Publications (38)119.24 Total impact
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Article: Identification of regulatory elements in the glucoamylase-encoding gene (glaB) promoter from Aspergillus oryzae.
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ABSTRACT: The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from -382 to -353 and from -332 to -313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using β-glucuronidase as a reporter. The results of the deletion analysis were consistent with the EMSA results. The promoter missing the -332 to -313 element was not induced by low water activity stress during SSC.Applied Microbiology and Biotechnology 12/2012; · 3.42 Impact Factor -
Article: High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.
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ABSTRACT: Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.Applied Microbiology and Biotechnology 07/2012; · 3.42 Impact Factor -
Article: Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.
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ABSTRACT: A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.Applied Microbiology and Biotechnology 07/2011; 92(3):561-9. · 3.42 Impact Factor -
Article: The construction and application of diploid sake yeast with a homozygous mutation in the FAS2 gene.
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ABSTRACT: In Japanese sake brewing, cerulenin-resistant sake yeasts produce elevated levels of ethyl caproate, an important flavor component. The FAS2 mutation FAS2-1250S of Saccharomyces cerevisiae generates a cerulenin-resistant phenotype. This mutation is dominant, and, in general, cerulenin-resistant diploid sake yeast strains carry this mutation heterozygously. Here we constructed diploid sake yeast with a homozygous mutation of FAS2 using the high-efficiency loss of heterozygosity method. The homozygous mutants grew more slowly in YPD medium than did the wild-type and heterozygous mutants, and they produced more ethyl caproate during sake brewing. In addition, although both the wild-type and heterozygous mutant were sensitive to 4 mg/l cerulenin, the homozygous mutant was resistant to more than 4 mg/l cerulenin. Next, we obtained a homozygous mutant of FAS2 without inducing genetic modification. After cultivating the heterozygous FAS2 mutant K-1801 in YPD, homozygous mutants were selected on medium containing high concentrations of cerulenin. Non-genetically modified yeast with a homozygous mutation of FAS2 produced 2.2-fold more ethyl caproate than did heterozygous yeast. Moreover, high-quality Japanese sake with a very rich flavor could be brewed using yeast containing a homozygous mutation in the FAS2 gene.Journal of Bioscience and Bioengineering 12/2010; 110(6):675-8. · 1.79 Impact Factor -
Article: Enhancement of beta-glucosidase activity on the cell-surface of sake yeast by disruption of SED1.
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ABSTRACT: We determined the genetic background that would result in a more optimal display of heterologously expressed beta-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Delta sed1 and Delta tos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type.Journal of Bioscience and Bioengineering 05/2010; 109(5):442-6. · 1.79 Impact Factor -
Article: Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module.
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ABSTRACT: A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.Applied Microbiology and Biotechnology 05/2010; 87(5):1783-9. · 3.42 Impact Factor -
Article: Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate.
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ABSTRACT: By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387-395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.Journal of Bioscience and Bioengineering 11/2009; 108(5):359-64. · 1.79 Impact Factor -
Article: Evaluation of cell surface-displayed protein stability against simulated gastric fluid.
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ABSTRACT: A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.Biotechnology Letters 06/2009; 31(8):1259-64. · 1.68 Impact Factor -
Article: Crawler, a novel Tc1/mariner-type transposable element in Aspergillus oryzae transposes under stress conditions.
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ABSTRACT: A novel active transposable element, designated Crawler, has been isolated from an industrial strain (OSI1013) of Aspergillus oryzae as an insertion sequence within the niaD gene encoding nitrate reductase. It is 1290bp in length with imperfect terminal inverted repeats of 28bp and is flanked by 2bp (TA) target site duplications. It contains an open reading frame with no introns that encodes a putative transposase (AotA) of 357 amino acid residues, which is highly homologous to the transposase existing in impala, a member of Tc1/mariner superfamily class II DNA transposon from Fusarium oxysporum. Southern blot analysis revealed that the OSI1013 strain has multiple copies (at least 16) of the element in the genome. Transcription of Crawler occurred under standard growth conditions, and was up-regulated in the presence of CuSO(4) or by heat shock at 42 degrees C. Moreover, transposition events of Crawler induced by various stress treatments were observed by transposon trapping, in which crnA and niaD genes were used as targets for insertion of the element. The excision analysis of Crawler inserted within promoter regions of the crnA gene revealed that CuSO(4) stress and heat shock treatment for conidia were most effective on its excision/transposition, and that acidic environment, oxidative stress, and UV irradiation also slightly induced transposition. To our knowledge, this is the first study reporting the observation of active transpositions of a resident class II transposon under various stress conditions in filamentous fungi.Fungal Genetics and Biology 04/2009; 46(6-7):441-9. · 3.74 Impact Factor -
Article: Enhancement of display efficiency in yeast display system by vector engineering and gene disruption.
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ABSTRACT: Vector engineering and gene disruption in host cells were attempted for the enhancement of alpha-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.Applied Microbiology and Biotechnology 02/2009; 82(4):713-9. · 3.42 Impact Factor -
Article: Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity.
