levels coinciding with Sake intake in mice. To identify the Sake components involved in the physiological preference, the relationships between the order of preference for five Sake and various Sake components levels were analyzed and a CE-TOFMS analysis of three Sake was performed. Suggested possible components involved in the Sake palatability were added to Sake and their preferences were tested in a two-bottle choice test in mice. As a result, glucose, Lys, and His increased the Sake preference and Gln, octanoic acid, pyruvic acid, and isoamyl acetate decreased that. However, the sole addition of Lys or His did not increase the Sake palatability in other cases; this indicated that the choice of Sake was based on more than one ingredient. The effect of Sake administration on blood glucose was tested, and a higher level of blood glucose was observed in mice administered with preferable Sake and unpreferable Sake with Lys added than for unpreferable Sake, suggesting a correlation between Sake physiological preference and high blood glucose levels.
Source: J. Brew. Soc. Japan, 106, 675-686 (2011)

12. Analysis of Element Composition of Japanese and Other Wine and Their Classification

Author: Sachie Horii, Tomokazu Hashiguchi, Hanae Izu and Shigetoshi Sudo
Abstract: A technique to determine the geographic origin of wine was examined using 99 samples from Japan and 4 other countries. The contents of 21 elements (Al, B, Ba, Ca, Cd, Cr, Cs, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Sr, V, and Zn) were determined by inductively coupled plasma optical emission spectrometry. A linear discriminate analysis (LDA) model was created to identify wine from Japan and other countries. With these parameters, 99% correct classification was achieved. We developed an LDA model calibrated with the analytical results of 5 elements (B, Ca, Cr, K, and Mg) chosen by a backward stepwise procedure. This LDA model identified the origin of wines as Japan with 91% certainty. This result suggests that discriminating by inorganic element composition may be useful to identify Japanese wine for import clearance.
Source: Journal of the Japanese Society for Horticultural Science、80、506-511、(2011)

13. Distribution of minerals in rice grains used for sake brewing

Author: Isao Aramaki, Masahide Ebe, Takeshi Miura, Mika Yoshii
Abstract: Mineral (K, P, Mg, S, Si, Fe, Ca, Mn, Na, Co, Ni ) contents of rice grains used for sake brewing were analysed. The contents of Co and Ni were very low ( < 0.1ppm) even in brown rice grains. Gohyakumangoku, Okuhomare, and Hyogokitanishiki showed higher contents of K, P and Mg in rice grains than other varieties. Yamadanishiki showed the lowest content of S among varieties used in this study, and it showed lower contents of Fe and S than those Nipponbare even cultivated in the same field. As the milling ratio was lower, the contents of K, P, Ca, Mg, Mn, and Si decreased rapidly from the brown rice to 70% of the milling ratio, but those did not change from 70% to 30% of the milling ratio. On the other hand, the content of S decreased linearly from brown rice to 30% of the milling ratio. There were no significant differences in mineral concentrations between white core rice grains and non-white core rice grains in Yamadanishiki and Gohyakumangoku, as the milling ratio was lower. Energy Dispersive X-Ray (EDX) micrograph observation showed P, K, and Mg distributed abundantly in the aleurone layer of rice grains and Si distributed abundantly in the pericarp of rice grains.
Source: J. Brew. Soc. Japan, 106, 837-847 (2011)

14. Development of an efficient gene-targeting system in Aspergillus luchuensis by deletion of the non-homologous end joining system

