Noda Institute for Scientific Research

Noda, Japan

Noda Institute for Scientific Research

Noda, Japan

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Ito K.,Kikkoman Corporation | Ito K.,Noda Institute for Scientific Research | Matsushima K.,Kikkoman Corporation | Koyama Y.,Noda Institute for Scientific Research
Applied and Environmental Microbiology | Year: 2012

Glutaminase is an enzyme that catalyzes the hydrolysis of L-glutamine to L-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available for Aspergillus oryzae RIB40, we cloned a novel glutaminase gene (AsgahA) from Aspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis (CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahA into A. oryzae RIB40 resulted in the overexpression of glutaminase activity. As- GahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only free L-glutamine and L-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase- asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase. © 2012, American Society for Microbiology.


Ogawa M.,Noda Institute for Scientific Research | Kobayashi T.,Nagoya University | Koyama Y.,Noda Institute for Scientific Research | Koyama Y.,Kikkoman Corporation
Fungal Genetics and Biology | Year: 2012

Fungal endo-β-mannanases (β-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of β-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of β-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced β-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)2Cys6 type DNA binding protein was identified. Moreover, a manR-overexpressing strain showed significantly increased β-mannanase activity. DNA microarray analysis of the manR-disruptant strain further indicated that when konjac glucomannan is used as the carbon source, ManR positively regulates the gene expression of not only β-mannanase, but also the enzymes involved in the degradation of galactomannans and glucomannans such as α-galactosidase, β-mannosidase, acetylmannan esterase, and β-glucosidase. Furthermore, we demonstrated that the presence of 1,4-β-d-mannobiose increased the expression of the endo-β-mannanase gene (manG, AO090010000122), and that ManR plays a key role in the inducible expression of manG in A. oryzae. Therefore, we conclude that ManR is a positive regulator of the β-mannan utilization system in A. oryzae. This is the first study to identify a transcriptional regulator of this system in eukaryotic cells. © 2012 Elsevier Inc.


Ito K.,Noda Institute for Scientific Research | Ito K.,Kikkoman Corporation | Hanya Y.,Kikkoman Corporation | Koyama Y.,Noda Institute for Scientific Research
Applied Microbiology and Biotechnology | Year: 2013

Glutaminase, an enzyme that hydrolyzes l-glutamine to l-glutamate, plays an important role in the production of fermented foods by enhancing the umami taste. In this study, we found ten glutaminase genes in the Aspergillus sojae genome by conducting a BLAST search of the characterized glutaminase sequence. We subsequently constructed glutaminase gene disruptants. The glutaminase activity of the gahB disruptant was decreased by approximately 90 % in A. sojae and Aspergillus oryzae, indicating that this enzyme (GahB) accounted for the majority of the glutaminase activity in Aspergillus species. Subsequently, GahB protein was purified from the AsgahB-overexpressing transformant and characterized. The molecular mass was estimated to be approximately 110 and 259 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahB was a dimer. The optimal pH was 9.0, and the optimal temperature was 50 C. Analysis of substrate specificity revealed that AsGahB had peptidoglutaminase-asparaginase activity, similar to AsGahA, but preferred free l-glutamine to free l-asparagine, C-terminal glutaminyl, and asparaginyl residues in peptides. © 2013 Springer-Verlag Berlin Heidelberg.


Ito K.,Kikkoman Corporation | Koyama Y.,Noda Institute for Scientific Research | Hanya Y.,Kikkoman Corporation
Bioscience, Biotechnology and Biochemistry | Year: 2013

Glutaminase, an enzyme that catalyzes the conversion of L-glutamine to L-glutamate, enhances the umami taste in soy sauce. The Aspergillus sojae genome contains 10 glutaminase genes. In this study, we estimated that approximately 60% of the glutamate in soy sauce is produced through the glutaminase reaction. To determine which glutaminase is involved in soy sauce glutamate production, we prepared soy sauces using single and multiple glutaminase gene disruptants of A. sojae. The glutamate concentration in soy sauce prepared using the δgahA-δgahB-δggtA-δgls disruptant was approximately 60% lower than that in the control strain, whereas it was decreased by approximately 20-30% in the δgahA-δgahB disruptant. However, the glutamate concentration was unchanged in the soy sauces prepared using the δgahA-δggtA-δgls and δgahB-δggtA-δgls disruptants. These results indicate that four glutaminases are involved in glutamate production in soy sauce, and that the peptidoglutaminase activities of GahA and GahB increase the glutamate concentration in soy sauce.


Patent
Noda Institute For Scientific Research and Kikkoman Corporation | Date: 2010-04-21

To improve the activity of a Koji mold protease in a solid or liquid culture medium in the production of foods (e.g., a seasoning), pharmaceuticals (e.g., a digestive agent), protease for use in a detergent and the like. Disclosed are a recombinant vector having capability of increasing the secretion of the Koji mold protease, a Koji mold which is transformed with the vector and has an increased expression of a gene for a protease or an increase secretion of the same, a method for the production of a protease by using the transformed Koji mold, and the like.


Hara S.,Noda Institute for Scientific Research | Jin F.J.,Noda Institute for Scientific Research | Takahashi T.,Noda Institute for Scientific Research | Koyama Y.,Noda Institute for Scientific Research
Molecular Genetics and Genomics | Year: 2012

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1-12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain. © Springer-Verlag 2011.


