Y Hayashi

Kao Corporation, Edo, Tōkyō, Japan

Are you Y Hayashi?

Claim your profile

Publications (8)25.03 Total impact

  • Source
    [show abstract] [hide abstract]
    ABSTRACT: Alkaline alpha-amylase (AmyK38) from the alkaliphilic Bacillus sp. strain KSM-K38 is a unique enzyme in that it is highly chelator-resistant and oxidatively stable [Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa-Kitayama, K., Ozaki, K., Kawai, S. & Ito, S. (2001) Appl. Environ. Microbiol. 67, 1744-1750]. This enzyme was found to contain no Ca and require Na (or monovalent cations) for manifestation of activity. The nucleotide sequence of the gene for the novel enzyme was determined, and it harbored an ORF of 1503 bp encoding the enzyme of 501 amino acids, including a 21-amino-acid signal peptide. The deduced amino-acid sequence of the mature enzyme (55 097 Da) showed moderate homology to those of alpha-amylases from Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, with approximately 63% identity. A methionine residue, which is conserved and susceptible to chemical oxidation, was replaced with leucine in AmyK38. Moreover, many conserved residues that are crucial ligands for Ca were replaced with other amino acids, thereby leading to loss of the Ca coordination geometries. By building a molecular model, we showed the calcium-independent, oxidatively stable active-site topology and structural integrity of AmyK38.
    European Journal of Biochemistry 08/2001; 268(14):3974-82. · 3.58 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: A novel alpha-amylase (AmyK38) was found in cultures of an alkaliphilic Bacillus isolate designated KSM-K38. Based on the morphological and physiological characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing and DNA-DNA reassociation analysis, it was suggested that the isolate was a new species of the genus Bacillus. The enzyme had an optimal pH of 8.0 to 9.5 and displayed maximum catalytic activity at 55 to 60 degrees C. The apparent molecular mass was approximately 55 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was around pH 4.2. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltohexaose, maltoheptaose, and, in addition, maltose as major end products after completion of the reaction. The activity was not prevented at all by EDTA and EGTA at concentrations as high as 100 mM. Moreover, AmyK38 was highly resistant to chemical oxidation and maintained more than 80% of its original activity even after incubation for 1 h in the presence of excess H2O2 (1.8 M).
    Applied and Environmental Microbiology 05/2001; 67(4):1744-50. · 3.68 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: alpha-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving amino acid residues from 122 to 134. This is the first report that thermostability of an alpha-amylase is improved by proline substitution.
    Bioscience Biotechnology and Biochemistry 10/1999; 63(9):1535-40. · 1.27 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).
    Bioscience Biotechnology and Biochemistry 10/1998; 62(9):1720-5. · 1.27 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: Part of a 2.4-kb DNA fragment that encoded the amino-terminal 584 residues (65 kDa) of an alkaline endoglucanase from Bacillus sp. KSM-635 (941 amino acid residues; 105 kDa) was spontaneously deleted during subcloning of the fragment. The remaining 1.1-kb insert of the deleted plasmid encoded amino acids from Ala228 to Leu584 of the enzyme. However, Escherichia coli HB101 cells harboring this plasmid produced an active endoglucanase. After addition of a termination codon, TAA, immediately downstream of the codon for Leu584, the 1.1-kb fragment was inserted into an expression vector, pHSP64. The resultant plasmid was introduced into Bacillus subtilis ISW1214 for extracellular production of the truncated endoglucanase. The enzyme was then purified to homogeneity from a culture of the recombinant B. subtilis cells. Amino-terminal sequencing of the enzyme showed that the enzyme consisted of 7 amino acid residues encoded by the vector and 357 amino acid residues encoded by the truncated gene, with a molecular mass of 40.2 kDa. The purified enzyme was very active against carboxymethylcellulose and its pH and temperature profiles were almost identical to those of the enzyme produced by Bacillus sp. KSM-635.
    Bioscience Biotechnology and Biochemistry 10/1995; 59(9):1613-8. · 1.27 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.
    Journal of Biological Chemistry 12/1994; 269(46):28752-6. · 4.65 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: The roles of one Glu and four Asp residues of endoglucanase K from Bacillus sp. KSM-330, which are conserved in all the endo-beta-glucanases in the family D, were analyzed by site-directed mutagenesis. The gene for endoglucanase K was mutated to replace Asp-154, Asp-191, Asp-193 or Asp-300 by Asn, or to replace Glu-130 by Gln in the encoded enzyme. Mutant and wild-type genes were separately expressed in Bacillus subtilis and the resultant enzymes were purified from the culture broth. All mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis as the wild-type enzyme and gave similar circular dichroism spectra to that of the wild-type enzyme. Substitution of Glu-130, Asp-191, Asp-193 or Asp-300 significantly decreased the specific activity of the enzyme toward CM-cellulose. Kinetic analysis of the abilities of these mutant enzymes to liberate p-nitrophenol from p-nitrophenylcellotrioside revealed that all the mutant enzymes had very much lower kcat values than that of the wild-type enzyme, while the Km values of these mutant enzymes were almost the same as that of the wild-type enzyme. Of these Glu and Asp residues, Glu-130 and Asp-191 seem to be most likely to be catalytic residues because substitutions of these residues resulted in the lowest kcat values of the mutant enzymes.
    Biochimica et Biophysica Acta 01/1994; 1207:159-164. · 4.66 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.
    Journal of Biological Chemistry 01/1994; 269:28752-28756. · 4.65 Impact Factor