Author: Toru Takahashi, Osamu Mizutani, Youhei Shiraishi, Osamu Yamada
Abstract: The industrial fungus Aspergillus luchuensis is used to produce a distilled spirit in Okinawa Island, Japan. Recently, the genome sequence of A. luchuensis RIB2604 (Aspergillus awamori NBRC 4314) was revealed and many functional genes are now expected to be analysed. Gene targeting is necessary for analysing the function of a gene; however, gene targeting frequencies in A. luchuensis are very low. To develop a highly efficient gene-targeting system for A. luchuensis, we disrupted A. luchuensis ligD (ALligD) encoding the human DNA ligase IV (ligIV) homologue using an Agrobacterium mediated gene transformation method. Deletion of ALligD dramatically improved homologous recombination efficiency (reached 100%) compared to that in the wild-type strain (0.8%), when 1000-bp homologous flanking regions were used. The ALligD disruptant showed no apparent defect in vegetative growth, and it exhibited increased sensitivity to phleomycin and high methyl methanesulphonate concentrations compared to the wild-type strain. Furthermore, using this ALligD disruptant, we disrupted ALpksP encoding an Aspergillus fumigatus polyketide synthase P (alb1/pksP) orthologue. The ALpksP disruptant displayed a decolourized conidial phenotype. This result indicated that ALpksP is a key factor for conidial black pigmentation in A. luchuensis. Our results indicate that the ALligD mutant is an efficient host for targeted gene disruption in A. luchuensis.
Source: Journal of Bioscience and Bioengineering, Vol. 112 (6), P. 529-534 (2011)

15. Ethanol fermentation driven by elevated expression of the G1 cyclin gene CLN3 in sake yeast

Author: Daisuke Watanabe, Satoru Nogami, Yoshikazu Ohya, Yoichiro Kanno, Yan Zhou, Takeshi Akao, and Hitoshi Shimoi
Abstract: Cellular and subcellular morphology reflects the physiological state of a cell. To determine the physiological nature of sake yeast with superior fermentation properties, we quantitatively analyzed the morphology of sake yeast cells by using the CalMorph system. All the sake strains examined here exhibited common morphological traits that are typically observed in the well-characterized whiskey (whi) mutants that show accelerated G1/S transition. In agreement with this finding, the sake strain showed less efficient G0/G1 arrest and elevated expression of the G1 cyclin gene CLN3 throughout the fermentation period. Furthermore, deletion of CLN3 remarkably impaired the fermentation rate in both sake and laboratory strains. Disruption of the SWI6 gene, a transcriptional coactivator responsible for Cln3p-mediated G1/S transition, also resulted in a decreased fermentation rate, whereas whi mutants exhibited significant improvement in the fermentation rate, demonstrating positive roles of Cln3p and its downstream signalling pathway in facilitating ethanol fermentation. The combined results indicate that enhanced induction of CLN3 contributes to the high fermentation rate of sake yeast, which are natural whi mutants.
Source: Journal of Bioscience and Bioengineering, Vol. 112, No. 6, 577-582 (2011)

16. Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

Author: Takeshi Akao, Isao Yashiro, Akira Hosoyama, Hiroshi Kitagaki, Hiroshi Horikawa, Daisuke Watanabe, Rinji Akada, Yoshinori Ando, Satoshi Harashima, Toyohisa Inoue, Yoshiharu Inoue, Susumu Kajiwara, Katsuhiko Kitamoto, Noriyuki Kitamoto, Osamu Kobayashi, Satoru Kuhara, Takashi Masubuchi, Haruhiko Mizoguchi, Yoshihiro Nakao, Atsumi Nakazato, Masahiro Namise, Takahiro Oba, Tomoo Ogata, Akinori Ohta, Masahide Sato, Seiji Shibasaki, Yoshifumi Takatsume, Shota Tanimoto, Hirokazu Tsuboi, Akira Nishimura, Koji Yoda, Takeaki Ishikawa, Kazuhiro Iwashita, Nobuyuki Fujita, and Hitoshi Shimoi
Abstract: The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.
Source: DNA Res., 18, 423-434 (2011)

17. Lack of endoplasmic reticulum 1,2-α-mannosidase activity that trims N-glycan Man₉GlcNAc₂ to Man₈GlcNAc₂ isomer B in a manE gene disruptant of Aspergillus oryzae

Author: Takeshi Akao, Akinori Yahara, Kazutoshi Sakamoto, Osamu Yamada, Osamu Akita and Takashi Yoshida
Abstract: The gene manE, encoding a probable class I endoplasmic reticulum 1,2-α-mannosidases (ER-Man), was identified from the filamentous fungus Aspergillus oryzae due to similarity to orthologs. It removes a single mannose residue from Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B. Disruption of manE caused drastic decreases in ER-Man activity in A. oryzae microsomes.
Source: J. Biosci. Bioeng., 113(4), 438-441 (2012)

18. Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2.

