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Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei

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Abstract

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.

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Acknowledgements

We thank Merja Helanterä, Kirsti Leskinen, Jaana Oksanen and Riitta Tarkiainen for skillful technical assistance. Outi Nikkilä and Tarja Lahtinen are acknowledged for performing the laboratory-scale bleaching trials. Sirpa Holm, Sirpa Okko, Elke Parkkinen, Auli Sinnemäki and Tarja Sjöblom are acknowledged for performing the laboratory-scale fermentations. Nisse Kalkkinen is acknowledged for performing the peptide analyses and Martina Blomster-Andberg for preparing samples for C-terminal analysis. David Wilson is acknowledged for kindly providing the T. fusca TfxA antibody and the pTX101 plasmid

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Author notes

  1. S. Leskinen

    Present address: Genencor International, Oy Hanko Plant, 10901, Hanko, Finland

  2. A. Mäntylä

    Present address: UniCrop Oy, Viikinkaari 4, 00790, Helsinki, Finland

  3. R. Fagerström & R. Lantto

    Present address: VTT Biotechnology, 02044, VTT, Finland

  4. P. Suominen

    Present address: Cargill Dow, LLC15305, Minnetonka, MN 55345, USA

Authors and Affiliations

  1. Primalco Ltd. Biotec, Roal Oy, 05201, Rajamäki, Finland

    S. Leskinen, A. Mäntylä, R. Fagerström, J. Vehmaanperä, R. Lantto, M. Paloheimo & P. Suominen

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  1. S. Leskinen
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  2. A. Mäntylä
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Correspondence to A. Mäntylä.

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Leskinen, S., Mäntylä, A., Fagerström, R. et al. Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei. Appl Microbiol Biotechnol 67, 495–505 (2005). https://doi.org/10.1007/s00253-004-1797-x

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