N. Kirikyali
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 |
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