Masahiro Nogawa

Shinshu University, Shonai, Nagano, Japan

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Publications (30)53.65 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.
    Bioscience Biotechnology and Biochemistry 01/2012; 76(3):517-22. · 1.27 Impact Factor
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    ABSTRACT: Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.
    Journal of Bioscience and Bioengineering 12/2011; 113(3):293-9. · 1.74 Impact Factor
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    ABSTRACT: The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments.
    Journal of Bioscience and Bioengineering 05/2010; 109(5):472-8. · 1.74 Impact Factor
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    ABSTRACT: A stable bacterial community expressing strong chitinolytic activity was constructed by mixing and cultivating chitinolytic bacteria collected from different natural sources. The DNA fragment pattern, after PCR-denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes using total DNA prepared from whole cells, indicated that the community was composed of four dominant bacterial species. All four species were isolated on agar medium, and one strain, SAY3, was deduced to be a novel species belonging to a new genus based on the 16S rRNA nucleotide sequence. The other strains showed high similarity in their 16S rRNA sequences to those of previously identified bacteria (Acinetobacter and Microbacterium). Strain SAY3 degraded and utilized larger particles of chitin faster than the community, indicating that it plays an important role in the chitin degradation conferred by the community.
    Bioscience Biotechnology and Biochemistry 03/2010; 74(3):636-40. · 1.27 Impact Factor
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    ABSTRACT: A bacterial strain capable of degrading chitin, strain SAY3T, was isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. The strain was gram-negative, curved rod-shaped, facultatively anaerobic, and motile with a single polar flagellum. It grew well with chitin as a sole carbon source. The cellular fatty acids profiles showed the presence of C16:1 omega7c and C16:0 as the major components. The G+C content of DNA was 67.6 mol% and Q-8 was the major respiratory quinone. A 16S rRNA gene sequence-based phylogenetic analysis showed the strain belonged to the family Neisseriaceae but was distantly related (94% identity) to any previously known species. Since the strain was clearly distinct from closely related genera in phenotypic and chemotaxonomic characteristics, it should be classified under a new genus and a new species. We propose the name Chitiniphilus shinanonensis gen. nov., sp. nov. The type strain is SAY3T (=NBRC 104970T=NICMB 14509T).
    The Journal of General and Applied Microbiology 05/2009; 55(2):147-53. · 0.74 Impact Factor
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    ABSTRACT: In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5'-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.
    Applied Microbiology and Biotechnology 11/2006; 72(5):995-1003. · 3.69 Impact Factor
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    ABSTRACT: We have previously reported on purification and characterization of an exo-beta-D-glucosaminidase (Gls93) from culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetyl-D-glucosamine (GlcNAc). The corresponding gene of Gls93 was cloned and characterized in this work. To our knowledge, this is the first report on cloning of the gene encoding fungal exo-beta-D-glucosaminidase. This gene has no introns and encodes a polypeptide of 892 amino acids (aa) containing a secretion signal of 28 amino acids. Comparison of the amino acid sequence to known proteins and phylogenetic analysis indicated that gls93 belongs to the glycoside hydrolase family (GHF) 2 and should be further classified into a new subgroup, exo-beta-D-glucosaminidase subgroup. The gls93 transcription was biphasic when T. reesei was grown on GlcNAc, suggesting that the expression of this gene may be regulated by a complex mechanism, in which multiple regulatory proteins are involved. Furthermore, gls93 could be expressed in Pichia pastoris (ca. 0.49-mg/ml culture). The recombinant Gls93 had the two molecular forms, ca. 105 and 100 kDa, whose difference is caused by N-glycosylation. Both of them had the same properties such as specific activity and substrate specificity and showed only the activity of exo-beta-D-glucosaminidase but not those of beta-galactosidase, beta-glucuronidase, and beta-mannosidase belonging to GHF2.
    Applied Microbiology and Biotechnology 11/2006; 72(4):687-95. · 3.69 Impact Factor
