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Gene cloning and expression in Escherichia coli of a bi-functional β-d-xylosidase/α-l-arabinosidase from Sulfolobus solfataricus involved in xylan degradation

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Abstract

An open reading frame encoding a putative bi-functional β-d-xylosidase/α-l-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual β-d-xylosidases and α-l-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75°C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like d-glucose and l-arabinose.

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Acknowledgments

This work was supported by grants from MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca)-Decreto Direttoriale prot. no. 1105/2002 and by Centro Regionale di Competenza BioTekNet.

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Authors and Affiliations

  1. Istituto di Biochimica delle Proteine, CNR, Via Pietro Castellino 111, 80131, Naples, Italy

    Alessandra Morana, Ornella Paris, Luisa Maurelli, Mosé Rossi & Raffaele Cannio

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  1. Alessandra Morana
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  2. Ornella Paris
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  3. Luisa Maurelli
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  4. Mosé Rossi
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  5. Raffaele Cannio
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Correspondence to Raffaele Cannio.

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Communicated by G. Antranikian

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Morana, A., Paris, O., Maurelli, L. et al. Gene cloning and expression in Escherichia coli of a bi-functional β-d-xylosidase/α-l-arabinosidase from Sulfolobus solfataricus involved in xylan degradation. Extremophiles 11, 123–132 (2007). https://doi.org/10.1007/s00792-006-0020-7

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