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

Paper ID Volume ID Publish Year Pages File Format Full-Text
17062 42637 2014 6 PDF Available
Title
Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris
Abstract

•We report the secretion of Neurospora crassa β-xylosidase from Pichia pastoris.•Recombinant β-xylosidase has high catalytic capacity against natural substrates.•The reaction product xylose is a non-competitive inhibitor of the β-xylosidase.•Transxylosilation activity of the enzyme has been identified.

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 Ki 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 (Vmax and kcat) with increasing chain length.

Keywords
Neurospora crassa; Xylose; β-Xylosidase; Enzyme kinetics; Protein expression
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Heterologous expression and kinetic characterisation of Neurospora crassa β-xylosidase in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 57, 10 April 2014, Pages 63–68
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
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Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
Online Support
Any Questions? feel free to contact us