Author: Shengbin Rao, Osamu Mizutani, Takuya Hirano, Kazuo Masaki, Haruyuki Iefuji
Abstract: An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.
Source: J Biosci Bioeng. 2011 Nov;112(5):441-6

19. Sensitive Measurement of Thermal Stress in Beer and Beer-like Beverages Utilizing the 2-Thiobarbituric Acid (TBA) Reaction.

Author: Akihiro Mizuno, Yoshitsugu Nomura, and Hiroshi Iwata
Abstract: We examined the sensitivity of the 2-thiobarbituric acid (TBA) reaction for measuring thermal stress in beer, happoshu (a beer-like beverage with <67% malt content), and new genre beverages (beer-like beverages without malt or happoshu diluted with spirit). The thiobarbituric acid index (TBI) analytical procedure has been used to measure thermal stress in malt, wort, and beer. However, the TBI method is not as sensitive for beer. We compared the TBA reaction method used in shochu (a traditional Japanese distilled alcoholic beverage) with the TBI method. We confirmed that the TBA reaction method used for shochu is more suitable for measuring thermal stress in beer and beer-like beverages, whereas the TBI method is more suitable for measuring thermal stress in wort. The difference in the suitability of these methods seemed to be partly owing to the difference in the reaction of TBA with 5-hydroxymethylfurfural and 3-deoxyglucosone, both of which are major intermediates in the Maillard reaction. We concluded that the shochu TBA reaction method could produce a sensitive and practical index of thermal stress inflicted during storage of beer and beer-like beverages.
Source: J. Am. Soc. Brew. Chem., 69, 220-226 (2011)

20. Application of Combustion Method to the Determination of Total Nitrogen in Beer, Low-Malt Beer, and Third-Category Beer

Author

M. Nakahara (Kirin Business Expert Co. Ltd.), Chair; S. Furusho (Sapporo Breweries Ltd.); E. Hirabayashi (Suntory Liquors Ltd.); A. Isamoto (Sapporo Breweries Ltd.); Y. Kasama (LECO Japan Co.); K. Kusaka (National Research Institute of Brewing); K. Matsui (Kirin Business Expert Co. Ltd.); S. Matsui (Sumika Chemical Analysis Service); R. Ota (Sumika Chemical Analysis Service); T. Ozawa (Kirin Business Expert Co. Ltd.); D. Sato (DKSH Japan K.K.); Y. Sato (Asahi Breweries, Ltd.); A. Tanahara (Ryukyu University); T. Uehara (Orion Breweries, Ltd.); and T. Yamakawa (Suntory Liquors Ltd.)

Abstract

The amount of protein in food is calculated from total nitrogen. The conventional Kjeldahl method has several disadvantages: the time required for quantitative determination and the use of dangerous reagents such as concentrated sulfuric acid, etc. In contrast, the combustion method is capable of determining total nitrogen.This subcommittee was charged with evaluating the combustion method for analysis of total nitrogen in beer, low-malt beer, and third-category beer.

The collaborative work was carried out by 11 collaborators. Both repeatability and reproducibility were calculated from the results of the collaborative work.

Relative repeatability standard deviation (RSDr) and repeatability limit (r95) for determination of total nitrogen by the combustion method ranged from 0.8 to 4.1% and from 12.6 to 34.5 mg/L, respectively, and were judged acceptable.
Relative reproducibility standard deviation (RSDR) and reproducibility limit (R95) for determination of total nitrogen by the combustion method ranged from 3.0 to 11.9% and from 36.6 to 115.4 mg/L, respectively, and were judged acceptable.

