2013 Research results

1. Accelerated alcoholic fermentation caused by defective glucose derepression-related gene expression in Saccharomyces cerevisiae

Author

Daisuke Watanabe, Naoya Hashimoto, Megumi Mizuno, Yan Zhou, Takeshi Akao, and Hitoshi Shimoi

Abstract

Sake yeast strains maintain a high fermentation rate even after entering the stationary growth phase. To investigate the molecular mechanisms underlying this advantageous brewing property, we compared the gene expression profiles of sake and laboratory yeast strains of Saccharomyces cerevisiae during the stationary growth phase. DNA microarray analysis revealed that the examined sake yeast strain had defects in expression of the glucose derepression-related genes mediated by the transcription factors Adr1p and Cat8p. Furthermore, deletion of the ADR1 and CAT8 genes slightly, but statistically significantly, improved the fermentation rate of a laboratory yeast strain. We also identified two loss-of-function mutations in the ADR1 gene of existent sake yeast strains. Taken together, these results demonstrate that the glucose derepression-related gene expression program of yeast acts as an impediment to effective alcoholic fermentation in the glucose-rich fermentative conditions.

Source

Bioscience, Biotechnology, and Biochemistry, 77, 2255-2262 (2013)

2. Rim15p-mediated regulation of sucrose utilization during molasses fermentation using Saccharomyces cerevisiae strain PE-2

Author

Tomomi Inai, Daisuke Watanabe, Yan Zhou, Rie Fukada, Takeshi Akao, Jun Shima, Hiroshi Takagi, and Hitoshi Shimoi

Abstract

Inherited loss-of-function mutations in the Rim15p-mediated stress-response pathway contribute to the high fermentation rate of sake yeast strains. In the present study, we found that disruption of the RIM15 gene in ethanol-producing Saccharomyces cerevisiae strain PE-2 accelerated molasses fermentation through enhanced sucrose utilization following glucose starvation.

Source

Journal of Bioscience and Bioengineering, 116, 591-594 (2013)

3. An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses

Author

Takuya Inaba, Daisuke Watanabe, Yoko Yoshiyama, Koichi Tanaka, Jun Ogawa, Hiroshi Takagi, Hitoshi Shimoi, and Jun Shima

Abstract

Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MATa/α, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1 gene. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP.

Source

AMB Express., 3, 74 (2013)

4. Constant Enthalpy Change Value during Pyrophosphate Hydrolysis within the Physiological Limits of NaCl

Author

Satoshi Wakai, Shun-ichi Kidokoro, Kazuo Masaki, Kaoru Nakasone, Yoshihiro Sambongi

Abstract

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. Howeve, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at approximately 4 M NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 M NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of -35 kJ/mol was obtained for the halophile and non-halophiles at 1.5-4.0 and 0.1-2.0 M NaCl, respectively. These results show that solvation changes caused by up to 4.0 M NaCl (water activity of approximately 0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.

Source

The Journal of Biological Chemistry, 288, 29247-29251 (2013)

5. Treatment of, and Candida utilis biomass production from shochu wastewater; the effects of maintaining a low pH on DOC removal and feeding cultivation on biomass production

Author

Takashi Watanabe, Haruyuki Iefuji, Hiroko K. Kitamoto

Abstract

Shochu wastewater (alcoholic distillery wastewater) contains large amounts of organic compounds (25,000 - 60,000 COD mg/L), nitrogen (1,000 - 6,000 T-N mg/L), and phosphorus (500 - 1,000 T-P mg/L). Despite its high nutrient content, shochu wastewater is highly perishable, which limits its utilization for animal feed and fertilizer. Therefore, shochu wastewater is mainly treated by methane fermentation. On the other hand, a feed yeast, Candida utilis, can utilize various organic compounds and be utilized as a yeast extract source and animal feed. We previously bred a mutant, C. utilis UNA1, that accumulates a large amount of nitrogen. Here, we investigated the use of C. utilis UNA1 to treat highly concentrated shoshu wastewater. With fed-batch cultivation using a 5-L jar fermenter, controlling pH at 5.0 with H2SO4, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted barley shochu wastewater (BSW), and about 16.7 kg of freeze-dried yeast biomass was obtained. The yeast sludge biomass generated from BSW contains about 60% crude protein. Furthermore, using H2SO4 to control pH increased the sulfur content of wastewater, which increased the methionine composition of yeast sludge biomass.

