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:	http://www.sciencedirect.com
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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