Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis

Johannes Aarnikunnas, A Pihlajaniemi, Airi Palva, Matti Leisola, Antti Nyyssölä

    Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

    Sammanfattning

    "The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD(+) as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (> 80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found."
    Originalspråkengelska
    TidskriftApplied and Environmental Microbiology
    Volym72
    Utgåva1
    Sidor (från-till)368-377
    Antal sidor10
    ISSN0099-2240
    StatusPublicerad - 2006
    MoE-publikationstypA1 Tidskriftsartikel-refererad

    Vetenskapsgrenar

    • 413 Veterinärvetenskap
    • bakteerit
    • Pantoea ananatis
    • geenit

    Citera det här

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    title = "Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis",
    abstract = "{"}The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51{\%}) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD(+) as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (> 80{\%}) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found.{"}",
    keywords = "413 Veterinary science, bakteerit, Pantoea ananatis, geenit, bakteerit, Pantoea ananatis, geenit, bakteerit, Pantoea ananatis, geenit",
    author = "Johannes Aarnikunnas and A Pihlajaniemi and Airi Palva and Matti Leisola and Antti Nyyss{\"o}l{\"a}",
    year = "2006",
    language = "English",
    volume = "72",
    pages = "368--377",
    journal = "Applied and Environmental Microbiology",
    issn = "0099-2240",
    publisher = "American Society for Microbiology",
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    }

    Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis. / Aarnikunnas, Johannes; Pihlajaniemi, A; Palva, Airi; Leisola, Matti; Nyyssölä, Antti.

    I: Applied and Environmental Microbiology, Vol. 72, Nr. 1, 2006, s. 368-377.

    Forskningsoutput: TidskriftsbidragArtikelVetenskapligPeer review

    TY - JOUR

    T1 - Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis

    AU - Aarnikunnas, Johannes

    AU - Pihlajaniemi, A

    AU - Palva, Airi

    AU - Leisola, Matti

    AU - Nyyssölä, Antti

    PY - 2006

    Y1 - 2006

    N2 - "The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD(+) as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (> 80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found."

    AB - "The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD(+) as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (> 80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found."

    KW - 413 Veterinary science

    KW - bakteerit

    KW - Pantoea ananatis

    KW - geenit

    KW - bakteerit

    KW - Pantoea ananatis

    KW - geenit

    KW - bakteerit

    KW - Pantoea ananatis

    KW - geenit

    M3 - Article

    VL - 72

    SP - 368

    EP - 377

    JO - Applied and Environmental Microbiology

    JF - Applied and Environmental Microbiology

    SN - 0099-2240

    IS - 1

    ER -