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Takao Ojima - One of the best experts on this subject based on the ideXlab platform.

  • characterization of a ghf45 cellulase akeg21 from the common sea hare aplysia kurodai
    Frontiers in Chemistry, 2014
    Co-Authors: Akira Inoue, Mohammad Matiur Rahman, Takao Ojima
    Abstract:

    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four Cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three Cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic -1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 Cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 Cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 Cellulases.

  • isolation and primary structure of a cellulase from the japanese sea urchin strongylocentrotus nudus
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Abstract Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS–PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 °C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50–57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

  • Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus.
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

Matthias Hess - One of the best experts on this subject based on the ideXlab platform.

  • a 2 08 a resolution structure of hlb5 a novel cellulase from the anaerobic gut bacterium parabacteroides johnsonii dsm 18315
    Protein Science, 2019
    Co-Authors: Changsoo Chang, Charles G Brooke, Hailan Piao, Jamey Mack, Gyorgy Babnigg, Andrzej Joachimiak, Matthias Hess
    Abstract:

    Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut, anaerobic bacteria are essential to the well-being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here, we report the 2.08 A resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for glycosidases. We anticipate that the information presented here will facilitate the identification of additional Cellulases for which no homologues have been identified to date and enhance our understanding how these novel Cellulases bind and hydrolyze their substrates.

  • a 2 08 a resolution structure of hlb5 a novel cellulase from the anaerobic gut bacterium parabacteroides johnsonii dsm 18315
    bioRxiv, 2019
    Co-Authors: Changsoo Chang, Charles G Brooke, Hailan Piao, Jamey Mack, Gyorgy Babnigg, Andrzej Joachimiak, Matthias Hess
    Abstract:

    Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut anaerobic bacteria are essential to the well-being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here we report the 2.08 Angstrom resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for enzymes that degrade carbohydrates. We anticipate that the information presented here will facilitate the identification of additional Cellulases for which no homologues have been identified until to date and in enhancing our understanding how these novel Cellulases bind and hydrolyze their substrates.

Yukiko Nishida - One of the best experts on this subject based on the ideXlab platform.

  • isolation and primary structure of a cellulase from the japanese sea urchin strongylocentrotus nudus
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Abstract Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS–PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 °C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50–57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

  • Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus.
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

Akira Inoue - One of the best experts on this subject based on the ideXlab platform.

  • characterization of a ghf45 cellulase akeg21 from the common sea hare aplysia kurodai
    Frontiers in Chemistry, 2014
    Co-Authors: Akira Inoue, Mohammad Matiur Rahman, Takao Ojima
    Abstract:

    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four Cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three Cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic -1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 Cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 Cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 Cellulases.

  • isolation and primary structure of a cellulase from the japanese sea urchin strongylocentrotus nudus
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Abstract Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS–PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 °C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50–57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

  • Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus.
    Biochimie, 2007
    Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima
    Abstract:

    Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal Cellulases.

Changsoo Chang - One of the best experts on this subject based on the ideXlab platform.

  • a 2 08 a resolution structure of hlb5 a novel cellulase from the anaerobic gut bacterium parabacteroides johnsonii dsm 18315
    Protein Science, 2019
    Co-Authors: Changsoo Chang, Charles G Brooke, Hailan Piao, Jamey Mack, Gyorgy Babnigg, Andrzej Joachimiak, Matthias Hess
    Abstract:

    Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut, anaerobic bacteria are essential to the well-being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here, we report the 2.08 A resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for glycosidases. We anticipate that the information presented here will facilitate the identification of additional Cellulases for which no homologues have been identified to date and enhance our understanding how these novel Cellulases bind and hydrolyze their substrates.

  • a 2 08 a resolution structure of hlb5 a novel cellulase from the anaerobic gut bacterium parabacteroides johnsonii dsm 18315
    bioRxiv, 2019
    Co-Authors: Changsoo Chang, Charles G Brooke, Hailan Piao, Jamey Mack, Gyorgy Babnigg, Andrzej Joachimiak, Matthias Hess
    Abstract:

    Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut anaerobic bacteria are essential to the well-being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here we report the 2.08 Angstrom resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for enzymes that degrade carbohydrates. We anticipate that the information presented here will facilitate the identification of additional Cellulases for which no homologues have been identified until to date and in enhancing our understanding how these novel Cellulases bind and hydrolyze their substrates.