Source

SpringerPlus, 2, 514 (2013)

6. Breeding of wastewater treatment yeasts having high nitrogen removal ability

Author

Takashi WATANABE, Haruyuki IEFUJI, Hiroko K. KITAMOTO

Abstract

The purpose of this study is to improve the nitrogen removal ability of wastewater treatment methods using yeasts. In the case of Saccharomyces cerevisiae, we previously confirmed that a positive selection of methylamine-resistant mutants to obtain Ure2 mutants was useful for improving the nitrogen removal ability. Therefore, we tried to improve the nitrogen removal abilities of wastewater treatment yeasts in the same manner and H. anomala IFO0963-HNA36 was obtained. Strain HNA36 removed 1.2-fold higher amounts of nitrogen from rice Shochu distillery wastewater than the wild type strain. We also developed another new breeding method to improve the nitrogen removal ability. A mutant constitutively secreting proteinase, Pseudozyma antarctica GB-4(0)-PNR26 was obtained. Strain PNR26 removed 1.14-fold higher amounts of nitrogen from rice Shochu distillery wastewater than the wild type strain. These results show that nitrogen removal abilities of wastewater treatment yeasts can be improved without recombinant techniques.

Source

J. Brew. Soc. Japan, 108, 823-829（2013）

7. Geochemical, stable isotope, and numerical modeling studies of sake and groundwater for identification of the location of sake production: A case study from Hakusan City, Japan

Author

Shingo Tomiyama, Tomokazu Hashiguchi, Akira Ueda, Akiko Kitai, Hanae Izu and Shigetoshi Sudo

Abstract

Understanding the source and chemical characteristics of groundwater used for sake production is an important strategy for the quality management of sake brewing. Oxygen isotope ratios (δ18O) of 96 sake samples from 19 prefectures in Japan were analyzed. A good correlation was found between the sake δ18O values and groundwater δ18O values taken from previously published data, where the δ18O of sake is similar to that of the groundwater used in the sake breweries. The combined geochemical and numerical modeling approach provides a good tool for determining the source of groundwater used in sake production.

Source

Geochemical Journal, 47, 591-608 (2013)

8. Characterization of Hokujun (Beichun) and Muscat Hamburg-Amurensis (MH-AM) by Simple Sequence Repeat (SSR) analysis

Author

Nami Goto-Yamamoto, Mineyo Numata, Junzo Furukawa

Abstract

Hokujun in Japanese, or Beichun in Chinese, is a grape variety bred in China by crossing between Vitis amurensis and Muscat Hamburg. Muscat Hamburg-Amurensis (MH-AM), cultivated in Hokkaido, the north part of Japan, is thought to be Beichun or its seedling, but it was not clear. This study showed that SSR data at 14 loci of MH-AM were identical to those of Beichun, and they are the same cultivar. Additionally, Beichun has both alleles from Muscat Hamburg except for 1 locus, and from V. amurensis or related wild grape accessions. Principal coordinate analysis was carried out based on a distance matrix calculated from SSR data, and the result showed that Beichun was plotted close to both of Muscat cultivars and V. amurensis or related wild grape accessions.

Source

Journal of ASEV Japan, 24, 4-8 (2013)

9. The transfer of radioactive cesium and potassium from rice to sake

Author

Masaki Okuda, Tomokazu Hashiguchi, Midori Joyo, Kaori Tsukamoto, Michiko Endo, Katsumi Matsumaru, Nami Goto-Yamamoto, Hiroshi Yamaoka, Kenji Suzuki, and Hitoshi Shimoi

Abstract

Using rice grains contaminated with radioactive cesium (134Cs and 137Cs) that was released by the Fukushima Daiichi Nuclear Power Plant Accident in March of 2011, we investigated the behaviors of the radioactive cesium and potassium (total K and 40K) during sake brewing. Cesium is a congener of K, and yeast cells have the ability to take up Cs using known K transporters. During rice polishing, the concentrations of radioactive Cs and K in the polished rice grains decreased gradually until a milling ratio (polished rice weight/brown rice weight) of 70% was reached. No significant changes were observed below this milling ratio. Sake was brewed on a small scale using the 70% polished rice. The transfer ratio of radioactive Cs to sake and to the sake cake was significantly different than the ratio of K. Approximately 36% and 23% of radioactive Cs in the polished rice was transferred to the sake and sake cake, respectively; however, 40% was removed by washing and steeping the rice grains. On the other hand, 25% and 40% of K in the polished rice was recovered in the sake and sake cake, respectively, and 35% was removed by washing and steeping the rice grains. From the present results, the concentration of radioactive Cs in sake would be 4 Bq/kg fresh weight, which is well below the regulation values (100 Bq/kg), even using brown rice containing 100 Bq/kg of radioactive Cs.