Ogawa M.,Noda Institute for Scientific Research | Tokuoka M.,Noda Institute for Scientific Research | Jin F.J.,Noda Institute for Scientific Research | Takahashi T.,Noda Institute for Scientific Research | Koyama Y.,Noda Institute for Scientific Research
Fungal Genetics and Biology | Year: 2010

Conidia of koji-mold Aspergillus oryzae are often used as starters in the fermented food industry. However, little is known about conidiation regulation in A. oryzae. To improve the productivity of conidia in A. oryzae, it is necessary to understand conidiation regulation in the strain. Therefore, we analyzed the conidiation regulatory system in A. oryzae using 10 kinds of conidiation regulatory gene disruptants. The phenotypes of AorfluG, AorflbA, AorflbB, AorflbC, AorflbD, AorflbE, AorbrlA, AorabaA, AorwetA, and AorfadA mutants are almost identical to those of the corresponding mutants in Aspergillus nidulans. The results indicated that the functions of conidiation regulatory genes are almost conserved between A. oryzae and A. nidulans. However, the severely reduced conidiation phenotype of the AorfluG disruptant in A. oryzae differs from the phenotype of the corresponding mutant in Aspergillus fumigatus in air-exposed culture conditions. These results suggest that A. oryzae, A. nidulans, and A. fumigatus have a G-protein signaling pathway and brlA orthologs in common, and only A. fumigatus has particular brlA activation pathways that are independent of the fluG ortholog. Furthermore, the analyses of AorflbA disruptant and AorfadA dominant-active mutants implicated that AorFadA-mediated G-protein signaling suppresses vegetative growth of A. oryzae. © 2009 Elsevier Inc. All rights reserved.


Ogawa M.,Noda Institute for Scientific Research | Kobayashi T.,Nagoya University | Koyama Y.,Noda Institute for Scientific Research
Bioscience, Biotechnology and Biochemistry | Year: 2013

ManR, a DNA-binding protein possessing the Zn(II)2Cys 6 binuclear cluster domain, acts as a positive regulator of mannanolytic enzyme genes in Aspergillus oryzae. In this study, we found that ManR controlled the expression not only of mannanolytic enzyme genes but also of cellulolytic enzyme genes in A. oryzae, based on DNA microarray analysis of a manR-disruptant and a manR-overexpressing strain. A new model for the regulation of cellulase and hemicellulase genes mediated by ManR and XlnR in A. oryzae is proposed.


Wada R.,University of Tokyo | Jin F.J.,University of Tokyo | Koyama Y.,Noda Institute for Scientific Research | Maruyama J.-I.,University of Tokyo | Kitamoto K.,University of Tokyo
Applied Microbiology and Biotechnology | Year: 2014

Heterokaryon formation by hyphal fusion occurs during a sexual/parasexual cycle in filamentous fungi, and therefore, it is biotechnologically important for crossbreeding. In the industrial filamentous fungus Aspergillus oryzae, a parasexual cycle has been reported, and it was recently suggested that sexual reproduction should be possible. However, as A. oryzae enters into hyphal fusion with a much lower frequency than Neurospora crassa, the process of heterokaryon formation has not been extensively characterized in A. oryzae. Here, we developed a detection system for heterokaryon formation by expressing red or green fluorescent proteins in nuclei and conferring uridine/uracil or adenine auxotrophy to MAT1-1 and MAT1-2 strains of A. oryzae. The heterokaryon formation of A. oryzae was investigated in paired culture using the genetically modified strains. No sclerotial formation was observed in the hyphal contact regions of the two strains with the same auxotrophy, whereas numerous sclerotia were formed between the strains with different auxotrophies. In most of the formed sclerotia, the uridine/uracil and adenine auxotrophies were complemented, and both red and green fluorescence were detected, indicating that heterokaryotic fusants were formed by hyphal fusion before or during sclerotial formation. Moreover, overexpressing the sclR gene, which encodes a transcription factor promoting sclerotial formation, increased the number of heterokaryotic sclerotia formed between the two auxotrophic strains. Notably, these effects in sclerotial formation of heterokaryotic fusants were observed independently of the mating type pairing combinations. Taken together, these findings demonstrated that paring of different auxotrophs and sclR overexpression promote the formation of heterokaryotic sclerotia in A. oryzae. © 2013 Springer-Verlag Berlin Heidelberg.


Disclosed are: transcription regulatory factors capable of regulating the transcription or expression of genes for mannanases or cellulases, as mentioned below; and others. Specifically disclosed is a protein selected from the following proteins (a), (b) and (c): (a) a protein comprising the amino acid sequence depicted in SEQ ID NO: 2; (b) a protein which comprises an amino acid sequence produced by deleting, substituting or adding one or several amino acid residues (e.g., 1 to 5 amino acid residues) in the amino acid sequence depicted in SEQ ID NO: 2 and which is capable of regulating the transcription of genes for mannanases or cellulases; and (c) a protein which comprises an amino acid sequence having a 70% or higher sequence identity to the amino acid sequence depicted in SEQ ID NO:2 and which is capable of regulating the transcription of genes for mannanases or cellulases, or a partial fragment of the protein. Also specifically disclosed are a gene encoding the protein, and others.

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