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    Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris


    Gaffney, Mark and Carberry, Stephen and Doyle, Sean and Murphy, Richard (2009) Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris. Enzyme and Microbial Technology, 45 (5). pp. 348-354. ISSN 0141-0229

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    Abstract

    A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 ◦C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

    Item Type: Article
    Keywords: Xylanase; Thermomyces lanuginosus; Protein purification; Physicochemical characterisation; Heterologous expression;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7392
    Identification Number: https://doi.org/10.1016/j.enzmictec.2009.07.010
    Depositing User: Dr. Sean Doyle
    Date Deposited: 26 Aug 2016 14:47
    Journal or Publication Title: Enzyme and Microbial Technology
    Publisher: Elsevier
    Refereed: Yes
    URI:

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