Source

Journal of Bioscience and Bioengineering, 116, 340-346, (2013)

10. Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS)
Saccharomyces cerevisiae

Author

Kou Wakabayashi, Atsuko Isogai, Daisuke Watanabe, Akiko Fujita, Shigetoshi Sudo

Abstract

Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called "hineka". Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Δmeu1, Δmri1, and Δmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Δmri1 and Δmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Δspe2 sake than in Δmri1 or Δmde1 sake, the DMTS producing potential of Δspe2 sake was as low as that of Δmri1 or Δmde1 sake. Sake brewing tests using BY4743 Δspe2 Δmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Δspe2 or Δmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake.

Source

Journal of Bioscience and Bioengineering, 116, 475-479 (2013)

11. Evaluation of Antioxidant Activity on Flavor Stability of Beer and Beer-like Beverages

Author

Akihiro Mizuno

Abstract

The relationships among flavor stability, antioxidant activity, and aging-related indices of beer were examined in traditional all-malt and adjunct beer, Happoshu (a beer-like beverage with less than 67% malt content), and New-genre (a beer-like beverage without malt, or Happoshu diluted with spirit) beverages. Flavor stability was evaluated by assessing flavor-staling degree using sensory analysis after an accelerated storage test at 30℃. Hydroxyl radical, the most reactive oxygen species, is considered a major determinant for flavor stability of beer. However, the staling degree measure exhibited a more significant correlation with 1,1'-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity than the hydroxyl radical-scavenging activity. The lag time of the hydroxyl radical generation analyzed by electron spin resonance spectroscopy is now used as an index of flavor stability of beer; it is thought that longer lag times indicate higher antioxidant activity of beer. However, a markedly longer lag time was observed in Happoshu and New-genre than in beer, although Happoshu and New-genre showed lower antioxidant activities than beer. Lag time did not appear to be applicable as an index of flavor stability for Happoshu and New-genre. DPPH-scavenging activity was found to be a more suitable and a common index for assessing flavor stability in not only traditional all-malt and adjunct beers but also Happoshu and New-genre, depending mostly on polyphenol-mediated antioxidant activity. In a brewing fermentation test, changes in DPPH-scavenging activity during fermentation were not observed, which suggests that the antioxidant activity of casting wort could be critical to the flavor stability of not only beer but also beer-like beverages.

Source

Journal of the American Society of Brewing Chemists, 71, 131-137, (2013)

12. Ferulic acid and ethyl ferulate in sake: Comparison of levels between sake and mirin and analysis of their sensory properties

Author

Katsumi HASHIZUME, Toshihiko ITO, Misako SHIMOHASHI, Takahiro ISHIZUKA, and Masaki OKUDA

Abstract

Ferulic acid (FA) and ethyl ferulate (EF) in sake and mirin samples were quantified. Concentrations of FA and EF in the sake and mirin samples showed high correlations (r=0.91 and 0.89, respectively). The highest level of EF in the sake samples was ca. 14-fold that of the mirin samples. Thresholds of FA and EF in a sake sample were estimated using a pipette method as 0.075 mg/L and 0.39 mg/L, respectively, by eight assessors in their twenties. The FA threshold was far lower than the highest level of FA in the sake samples, which suggested that FA might affect the sensory quality of sake. FA added to the sake sample showed unpleasant bitter, astringent, "egumi", or irritating taste characteristics. Sensory and instrumental analyses suggested that EF has the ability to mitigate the taste of FA in sake.

Source

Food Science and Technology Research, 19, 705-709 (2013)

13. Construction of a ligD disruptant for efficient gene targeting in white koji mold, Aspergillus kawachii

Author

Satoshi Tashiro, Taiki Futagami, Shotaro Wada, Yasuhiro Kajiwara, Hideharu Takashita, Toshiro Omori, Toru Takahashi, Osamu Yamada, Kaoru Takegawa, and Masatoshi Goto

Abstract

Aspergillus kawachii IFO4308 is a commonly used strain for research into shochu-making and enzyme production without the technique of genetic engineering. In the present study, we successfully constructed various A. kawachii host strains with the ability of highly efficient homologous recombination. In addition to the genome information about A. kawachii, these strains are useful for development of genetic study concerning shochu making and industrial productions of proteins and organic acids.