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ABSTRACT: Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.Applied Microbiology and Biotechnology 02/2009; 82(2):387-95. · 3.42 Impact Factor -
Article: Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography.
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ABSTRACT: Aleuria aurantia lectin (AAL) is widely used to estimate the extent of alpha1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of alpha1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K(d)) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for alpha1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K(a)) for alpha1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were alpha1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of alpha1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.Analytical Biochemistry 01/2009; 386(2):217-21. · 3.00 Impact Factor -
Article: Construction of an Aspergillus oryzae cell-surface display system using a putative GPI-anchored protein.
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ABSTRACT: A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of beta-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, beta-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.Applied Microbiology and Biotechnology 10/2008; 81(4):711-9. · 3.42 Impact Factor -
Article: Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases.
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ABSTRACT: Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.Bioscience Biotechnology and Biochemistry 06/2008; 72(5):1376-9. · 1.28 Impact Factor -
Article: Isoflavone aglycones production from isoflavone glycosides by display of beta-glucosidase from Aspergillus oryzae on yeast cell surface.
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ABSTRACT: For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of beta-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with alpha-agglutinin. Three beta-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these beta-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit beta-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying beta-glucosidase.Applied Microbiology and Biotechnology 06/2008; 79(1):51-60. · 3.42 Impact Factor -
Article: Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase.
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ABSTRACT: Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.Journal of Bioscience and Bioengineering 06/2008; 105(6):622-7. · 1.79 Impact Factor -
Article: Deletion analysis of the catalase-encoding gene (catB) promoter from Aspergillus oryzae.
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ABSTRACT: The catalase-encoding gene (catB) is expressed strongly in Aspergillus oryzae. To identify the transcription regulatory elements involved in strong expression, we did promoter deletion analysis using beta-glucuronidase (GUS) as a reporter and an electrophoretic gel mobility shift assay (EMSA) systematically. The deletion 200-bp sequence from -1,000 to -800 in the 1,400-bp catB promoter caused a drastic decrease in GUS activity. In addition, EMSA implicated a 45-bp element from -1,000 to -956 containing cis-elements. According to detailed promoter deletion analysis, a region from -1,000 to -975, which contains putative heat shock element (HSE) and the CCAAT-box, was involved in strong expression.Bioscience Biotechnology and Biochemistry 02/2008; 72(1):48-53. · 1.28 Impact Factor -
Article: Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.
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ABSTRACT: We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.Journal of Bioscience and Bioengineering 12/2007; 104(5):424-7. · 1.79 Impact Factor -
Article: Enhancement of substrate recognition ability by combinatorial mutation of beta-glucosidase displayed on the yeast cell surface.
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ABSTRACT: Recently, in family 3 beta-glucosidase (BGL), the catalytically important Asp nucleophile has been identified in the SDW segment of the SDWG sequence by site-directed mutagenesis. However, the details about the roles of each amino acid residue of the SDWG sequence have not been investigated. W293 of the SDW segment, which is the residue next to the nucleophile (D292) in family 3 BGL, is very important for hydrolytic reaction as a binder to a substrate. G294 of the SDWG sequence might play an important role in catalysis. In this study, to obtain a functional BGL1 mutant by the substitution of G294 using a genetic engineering method, the library of mutant BGL1 from Aspergillus oryzae was rapidly constructed by yeast cell surface engineering, and the hydrolytic activities of mutants were comprehensively detected. Consequently, G294F, G294W, and G294Y, in which G was substituted with aromatic amino acids, showed higher activities for substrate recognition than the parent strain (1.5-, 1.5-, and 1.6-fold, respectively). These results suggest the presence of some interaction between the sugar rings and aromatic ring of W293 at the entrance of the catalytic pocket, which enhances the substrate recognition of beta-glucosidase.Applied Microbiology and Biotechnology 11/2007; 76(5):1027-33. · 3.42 Impact Factor -
Article: The glucoamylase-encoding gene (glaB) is expressed in solid-state culture with a low water content.
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ABSTRACT: Solid-state culture encourages high-level enzyme secretion by Aspergillus oryzae. Using the real-time quantitative reverse transcriptase-polymerase chain reaction, we confirmed that expression of the glucoamylase-encoding gene in A. oryzae cultured in solid-state culture depends on the water content of the culture.Bioscience Biotechnology and Biochemistry 08/2007; 71(7):1797-9. · 1.28 Impact Factor
Top co-authors
Top Journals
Institutions
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2008–2010
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Kobe University
- Department of Chemical Science and Engineering
Kōbe-shi, Hyogo-ken, Japan -
Honda Research Institute Japan Co., Ltd.
Saitama, Saitama-ken, Japan
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2009
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Kyoto University
- Division of Applied Life Sciences
Kyoto, Kyoto-fu, Japan
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2007
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National Research Institute of Brewing
Tokyo, Tokyo-to, Japan -
University of Fukui
- Department of Applied Chemistry and Biotechnology
Fukui-shi, Fukui-ken, Japan
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