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    ABSTRACT: Wheat (Triticum aestivum L. var. Shiranekomugi) seeds were soaked in water at 22 degrees C for 1 d. Thereafter, the embryo of the soaked seeds was inoculated with Agrobacterium tumefaciens by piercing a region of the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were incubated at 22 degrees C for 2 d and sterilized by cefotaxime (Claforan) (1000 ppm water solution) treatment and then vernalized at 5 degrees C for 25 d. Finally, the seedlings were grown to maturation (T(0) plants) and allowed to pollinate naturally for seed setting (T(1) plants) in pots under nonsterile condition. To examine the transformation by various means, four different strains of A. tumefaciens were used for transformation. The following five lines of evidence proved the transformation: altered phenotype and its transmittance to the next generation, resistance of T(1) seed germination to geneticin or hygromycin B, the detection of a transgene in T(1) plants by PCR analysis and Southern hybridization and the rescue of the plasmid consisting of the integrated T-DNA and flanking wheat genome DNA from T(1) plants. The transformation efficiency of T(1) plants, which were transformed using different A. tumefaciens strains, was estimated to be 33% by PCR analysis, 75% by Southern hybridization and 40% by plasmid rescue.
    Journal of Bioscience and Bioengineering 10/2006; 102(3):162-70. · 1.74 Impact Factor
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    ABSTRACT: We purified a chitinase (named Chi46), with a molecular mass of 46 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from the culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetylglucosamine (GlcNAc). The relative activity of this enzyme reduced when the degrees of acetylation (DA) of chitosan decreased. Furthermore, the enzyme was able to hydrolyze colloidal chitin and ethylene glycol chitin. The gene chi46 was cloned and sequenced. chi46 encodes a protein of 424 amino acid residues containing a 35-amino acid prepro-type secretion signal peptide. The molecular mass of mature Chi46 calculated from deduced amino acid sequence was 42,265 Da. The chi46 transcript was biphasic when the mycelia were grown on GlcNAc, suggesting that the multiple regulatory proteins are involved in the chi46 expression. The chi46 cDNA was expressed in Escherichia coli (ca. 0.23 mg/ml culture). To determine substrate cleavage fashion of Chi46 in more detail, we carried out high-performance liquid chromatography analysis and viscosimetric assay using recombinant Chi46 (rChi46). Chi46 was shown to release mainly (GlcNAc)(2) from colloidal chitin (insoluble chitin) as an exo-type manner and to act on chitosan 7B (DA ca. 30%) and N-acetylchitooligosaccharides (soluble chitins) in an endo-type one.
    Applied Microbiology and Biotechnology 08/2006; 71(3):294-303. · 3.69 Impact Factor
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    ABSTRACT: Abstract A mycovirus (named FusoV) from the plant pathogenic fungus Fusarium solani possessed two types of double-stranded (ds) RNA genome, designated Ml and M2. RNA-dependent RNA polymerase activity was detected in FusoV particle fractions. An in vitro RNA polymerase reaction using purified FusoV particles that was supplemented with NTPs revealed the synthesis of single-stranded (ss) RNA species and a subsequent formation of dsRNAs having the same size as Ml and M2. The ssRNA species synthesized in the first stage were proved to be of positive polarity (coding strand) for both M1 and M2 by dot blot hybridization analysis. These results suggest that FusoV genomic dsRNA replicates in a conservative manner.
    FEMS Microbiology Letters 01/2006; 137(1):45 - 49. · 2.05 Impact Factor
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    ABSTRACT: Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2 d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T0 plants) and allowed to pollinate naturally to set seeds (T1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation: an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation: the altered phenotype and its inheritance by the next generation, histochemical detection of beta-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of beta-glucuronidase, respectively.
    Journal of Bioscience and Bioengineering 11/2005; 100(4):391-7. · 1.74 Impact Factor
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    ABSTRACT: Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.
    Journal of Bioscience and Bioengineering 02/2004; 98(2):136-9. · 1.74 Impact Factor
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    ABSTRACT: A 24 kDa vacuolar protein (VP24) was found to accumulate abundantly in anthocyanic vacuolar inclusions (AVIs) in anthocyanin-producing sweet potato cells grown in suspension culture. To clarify the function of VP24, we have isolated and characterized a cDNA clone encoding VP24 (Accession No. AB025531). Sequence analysis revealed that the 2.9 kbp VP24 cDNA encodes a protein of 893 amino acid residues with a predicted molecular mass of 96.3 kDa. VP24 is synthesized as a large precursor protein which possesses an N-terminal extension composed of a signal peptide and a propeptide, plus the polypeptide of the mature VP24 and its C-terminal propeptide, that contains the multiple transmembrane domains [Plant Physiol. 125 (2001) 447]. Based on sequence identities, the mature VP24 domain most likely belongs to the zinc metalloprotease family. In order to confirm whether VP24 actually has peptidase activity we overexpressed the mature VP24 domain in Escherichia coli. The expressed fusion protein clearly showed aminopeptidase activity in vitro. Native VP24 purified from the anthocyanin-accumulating cells also showed aminopeptidase activity. These results suggest that VP24 expressed in the anthrocyanin-producing sweet potato cells could be a novel vacuolar localized aminopeptidase.