Source

J. Am. Soc. Brew. Chem., 69, 308-309 (2011)

21. Analysis of Sake Component Presented to the Sake Contests in 2010

Author: S. Sudo, A. Isogai, A. Fujita, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 183, 1-15 (2011)

22. Analysis of Traditional Shochu Presented to the 33rd Contest in 2010

Author: H. Fukuda, K. Kobayashi, K. Sakamoto, O. Mizutani, M. Kanai, J. Hiramatsu
Source: Report of the Research Institute of Brewing, 183, 16-25 (2011)

23. Contents of amines in Sake

Author: S. Horii, T. Hashiguchi, H. Izu, S. Sudo
Source: Report of the Research Institute of Brewing, 183, 26-30 (2011)

24. Search for an O-methyltransferase gene of koji-mold involved in the formation of a musty/moldy off-odor

Author: Michiko ENDO, Akiko FUJITA, Atsuko ISOGAI, Ryoko KANDA, Kazuhiro IWASHITA, Osamu YAMADA, Hiroshi IWATA and Shigetoshi SUDO
Abstract: To search for an O-methyltransferase gene of koji-mold involved in the conversion of 2,4,6-trichlorophenol (TCP) to 2,4,6-trichloroanisole (TCA, a main cause of a musty/moldy off-odor), we performed gene disruptions in Aspergillus oryzae and screened a disruptant weakened in the conversion ability. First, 76 genes assigned as SAM-dependent methyltransferase or O-methyltransferase were extracted from the Aspergillus oryzae RIB40 genome database. Among them, 34 genes were individually disrupted. Rice-koji was then made using the resulting disruptants in the presence of TCP. The conversion ability of TCP to TCA in the rice-koji of the AO080521000231 disruptant was much lower (12%) than that of non-disrupted transformant while the rice-koji retained sufficient enzyme productivities of α-amylase, glucoamylase, acid carboxypeptidase, and acid protease. These results suggest that AO080521000231 encodes the primary O-methyltransferase involved in the conversion of TCP to TCA.
Source: J. Brew. Soc. Japan, 106（8）, 556-561 （2011）

25. Sake yeast strains have difficulty in entering a quiescent state after cell growth cessation

Author: Henryk Urbanczyk, Chiemi Noguchi, Hong Wu, Daisuke Watanabe, Takeshi Akao, Hiroshi Takagi, and Hitoshi Shimoi
Abstract: Sake yeast strains produce a high concentration of ethanol during sake brewing compared to laboratory yeast strains. As ethanol fermentation by yeast cells continues even after cell growth stops, analysis of the physiological state of the stationary phase cells is very important for understanding the mechanism of producing higher concentrations of ethanol. We compared the physiological characteristics of stationary phase cells of both sake and laboratory yeast strains in an aerobic batch culture and under sake brewing conditions. We unexpectedly found that sake yeast cells in the stationary phase had a lower buoyant density and stress tolerance than did the laboratory yeast cells under both experimental conditions. These results suggest that it is difficult for sake yeast cells to enter a quiescent state after cell growth has stopped, which may be one reason for the higher fermentation rate of sake yeast compared to laboratory yeast strains.
Source: Journal of Bioscience and Bioengineering, Vol. 112, No. 1, 44-48 (2011)

26. Automatic measurement of sake fermentation kinetics using a multi-channel gas monitor system

Author: Daisuke Watanabe, Tomonori Ota, Fusajiro Nitta, Takeshi Akao, and Hitoshi Shimoi
Abstract: In small-scale sake brewing tests, progression of ethanol fermentation is examined by monitoring carbon dioxide emission using the Fermograph, an automated multi-channel gas monitor instrument. The Fermograph system enables automatic measurements of fermentation profiles with high accuracy, reproducibility, and resolution, and facilitates comprehensive and quantitative sake fermentation kinetic analyses.
Source: Journal of Bioscience and Bioengineering, Vol. 112, No. 1, 54-57 (2011)

