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Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering

Paper ID Volume ID Publish Year Pages File Format Full-Text
25435 43574 2006 15 PDF Available
Title
Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering
Abstract

Native pyranose 2-oxidase (P2Ox) was purified from Peniophora sp. and characterized. To improve its catalytic efficiencies and stabilities by protein engineering, we cloned and expressed the P2Ox gene in Escherichia coli and received active, fully flavinylated recombinant P2OxA. Selenomethionine-labeled P2OxA was used for X-ray analysis and the resulting crystal structure enabled the rational design using variant P2OxA1 with the substitution E542K as template. Besides increased thermal and pH stabilities this variant showed improved catalytic efficiencies (kcat/Km) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His6-tag exhibited significantly decreased apparent Km values for d-glucose (0.47 mM), l-sorbose (1.79 mM), and d-xylose (1.35 mM). Compared to native P2Ox, the catalytic efficiencies were substantially improved for d-glucose (230-fold), l-sorbose (874-fold), and d-xylose (1751-fold). This P2Ox variant was used for the bioconversion of l-sorbose under O2-saturation in a molar scale. The structure–activity relationships of the amino acid substitutions were analyzed by modelling of the mutated P2Ox structures. Molecular docking calculations of various carbohydrates into the crystal structure of P2OxA and the analysis of the protein–ligand interactions in the docked complexes enabled us to explain the substrate specificity of the enzyme by a conserved hydrogen bond pattern which is formed between the protein and all substrates.

Keywords
Pyranose 2-oxidase gene; Rational protein design; Improved enzyme variants; Bioconversion; Structure–activity relationship; Molecular docking; Enzyme–ligand interactions
First Page Preview
Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 124, Issue 1, 25 June 2006, Pages 26–40
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering