Shiono Y

References (9)

Title : Identification and characterization of an acetyl xylan esterase from Aspergillus oryzae - Kato_2021_J.Biosci.Bioeng__
Author(s) : Kato T , Shiono Y , Koseki T
Ref : J Biosci Bioeng , : , 2021
Abstract : In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with alpha-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZalphaC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 degreesC, respectively, and was stable up to 50 degreesC. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of alpha-naphthyl esters (C2-C16), was alpha-naphthyl propionate (C3), with an activity of 0.35 +/- 0.006 units/mg protein. No significant difference of the K(m) value was observed between C3 (2.3 +/- 0.7 mM) and C2 (1.9 +/- 0.4 mM). In contrast, k(cat) value for C3 (18 +/- 3.9 s(-1)) was higher compared to C2 (4.5 +/- 0.7 s(-1)). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.
ESTHER : Kato_2021_J.Biosci.Bioeng__
PubMedSearch : Kato_2021_J.Biosci.Bioeng__
PubMedID: 34376338
Gene_locus related to this paper: aspfu-faec , aspor-faec

Title : Biochemical Characterization of a Lipolytic Enzyme From Aspergillus oryzae That Hydrolyzes Triacylglycerol and Sterol Esters - Ichikawa_2020_Appl.Biochem.Biotechnol_192_910
Author(s) : Ichikawa K , Yoshida A , Shiono Y , Koseki T
Ref : Appl Biochem Biotechnol , 192 :910 , 2020
Abstract : A novel lipolytic enzyme-encoding gene, lipO745, from Aspergillus oryzae RIB40 was cloned and expressed in Pichia pastoris. Purified recombinant LipO745 (rLipO745) had a molecular mass of approximately 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. rLipO745 exhibited maximum activity at 40 degreesC and pH 7.0 and was stable at temperatures >= 40 degreesC. The substrate specificity of purified rLipO745 was analyzed using alpha-naphthyl esters as artificial substrates and various triacylglycerol and sterol esters as natural substrates. From among a panel of alpha-naphthyl esters (C2-C16), alpha-naphthyl butyrate (C4), with an activity of 269 +/- 3.3 units/mg protein, was the optimal substrate for hydrolysis by the purified recombinant protein. The K(m) and k(cat) values of rLiO745 for the C4 substrate were 0.073 +/- 0.0012 mM and 608 +/- 108 s(-1), respectively. The purified recombinant enzyme had considerable hydrolytic activity toward tributyrin, tripalmitin, and triolein, indicating lipase activity, and toward cholesteryl acetate, butyrate, palmitate, and oleate, indicating sterol esterase activity. Transesterification activities between tributyrin and cholesterol or between tributyrin and campesterol were also determined.
ESTHER : Ichikawa_2020_Appl.Biochem.Biotechnol_192_910
PubMedSearch : Ichikawa_2020_Appl.Biochem.Biotechnol_192_910
PubMedID: 32617843
Gene_locus related to this paper: aspor-q2tw11

Title : Characterization of a novel Aspergillus oryzae tannase expressed in Pichia pastoris - Koseki_2018_J.Biosci.Bioeng_126_553
Author(s) : Koseki T , Ichikawa K , Sasaki K , Shiono Y
Ref : J Biosci Bioeng , 126 :553 , 2018
Abstract : We report the characterization of tannase-encoding gene, AotanB, from Aspergillus oryzae and its recombinant enzyme expressed in Pichia pastoris. The gene except for the signal sequence was cloned into a vector pPICZalphaA and the recombinant protein was secreted into the medium as an active enzyme. Recombinant AoTanB highly expressed in the incubation at 18 degrees C compared to 30 degrees C. Purified recombinant protein exhibited smeared band with molecular mass of approximately 90-120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant protein yielded molecular mass of 65 kDa after N-deglycosylation. Purified recombinant enzyme had a pH and a temperature optima of 6.0 and 30-35 degrees C, respectively, and was stable up to 40 degrees C. Recombinant AoTanB was able to release gallic acid from natural substrates, such as (-)-catechin gallate, (-)-epicatechin gallate, (-)-gallochatechin gallate, and (-)-epigallocatechin gallate. The enzyme also hydrolyzed ethyl protocatechuate. Meanwhile, no activity was detected toward ethyl 4-hydroxybenzoate. The activity of recombinant AoTanB was lower toward natural substrates compared to that of AoTanA from A. oryzae. The lower catalytic efficiency (kcat/Km value) toward ethyl protocatechuate was due to a combination of increased Km and considerably decreased kcat. Kinetic analysis of the recombinant AoTanB showed that kcat values toward natural substrates decreased compared to those of recombinant AoTanA. Therefore, recombinant AoTanB showed a decrease in catalytic efficiency (kcat/Km value) compared to recombinant AoTanA was due to considerably lower kcat value.
ESTHER : Koseki_2018_J.Biosci.Bioeng_126_553
PubMedSearch : Koseki_2018_J.Biosci.Bioeng_126_553
PubMedID: 29859669
Gene_locus related to this paper: aspor-q2uii1

Title : Mutational analysis of Kex2 recognition sites and a disulfide bond in tannase from Aspergillus oryzae - Koseki_2017_Biochem.Biophys.Res.Commun_482_1165
Author(s) : Koseki T , Otsuka M , Mizuno T , Shiono Y
Ref : Biochemical & Biophysical Research Communications , 482 :1165 , 2017
Abstract : Aspergillus oryzae tannase (AoTanA), which contains two Kex2 recognition sites at positions Arg311 and Arg316, consists of two subunits that are generated by the cleavage of tannase gene product by the Kex2 protease. Based on the crystal structure of feruloyl esterase from Aspergillus oryzae (AoFaeB), which has been classified as a member of the fungal tannase family, the catalytic triad residues of AoTanA are predicted to be Ser195, Asp455, and His501, with the serine and histidine residues brought together by a disulfide bond of the neighboring cysteines, Cys194 and Cys502. In this study, we investigated the functional role of the Kex2 recognition sites and disulfide bond between the neighboring cysteines in AoTanA. We constructed a double variant (R311A/R316A), a seven amino-acid deletion variant of region Lys310-Arg316 (DeltaKR), and two single variants (C194A and C502A). While the R311A/R316A variant exhibited the two bands similar to wild type by SDS-PAGE after treatment with endoglycosidase H, the DeltaKR variant exhibited only one band. R311A/R316A variation had no effect on tannase activity and stability. Meanwhile, the DeltaKR variant exhibited higher activity compared to the wild-type. The activities of the C194A and C502A variants decreased considerably (<0.24% of the wild-type) toward methyl gallate.
ESTHER : Koseki_2017_Biochem.Biophys.Res.Commun_482_1165
PubMedSearch : Koseki_2017_Biochem.Biophys.Res.Commun_482_1165
PubMedID: 27919681
Gene_locus related to this paper: aspor-tan

Title : An unusual feruloyl esterase from Aspergillus oryzae: two tryptophan residues play a crucial role for the activity - Koseki_2016_J.Mol.Catal.B.Enzym_133_S560
Author(s) : Koseki T , Handa H , Watanabe YY , Ohtsuka M , Shiono Y
Ref : J Mol Catal B Enzym , 133 :S560\ , 2016
Abstract : The genome of Aspergillus oryzae encodes a hypothetical protein (annotated as AO090701000884), designated AoFaeD, which exhibits sequence identity to feruloyl esterases from Aspergillus clavatus, Coprinopsis cinerea, Neurospora crassa, and Pseudomonas fluorescens subsp. cellulosa. In this study, we cloned the AofaeD coding sequence into Pichia pastoris and demonstrated heterologous expression and secretion of active recombinant enzyme (rAoFaeD) as a 30-kDa protein in the culture medium. Purified rAoFaeD exhibited optimal activity at pH 7.0 and at 45 C, but was stable at a pH range of 7.0-10.0 and a temperature of 40 C. While wild-type rAoFaeD failed to cleave the methyl esters of ferulic acid (MFA), p-coumaric acid (MpCA), caffeic acid (MCA), and sinapic acid (MSA) and ethyl ester of ferulic acid (EFA), enzyme exhibited esterase activity when wheat arabinoxylan was used as a substrate, and this reaction was enhanced synergistically by the addition of xylanase. Notably, rAoFaeD variants in which one of the tryptophan residues (Trp142 or Trp144) located adjacent to the putative catalytic serine residue at position 143 (Ser143) was replaced with phenylalanine and tyrosine (W142F and W144Y), respectively, exhibited hydrolytic activity toward MFA, MpCA, MCA, MSA, and EFA. Moreover, these variants exhibited enhanced production of ferulic acid from wheat arabinoxylan compared to the wild-type protein, and the activity of these proteins was enhanced by the addition of xylanase.
ESTHER : Koseki_2016_J.Mol.Catal.B.Enzym_133_S560
PubMedSearch : Koseki_2016_J.Mol.Catal.B.Enzym_133_S560
PubMedID:
Gene_locus related to this paper: aspor-q2u7d3