27. Analyses of peptides in sake mash: Forming a profile of bitter-tasting peptides

Author: Yukiko Maeda, Masaki Okuda, Katsumi Hashizume, Midori Joyo, Shigeaki Mikami and Nami Goto-Yamamoto
Abstract: Some oligopeptides and amino acids have a strong influence on the sensory qualities of sake, but the formation process of such compounds in sake mash has not been well elucidated. In this study, we investigated the formation process of bitter-tasting peptides derived from rice proteins in sake mash, because knowledge about their formation may contribute to the quality control of sake. We analyzed rice protein hydrolysates in sake mash, as well as in the enzymatic digest of steamed rice grains digested by either sake-koji or by crude enzyme extracted from sake- koji. SDS-PAGE showed that a smaller amount of polypeptides (>M.W. 10,000) accumulated in the supernatant of sake mash than in either enzymatic digest. The concentration of peptides in the supernatant of sake mash increased gradually from the early stages of fermentation. Five bitter-tasting peptides (No. 9, <QLFNPS; No. 13, <QLFNPSTNP; No. 17, <QLFNPSTNPWH; No. 18, <QLFNPSTNPWHSP; No. 20, <QLFGPNVNPWHNP), which were previously found in sake mash, were not found in significant amounts in sake- koji. On the other hand, these peptides accumulated at the early stages of both sake mash fermentation and the enzymatic digests, although the levels in sake mash were higher than those in the digests. The present study demonstrated that the 5 bitter-tasting peptides formed in high concentrations when steamed rice grains were digested under conditions of sake mash fermentation with yeast.
Source: Journal of Bioscience and Bioengineering 112, 3, 238-246 (2011)

28. Simple method for the simultaneous quantification of medium-chain fatty acids and ethyl hexanoate in alcoholic beverages by gas chromatography-flame ionization detector: Development of a direct injection method

Author: Kei Takahashi and Nami Goto-Yamamoto
Abstract: Free medium-chain fatty acids (MCFAs) can negatively influence the fermentation process and taste quality in alcoholic beverages. Ethyl hexanoate is important in providing a fruit-like flavour to drinks, particularly in Japanese sake. In this study, we developed a direct injection method for a gas chromatography-flame ionization detector following the semi-purification of chemical components, such as esters, alcohols and MCFAs in alcoholic beverages. Evaluation of MCFAs by this method gave a limit of detection on the order of sub-ppm and relative standard deviations less than 10% in standard solution. Good repeatability and recovery rates against MCFAs and ethyl hexanoate were also obtained in non-distilled real alcoholic beverages. Because this method enabled us to simultaneously quantify the concentrations of MCFAs and ethyl hexanoate, the proportion of ester against MCFAs was proposed as a quality control index. This method could be suitable for routine analysis in the alcohol beverage industry.
Source: J Chromatogr. A 1218, 43, 7850-7856 (2011)

29. Accumulation of Co in Yeast Cells under Metabolically Active Condition - Implication for Role of Yeast in Migration of Radioactive Co -

Author: Naofumi KOZAI, Toshihiko OHNUKI, Fuminori SAKAMOTO, Yoshinori SUZUKI, Kazuya TANAKA, Haruyuki IEFUJI, and Takuro SAKAI
Abstract: The accumulation and change in the chemical states of Co have been studied to elucidate the role of the yeast Saccharomyces cerevisiae in the migration of radioactive cobalt in the environment. The yeast was grown in a solution containing Co(II) ions, a carbon source, and essential elements (metabolically active condition). For comparison, an adsorption experiment of Co(II) ions on the yeast cells under resting condition without essential elements was performed. Time courses of Co concentration in the solution, in the cells, and chemical states of the accumulated Co were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), particle-induced X-ray emission (PIXE), and X-ray absorption fine structure (XAFS) analyses. The time courses of Co concentration in the solution showed that a higher amount of Co was accumulated by the yeast cells under the metabolically active condition than under the resting one. PIXE analyses showed the concurrent accumulation of Fe with Co accumulation under the metabolically active condition, suggesting the intracellular accumulation of Co. XAFS analyses showed that the k3-weighted extended-XAFS functions and the radial structural function of Co accumulated by the yeast cells under the metabolically active condition are similar to those under the resting condition, indicating that the chemical states of the accumulated Co were nearly the same between both conditions. These results indicate that the yeast performs better retardation of the migration of Co under the metabolically active condition than under the resting one.
Source: Journal of Nuclear Science and Technology, 48, 1206-1213 (2011)

