Heterologous expression and kinetic characterisation of Neurospora crassa ?-xylosidase in Pichia pastoris
Kirikyali, N.; Connerton, I.F.
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.
Kirikyali, N., & Connerton, I. (2014). Heterologous expression and kinetic characterisation of Neurospora crassa ?-xylosidase in Pichia pastoris. Enzyme and Microbial Technology, 57, 63-68. doi: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|
|Peer Reviewed||Peer Reviewed|
|Keywords||Neurospora crassa; Xylose; ?-Xylosidase; Enzyme kinetics; Protein expression|
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