Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris

Kirikyali, N.; Connerton, I.F.

Abstract

To degrade plant hemicelluloses fungi employ β-xylosidases to hydrolyse xylooligosaccharides, released by endo-xylanases, into xylose. We have expressed the β-xylosidase from Neurospora crassa in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. The recombinant enzyme is optimally active at 50 °C and pH 5.0 with Km and Vmax values of 8.9 mM and 1052 μmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl β-xylopyranoside. Xylose is a non-competitive inhibitor with a K(i) of 1.72 mM. The enzyme is characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X₂, X₃ and X₄) but also capable of transxylosilation. Catalytic conversion of X₂, X₃ and X4 decreases (V(max) and k(cat)) with increasing chain length.

Citation

Kirikyali, N., & Connerton, I. (2014). Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris. Enzyme and Microbial Technology, 57, 63-68. https://doi.org/10.1016/j.enzmictec.2014.02.002

• Journal Article Type: Article
• Acceptance Date: Feb 5, 2014
• Online Publication Date: Feb 14, 2014
• Publication Date: Apr 10, 2014
• Deposit Date: Oct 27, 2018
• Journal: Enzyme and Microbial Technology
• Print ISSN: 0141-0229
• Electronic ISSN: 1879-0909
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 57
• Pages: 63-68
• DOI: https://doi.org/10.1016/j.enzmictec.2014.02.002
• Keywords: Neurospora crassa; Xylose; β-Xylosidase; Enzyme kinetics; Protein expression
• Public URL: https://nottingham-repository.worktribe.com/output/1201319
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0141022914000271