Source

Journal of General and Applied Microbiology, 59, 257-260 (2013)

14. Carbon stable isotope analysis for tracing the raw materials of commercially-supplied Mirin

Author

Hanae IZU, Tomokazu HASHIGUCHI, Tomoko HASHIMOTO, Katsumi MATSUMARU

Abstract

In this study, we determined the carbon stable isotope ratios of extract (δ13CEx) and alcohol (δ13CAlc) in commercially-supplied Mirin to confirm them as a potential index for verifying the raw material origins. The δ13CAlc values of the Mirin which brewing alcohol was added (-14.6 ± 4.0‰) are significantly higher than that of the Mirin which Shochu was added (-26.0 ± 1.7‰). The δ13CEx values of the Mirin are significantly higher by sugar addition (+sugar; -17.1 ± 3.3‰, -sugar; -26.1 ± 1.0‰). Old-style Mirin which was made from rice, koji, and Shochu shows lower δ13CAlc and δ13CEx values (δ13CAlc ; -25.8 ± 1.1‰, δ13CEx ; -26.6 ± 0.9‰) than those of new-style Mirin which includes brewing alcohol and sugar as raw materials (δ13CAlc ; -16.3 ± 0.8‰, δ13CEx ; -19.8 ± 0.7‰). Mirin-like seasonings (fermentation seasoning and Mirin-type seasoning) and Akumochizake used like Mirin are also analyzed, and their δ13CAlc and δ13CEx show higher values by addition of brewing alcohol and sugar, respectively. These results suggest that the δ13CAlc and δ13CEx values in commercially-supplied Mirin are useful for the confirmation of sugar, brewing alcohol, and Shochu addition as the raw material origins.

Source

BUNSEKI KAGAKU, 62, 841-844 (2013)

15. Quantitation of DMTS precursor, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) in alcoholic beverages and fermented solutions by a stable isotope dilution analysis

Author

Atsuko ISOGAI, Ryoko KANDA, Shigetoshi SUDO, Katsumi MATSUMARU

Abstract

A method for quantitation of DMTS-P1, a precursor of DMTS, was developed by using a stable isotope of DMTS-P1, DMTS-P1-d3, as an internal standard, in which the methyl group was labeled by three deuterium atoms. The recovery rate from various kinds of samples was improved to 100±10％ using this method. Application of the method to various commercial alcoholic beverages revealed that the DMTS-P1 concentration in sake was higher than other beverages. When sake, wine, and beer yeast were cultivated in the same medium, sake yeast produced higher amount of DMTS-P1 than other yeast. The correlation of DMTS-P1 concentration and DMTS producing potential (production of DMTS after storage) was observed for commercial sake, but not for wine, suggesting the presence of other components responsible for DMTS production. A small-scale sake brewing test was carried out and changes in the DMTS-P1 concentration were investigated. DMTS-P1 increased during fermentation and a slight increase was observed after filtration. High fermentation temperature increased both DMTS-P1 concentration and DMTS producing potential.

Source

J. Brew. Soc. Japan, 108, 605-614 (2013)

16. Analysis of Sake Component Presented to Sake Contests in 2012

Author

Katsumi MATSUMARU, Atsuko ISOGAI, Hanae IZU, Ryoko KANDA and Yasuzo KIZAKI

Source

Report of the Research Institute of Brewing, 185, 1-16 (2013)

17. Analysis of Traditional Shochu Presented to the 35ｓｔ Contest in 2012

Author

Osamu YAMADA, Osamu MIZUTANI, Muneyoshi KANAI, and Yasuzo KIZAKI

Source

Report of the Research Institute of Brewing, 185, 17-26 (2013)

18. Survey Results of Radiocesium in Alcoholic Beverages and Brewing Water

Author

Tomokazu HASHIGUCHI, Hanae IZU, and Katsumi MATSUMARU

Source

Report of the Research Institute of Brewing, 185, 27-31 (2013)