30. Molecular biological researches of Kuro-Koji molds, their classification and safety

Author: Osamu Yamada, Ryo Takara, Ryoko Hamada, Risa Hayashi, Masatoshi Tsukahara, and Shigeaki Mikami
Abstract: To assess the position of Kuro-Koji molds in black Aspergillus, we performed sequence analysis of approximately 2500 nucleotides of partial gene fragments, such as histone 3, on a total of 57 Aspergillus strains, including Aspergillus kawachii NBRC 4308, 12 Kuro-Koji molds isolated from awamori breweries in Japan, Aspergillus niger ATCC 1015, and A. tubingensis ATCC10550. Sequence results showed that all black Aspergillus strains could be classified into 3 types, type N which includes A. niger ATCC 1015, type T which includes A. tubingensis ATCC 10550, and type L which includes A. kawachii NBRC 4308. Phylogenetic analysis showed these three types belong to different clusters. All 12 Kuro-Koji molds isolated from awamori breweries were classified as type L, thus we concluded type L represents the industrial Kuro-Koji molds. We found all type L strains lack the An15g07920 gene which is required for ochratoxin A biosynthesis in black Aspergillus. This sequence is present in the genome of A. niger CBS 513.88 and has homology to the polyketide synthase fragment of A. ochraceus which is involved in ochratoxin A biosynthesis. Based on the industrial importance and the safety of Kuro-Koji molds, we propose to classify the type L strains as Aspergillus luchuensis, as initially reported by Dr. Inui.
Source: Journal of Bioscience and Bioengineering, 112, 233-237 (2011)

31. Aflatoxin non-productivity of Aspergillus oryzae caused by loss of function in the aflJ gene product

Author: Takuro Kiyota, Ryoko Hamada, Kazutoshi Sakamoto, Kazuhiro Iwashita, Osamu Yamada, and Shigeaki Mikami
Abstract: Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1-3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level.
Source: Journal of Bioscience and Bioengineering, 111, 512-517 (2011)

32. In silico Analysis of 3'-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data

Author: MIZUKI Tanaka, YOSHIFUMI Sakai, OSAMU Yamada, TAKAHIRO Shintani, and KATSUYA Gomi
Abstract: To investigate 3'-end-processing signals in Aspergillus oryzae, we created a nucleotide sequence data set of the 3'-untranslated region (3'UTR) plus 100 nucleotides (nt) sequence downstream of the poly(A) site using A. oryzae expressed sequence tags and genomic sequencing data. This data set comprised 1065 sequences derived from 1042 unique genes. The average 3'UTR length in A. oryzae was 241 nt, which is greater than that in yeast but similar to that in plants. The 3'UTR and 100 nt sequence downstream of the poly(A) site is notably U-rich, while the region located 15-30 nt upstream of the poly(A) site is markedly A-rich. The most frequently found hexanucleotide in this A-rich region is AAUGAA, although this sequence accounts for only 6% of all transcripts. These data suggested that A. oryzae has no highly conserved sequence element equivalent to AAUAAA, a mammalian polyadenylation signal. We identified that putative 3'-end-processing signals in A. oryzae, while less well conserved than those in mammals, comprised four sequence elements: the furthest upstream U-rich element, A-rich sequence, cleavage site, and downstream U-rich element flanking the cleavage site. Although these putative 3'-end-processing signals are similar to those in yeast and plants, some notable differences exist between them.
Source: DNA RESEARCH, 18, 189-200 (2011)

