Purification, characterization and gene identification of a α-Neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601

Teruhiko Watanabe, Kana Kashimura, Kotaro Kirimura

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    4 Citations (Scopus)

    Abstract

    Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42. kDa by SDS-PAGE and 84. kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-l-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and d-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25°. C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092. bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41. kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active.

    Original languageEnglish
    JournalJournal of Molecular Catalysis B: Enzymatic
    DOIs
    Publication statusAccepted/In press - 2016 Oct 15

    Fingerprint

    Cellvibrio
    Hydrolases
    Purification
    Bacteria
    Genes
    Galactose
    Nucleotides
    Cell Extracts

    Keywords

    • Agarase
    • Cellvibrio sp. WU-0601
    • Glycoside hydrolase family 117
    • Neoagarobiose
    • α-Neoagarooligosaccharide hydrolase

    ASJC Scopus subject areas

    • Catalysis
    • Bioengineering
    • Biochemistry
    • Process Chemistry and Technology

    Cite this

    @article{a7028b85f2164c6dbca693092506bc60,
    title = "Purification, characterization and gene identification of a α-Neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601",
    abstract = "Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42. kDa by SDS-PAGE and 84. kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-l-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and d-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25°. C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092. bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41. kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active.",
    keywords = "Agarase, Cellvibrio sp. WU-0601, Glycoside hydrolase family 117, Neoagarobiose, α-Neoagarooligosaccharide hydrolase",
    author = "Teruhiko Watanabe and Kana Kashimura and Kotaro Kirimura",
    year = "2016",
    month = "10",
    day = "15",
    doi = "10.1016/j.molcatb.2017.02.003",
    language = "English",
    journal = "Journal of Molecular Catalysis - B Enzymatic",
    issn = "1381-1177",
    publisher = "Elsevier",

    }

    TY - JOUR

    T1 - Purification, characterization and gene identification of a α-Neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601

    AU - Watanabe, Teruhiko

    AU - Kashimura, Kana

    AU - Kirimura, Kotaro

    PY - 2016/10/15

    Y1 - 2016/10/15

    N2 - Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42. kDa by SDS-PAGE and 84. kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-l-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and d-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25°. C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092. bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41. kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active.

    AB - Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42. kDa by SDS-PAGE and 84. kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-l-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and d-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25°. C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092. bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41. kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active.

    KW - Agarase

    KW - Cellvibrio sp. WU-0601

    KW - Glycoside hydrolase family 117

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    KW - α-Neoagarooligosaccharide hydrolase

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    U2 - 10.1016/j.molcatb.2017.02.003

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