19. Fungal-specific sirtuin HstD coordinates secondary metabolism and development through control of LaeA

Author

Moriyuki Kawauchi, Mika Nishiura, Kazuhiro Iwashita

Abstract

The sirtuins are members of the NAD+-dependent histone deacetylase family that contributes to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungal-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungal-specific sirtuin hstD/Aohst4, which is a homolog of yeast hst4 in Aspergillus oryzae. The deletion of all histone deacetylases in A. oryzae demonstrated that fungal-specific sirtuin hstD/Aohst4 is required for the coordination of fungal development and secondary metabolite production. We also showed that the gene expression of laeA, which is the most studied fungal-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in the ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that hstD/Aohst4 works upstream of laeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungal-specific but conserved in the vast family of filamentous fungi. Thus, we concluded that the fungal-specific sirtuin hstD/Aohst4 coordinates fungal development and secondary metabolism via the regulation of laeA in filamentous fungi.

Source

Eukaryotic Cell, 12, 1087-1096 (2013)

20. SSR Genotyping of Wild Grape Species and Grape Cultivars of Vitis vinifera and V. vinifera × V. labrusca

Author

Nami Goto-Yamamoto, Akifumi Azuma, Nobuhito Mitani and Shozo Kobayashi

Abstract

To determine the relationship among wild grape species and grape cultivars, genotyping with 8 SSR loci was carried out using a wide range of grape species and cultivars, i.e., North American species, East Asian species, cultivars of Vitis vinifera, and those of V. vinifera × V. labrusca. SSR genotyping showed that wild grape species are more highly diverse than cultivated grapes. A principal coordinate analysis based on the distance (1- proportion of shared alleles) among accessions showed a clear separation between wild and cultivated grapes. A native Japanese cultivar, 'Koshu', and a Chinese one, 'Niunai', were plotted among the cultivars of V. vinifera, even though these 2 Far East cultivars have some unique alleles.

Source

Journal of Japanese Society for Horticultural Science, 82, 125-130 (2013)

21. Aspergillus luchuensis, an Industrially Important Black Aspergillus in East Asia.

Author

Seung-Beom Hong, Mina Lee, Dae-Ho Kim, Janos Varga, Jens C. Frisvad, Giancarlo Perrone, Katsuya Gomi, Osamu Yamada, Masayuki Machida, Jos Houbraken, Robert A. Samson

Abstract

Aspergilli known as black- and white-koji molds which are used for awamori, shochu, makgeolli and other food and beverage fermentations, are reported in the literature as A. luchuensis, A. awamori, A. kawachii, or A. acidus. In order to elucidate the taxonomic position of these species, available ex-type cultures were compared based on morphology and molecular characters. A. luchuensis, A. kawachii and A. acidus showed the same banding patterns in RAPD, and the three species had the same rDNA-ITS, β-tubulin and calmodulin sequences and these differed from those of the closely related A.niger and A. tubingensis. Morphologically, the three species are not significantly different from each other or from A. niger and A. tubingensis. It is concluded that A. luchuensis, A. kawachii and A. acidus are the same species, and A. luchuensis is selected as the correct name based on priority. Strains of A. awamori which are stored in National Research Institute of Brewing in Japan, represent A. niger (n = 14) and A. luchuensis (n = 6). The neotype of A. awamori (CBS 557.65 = NRRL 4948) does not originate from awamori fermentation and it is shown to be identical with the unknown taxon Aspergillus welwitschiae. Extrolite analysis of strains of A. luchuensis showed that they do not produce mycotoxins and therefore can be considered safe for food and beverage fermentations. A. luchuensis is also frequently isolated from meju and nuruk in Korea and Puerh tea in China and the species is probably common in the fermentation environment of East Asia. A re-descriptionof A. luchuensis is provided because the incomplete data in the original literature.

Source

PLOS ONE, 8, e63769 (2013)

22. Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Author

Jantaporn Thongekkaew, Hiroko Ikeda, Kazuo Masaki, Haruyuki Iefuji

Abstract

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.

Source

Enzyme and Microbial Technology, 52, 241-246 (2013)

23. Genome-wide screening to study breeding methods to improve the nitrogen accumulation ability of yeast without gene recombinant techniques.

Author

Takashi Watanabe, Haruyuki Iefuji, and Hiroko K. Kitamoto.

Abstract

To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD₆₆₀) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.