Title : Biochemical characterization of Aspergillus oryzae native tannase and the recombinant enzyme expressed in Pichia pastoris - Mizuno_2014_J.Biosci.Bioeng_118_392
Author(s) : Mizuno T , Shiono Y , Koseki T
Ref : J Biosci Bioeng , 118 :392 , 2014
Abstract : In this study, the biochemical properties of the recombinant tannase from Aspegillus oryzae were compared with those of the native enzyme. Extracellular native tannase was purified from a commercial enzyme source. Recombinant tannase highly expressed in Pichia pastoris was prepared as an active extracellular protein. Purified native and recombinant tannases produced smeared bands with apparent molecular masses of 45-80 kDa and 45-75 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, the native enzyme yielded molecular masses of 33 kDa and 30 kDa, whereas the recombinant enzyme yielded molecular masses of 34 kDa and 30 kDa. Purified native and recombinant tannases had an optimum pH of 4.0-5.0 and 5.0, respectively, and were stable up to 40 degrees C. After N-deglycosylation, both enzymes exhibited reduced thermostability. Catalytic efficiencies of both purified enzymes were greater with natural substrates, such as (-)-catechin, (-)-epicatechin, and (-)-epigallocatechin gallates, than those with synthetic substrates, such as methyl, ethyl, and propyl gallates. However, there were no activities against the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids, which indicate feruloyl esterase activity, or the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid, which indicate paraben hydrolase activity.
ESTHER : Mizuno_2014_J.Biosci.Bioeng_118_392
PubMedSearch : Mizuno_2014_J.Biosci.Bioeng_118_392
PubMedID: 24856589
Gene_locus related to this paper: aspor-tan

Title : Characterization of a novel lipolytic enzyme from Aspergillus oryzae - Koseki_2013_Appl.Microbiol.Biotechnol_97_5351
Author(s) : Koseki T , Asai S , Saito N , Mori M , Sakaguchi Y , Ikeda K , Shiono Y
Ref : Applied Microbiology & Biotechnology , 97 :5351 , 2013
Abstract : In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZalphaC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0-8.0 and 30 degrees C, respectively, and was stable at the pH range of 7.0-10.0 and up to 40 degrees C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of alpha-naphthyl esters (C2-C16), was alpha-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity.
ESTHER : Koseki_2013_Appl.Microbiol.Biotechnol_97_5351
PubMedSearch : Koseki_2013_Appl.Microbiol.Biotechnol_97_5351
PubMedID: 23001008
Gene_locus related to this paper: aspor-q2uf27

Title : A novel Aspergillus oryzae esterase that hydrolyzes 4-hydroxybenzoic acid esters - Koseki_2010_FEBS.Lett_584_4032
Author(s) : Koseki T , Mihara K , Murayama T , Shiono Y
Ref : FEBS Letters , 584 :4032 , 2010
Abstract : In this study we report the biochemical characterization of a hypothetical protein from Aspergillus oryzae exhibiting sequence identity with feruloyl esterase and tannase from the genus Aspergillus. The purified recombinant protein showed a hydrolytic activity toward the ethyl, propyl, or butyl esters of 4-hydroxybenzoic acid, but did not show feruloyl esterase or tannase activity. Finally, the enzyme decreased the antimicrobial activity of parabens against A. oryzae via hydrolysis of the ester bond present in butyl 4-hydroxybenzoic acid.
ESTHER : Koseki_2010_FEBS.Lett_584_4032
PubMedSearch : Koseki_2010_FEBS.Lett_584_4032
PubMedID: 20728445
Gene_locus related to this paper: aspor-q2uh24

Title : Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae - Koseki_2009_Appl.Microbiol.Biotechnol_83_689
Author(s) : Koseki T , Hori A , Seki S , Murayama T , Shiono Y
Ref : Applied Microbiology & Biotechnology , 83 :689 , 2009
Abstract : Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0-10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.
ESTHER : Koseki_2009_Appl.Microbiol.Biotechnol_83_689
PubMedSearch : Koseki_2009_Appl.Microbiol.Biotechnol_83_689
PubMedID: 19242690
Gene_locus related to this paper: aspor-q2umx6 , aspor-q2up89