    Biochemical Engineering Journal. 01/2003; 14(3):199-205.
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    ABSTRACT: A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.
    Bioscience Biotechnology and Biochemistry 05/2001; 65(4):977-81. · 1.27 Impact Factor
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    ABSTRACT: The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region.
    Journal of Bioscience and Bioengineering 02/2001; 91(4):359-62. · 1.74 Impact Factor
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    ABSTRACT: Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.
    Journal of Bioscience and Bioengineering 02/2001; · 1.74 Impact Factor
  • Bioscience Biotechnology and Biochemistry - BIOSCI BIOTECHNOL BIOCHEM. 01/2001; 65(4):977-981.
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    ABSTRACT: l-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, l-sorbose was found to regulate coordinately six cellulase genes (including egl3, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when l-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45–60% and 20–30% of the cbh1 transcript, whereas small amounts of mRNA, 1–2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and l-sorbose, respectively.
    Current Genetics 12/2000; 38(6):329-334. · 2.41 Impact Factor
  • J Xu, M Nogawa, H Okada, Y Morikawa
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    ABSTRACT: The characteristics of regulation of the gene encoding the third xylanase (Xyn III) of a filamentous fungus, Trichoderma reesei PC-3-7, were studied by Northern blot analysis. A partial DNA sequence (185 bp) of the xyn3 gene was obtained by PCR amplification of genomic DNA of T. reesei PC-3-7 and sequenced. This xyn3 gene fragment was used as a probe for Southern and Northern blot analysis. The expression of the xyn3 gene was regulated at the transcriptional level. The xyn3 mRNA was expressed in mycelia of T. reesei PC-3-7 induced by Avicel, L-sorbose and sophorose, but not by xylose, xylooligosaccharides and birchwood xylan. Furthermore, it was observed that xyn3 was synchronously expressed with egll but not with xyn1 and xyn2 by L-sorbose, indicating that the xyn3 gene may be coordinately expressed with cellulase genes. By Southern blot analysis, the xyn3 gene was confirmed to exist as a single copy in both strains of T. reesei PC-3-7 and QM9414. However, no xyn3 mRNA appeared in the mycelia induced by any kind of inducers in T. reesei QM9414 even when total RNA was used in large excess, suggesting that the xyn3 gene in T. reesei QM9414 is in the dormant state and cannot be expressed. Therefore, T. reesei PC-3-7 may be a very useful strain for elucidating the induction mechanism of xylanase biosynthesis by cellulosic and xylanosic substrates, and also the regulatory correlation between cellulase and xylanase induction.
    Applied Microbiology and Biotechnology 10/2000; 54(3):370-5. · 3.69 Impact Factor
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    ABSTRACT: cDNA of Cel12A (formerly EG III), one of five endoglucanases of Trichoderma reesei, was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pAGmegl3 containing mature form Cel12A cDNA produced Cel12A protein largely as insoluble inclusion bodies in the cytoplasm of the cells. The insoluble fraction was solubilized with urea from which Cel12A was purified by cation chromatography to electrophoretic homogeneity. The purified enzyme was immunologically and enzymologically identical to that of Cel12A purified from T. reesei. E116 and E200 of Cel12A of T. reesei are completely conserved in family-12 cellulases. In order to investigate the role of these two glutamate residues in the enzymic function of Cel12A, two mutant enzymes were produced at each position, namely E116D/Q and E200D/Q. The specific activity of these mutant enzymes is reduced by more than 98%, revealing the importance of these two residues to the catalytic function of Cel12A. The data demonstrated that E116 and E200 are the nucleophilic and acid-base residues, respectively.
    Journal of Molecular Catalysis B-enzymatic - J MOL CATAL B-ENZYM. 01/2000; 10(1):249-255.

Publication Stats

393 Citations
53.65 Total Impact Points

Institutions

  • 1993–2012
    • Shinshu University
      • Division of Applied Biology
      Shonai, Nagano, Japan
  • 1998–2006
    • Nagaoka University of Technology
      • Department of Bioengineering
      Nagaoka, Niigata-ken, Japan