33. Different enantioselectivity of two types of poly(lactic acid) depolymerases toward poly(L-lactic acid) and poly(D-lactic acid)

Author: Fusako Kawai, Kosuke Nakadai, Emiko Nishioka, Hajime Nakajima, Hitomi Ohara, Kazuo Masaki, Haruyuki Iefuji
Abstract: Poly(lactic acid) (PLA) depolymerases are categorized into protease-type and lipase-type. Protease-types can hydrolyze poly(L-lactic acid) (PLLA) but not poly(D-lactic acid) (PDLA). Lipase-types, including cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2 preferentially hydrolyze PDLA. Both enzymes degraded not only PLA emulsion but also PLA film, in which amorphous region is preferentially attacked, but crystalline region can be also attacked. Stereocomplex PLA (sc-PLA) formed by 50:50 blending of PLLA and PDLA included no homo crystals, but a tiny homo crystallization peak appeared and crystallinity increased by 5% when attacked by CLE, although no significant change of molecular weight and crystalline size was found. Enantioselective degradation must occur in amorphous region of PLLA/PDLA film and preferentially hydrolyzed PDLA, resulting in a slightly excess amount of PLLA remained, which must be crystallized.
Source: Polym. Degrad. Stab. 96 (2011) 1342-1348

34. Calcineurin ensures a link between the DNA replication checkpoint and microtubule-dependent polarized growth

Author: Kazunori Kume, Takayuki Koyano, Muneyoshi Kanai, Takashi Toda and Dai Hirata
Abstract: Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (CTIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signaling pathway linking CTIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The CTIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.
Source: Nat. Cell Biol., 13(3), 234-42, (2011)

35. Purification and Characterization of porcine skeletal muscle aminopeptidase T, a novel metallopeptidase homologous to Leukotriene A4 hydrolase

Author: Mohammed Alamgir SARKER, Shinji MATSUDA, Osamu MIZUTANI, Shengbin RAO, Koshiro MIGITA, Nami GOTO-YAMAMOTO, Haruyuki IEFUJI, Toshihide NISHIMURA
Abstract: A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.
Source: Biosci. Biotechnol. Biochem., 2011 Jun 23;75(6):1154-1159

36. Isolation and characterization of glucoamylase from a wastewater treatment yeast Hansenula fabianii J640, and construction of expression vector.

Author: Miyoshi Kato, Teruhito Kitajima, Haruyuki Iefuji
Abstract: Hansenula fabianii J640 highly expresses an extracellular glucoamylase (GA). Here, we purified the GA and showed that it has pH and temperature optima of 5.0 and 50 ℃, is stable at temperatures up to 50 ℃, is inhibited by Ag²⁺, Hg²⁺ and Cu²⁺. The gene was found in an expression library with anti-GA antibodies. A cDNA was found to encode 491 amino acids, including a putative signal peptide of 21 amino acids. Because of the gene's high expression, we used its promoter and terminator regions to improve a previously developed H. fabianii J640 expression system.
Source: Appl. Microbiol. Biotech. 90, 981-987 (2011)

37. Breeding of a new wastewater treatment yeast by genetic engineering

Author: Miyoshi Kato and Haruyuki Iefuji
Abstract: We previously developed a host vector system for the wastewater treatment yeast Hansenula fabianii J640. The promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640 were used for a new expression vector, pHFGE-1. The performance of pHFGE-1 was compared with that of the widely used pG-1 transformant vector. H. fabianii J640 (HF-TAMY) cells were transformed with pHFGE-1, and Saccharomyces cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of which carried the Taka-amylase. Expression of Taka-amylase by HF-TAMY showed higher than that by SC-TAMY. By using this new system, we bred the new wastewater treatment yeast that shows α-amylase activity. This yeast appears to grow well under experimental wastewater conditions, and is effective in treating model wastewater containing soluble and insoluble starch.
Source: AMB Express. 1: 7 (2011)