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 (k(cat)/K(m)) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His(6)-tag exhibited significantly decreased apparent K(m) 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 O(2)-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.
Subject(s)
Carbohydrate Dehydrogenases/chemistry , Carbohydrate Dehydrogenases/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Protein Engineering , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Basidiomycota/enzymology , Basidiomycota/growth & development , Binding Sites , Carbohydrate Dehydrogenases/genetics , Carbohydrate Dehydrogenases/isolation & purification , Catalysis , Crystallography, X-Ray , Enzyme Stability/genetics , Escherichia coli/genetics , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Genetic Variation , Histidine/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Phylogeny , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structure-Activity Relationship , Substrate SpecificityABSTRACT
To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k(cat)/K(m)) for D-xylose and L-sorbose. P2OxB1 was provided with a C-terminal His(6)-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k(cat)/K(m) for D-glucose (5.3-fold), methyl-beta-D-glucoside (2.0-fold), D-galactose (4.8-fold), D-xylose (59.9-fold), and L-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of L-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.
Subject(s)
Basidiomycota/enzymology , Carbohydrate Dehydrogenases/chemistry , Carbohydrate Dehydrogenases/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Protein Engineering , Amino Acid Substitution , Carbohydrate Dehydrogenases/genetics , Cloning, Molecular , DNA, Fungal/chemistry , DNA, Fungal/genetics , Directed Molecular Evolution , Enzyme Stability/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Galactose/metabolism , Gene Expression , Glucose/metabolism , Hydrogen-Ion Concentration , Methylglucosides/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation, Missense , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sorbose/metabolism , Temperature , Xylose/metabolismABSTRACT
Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.