Source

Bioscience, Biotechnology, and Biochemistry, 77, 917-922. (2013)

24. Identification of the sulphate ion as one of the key components of yeast spoilage of a sports drink through genome-wide expression analysis

Author

Lahiru N. Jayakody, Keisuke Tsuge, Akihiro Suzuki, Hitoshi Shimoi, Hiroshi Kitagaki

Abstract

Because of the growing market for sports drinks, prevention of yeast contamination of these beverages is of significant concern. This research was performed to achieve insight into the physiology of yeast growing in sports drinks through a genome-wide approach to prevent microbial spoilage of sports drinks. The genome-wide gene expression profile of Saccharomyces cerevisiae growing in the representative sports drink was investigated. Genes that were relevant to sulphate ion starvation response were upregulated in the yeast cells growing in the drink. These results suggest that yeast cells are suffering from deficiency of extracellular sulphate ions during growth in the sports drink. Indeed, the concentration of sulphate ions was far lower in the sports drink than in a medium that allows the optimal growth of yeast. To prove the starvation of sulphate ions of yeast, several ions were added to the beverage and its effects were investigated. The addition of sulphate ions, but not chloride ions or sodium ions, to the beverage stimulated yeast growth in the beverage in a dose-dependent manner. Moreover, the addition of sulphate ions to the sports drink increased the biosynthesis of sulphur-containing amino acids in yeast cells and hydrogen sulphide in the beverage. These results indicate that sulphate ion concentration should be regulated to prevent microbial spoilage of sports drinks.

Source

The Journal of General and Applied Microbiology, 59, 227-238 (2013)

25. Fission yeast leucine-rich repeat protein Lrp1 is essential for cell morphogenesis as a component of the morphogenesis Orb6 network (MOR)

Author

Kazunori Kume, Shunsuke Kubota, Takayuki Koyano, Muneyoshi Kanai, Masaki Mizunuma, Takashi Toda, Dai Hirata

Abstract

In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.

Source

Bioscience, Biotechnology, and Biochemistry, 77, 1086-1091 (2013)

26. Starch characteristics and enzyme digestibility of steamed rice grains grown at high air temperature during grain filling in 2010

Author

Masaki Okuda, Midori Joyo, Kei Takahashi, Nami Goto-Yamamoto, Yukio Takagaki, Masaru Ikegami and Yoshihito Nabekura

Abstract

The mean temperature in August 2010 was the highest ever recorded in Japan. Previous studies showed that higher temperatures during rice grain filling cause a higher retrogradation of gelatinized starch due to longer side chains of amylopectin, as well as lower enzymatic digestibility during sake making. Thus, the effect of extremely high temperatures during the 2010 season on starch characteristics of rice grains for sake making and the enzyme digestibility of steamed rice grains was examined. The gelatinization temperatures of rice samples harvested during the 2010 season were higher than those harvested in the 2007 (a hot summer but cooler than 2010) and 2009(a cooler summer) seasons. The decreasing degree of enzymatic digestibility of steamed rice grains after partial retrogradation of starch was higher in 2010 than in 2007 and 2009. On the other hand, even in the rice samples harvested in the same prefectures, gelatinization temperatures and enzyme digestibility of rice grains varied among samples. Especially, the rice samples whose heading date was late showed lower gelatinization temperature and higher enzyme digestibility of steamed rice grains than early heading samples. Moreover, the polished rice grains harvested during the 2010 season showed lower alkali solubility than those harvested in 2009. The present study suggests that the enzyme digestibility of steamed rice grains can be predicted by mean air temperature after heading even in historic high air temperature such as the 2010 summer, and the alkali solubility method can be useful for examining the starch characteristics of rice grains conveniently.

Source

J. Brew. Soc. Japan, 108, 368-376 (2013)

27. Relationship between sake cake ratios and atmospheric temperature conditions (part 2)

Author

Masaki Okuda, Midori Joyo, Katsumi Hashizume, Kei Takahashi and Nami Goto-Yamamoto

Abstract

Monthly mean temperature anomalies in August and September from 1998 to 2010 showed a positive correlation with the national mean data of sake cake ratios. The average enzyme digestibility of 7 rice cultivars showed negative correlations with the national mean data of sake cake ratios. Gelatinization temperatures measured by differential scanning calorimetry (DSC) showed positive correlations with the national mean data of sake cake ratios. The obtained results suggest that atmospheric temperature conditions during the grain-filling period of material rice strongly affect the digestibility of steamed rice grains in sake mash because the structure of amylopectin is influenced by atmospheric temperature conditions during grain-filling.

Source

J. Brew. Soc. Japan, 108, 461-466 (2013)