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Direct amplification of new cellulase genes from woodland soil purified DNA

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Abstract

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.

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Acknowledgments

This work was supported by “Fondo per la promozione delle espressioni di interesse dei privati e azioni conseguenenti” instituted by Lombardia region (Italy) (Project number: 12771762).

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Authors and Affiliations

  1. Plantechno s.r.l, via Staffolo 60, 26041, Vicomoscano, Cremona, Italy

    Marco Cucurachi

  2. Institute of Agronomy, Genetics, and Field Crops, Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122, Piacenza, Italy

    Matteo Busconi, Mariangela Marudelli, Giovanna Soffritti & Corrado Fogher

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  1. Marco Cucurachi
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  2. Matteo Busconi
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Correspondence to Matteo Busconi.

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Marco Cucurachi and Matteo Busconi contribute equally to this study.

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Cucurachi, M., Busconi, M., Marudelli, M. et al. Direct amplification of new cellulase genes from woodland soil purified DNA. Mol Biol Rep 40, 4317–4325 (2013). https://doi.org/10.1007/s11033-013-2519-1

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  • DOI: https://doi.org/10.1007/s11033-013-2519-1

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