Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency.
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.
Bastian
Sabine
S
Lehrstuhl für Angewandte Mikrobiologie, Universität des Saarlandes, Postfach 15 11 50, Saarbrücken 66041, Germany.
Rekowski
Matthias J
MJ
Witte
Klaus
K
Heckmann-Pohl
Dorothée M
DM
Giffhorn
Friedrich
F
D019943:Amino Acid Substitution; D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013013:Sorbose / Q000378:metabolism; D013696:Temperature; D014994:Xylose / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D008757:Methylglucosides; D011994:Recombinant Proteins; C027020:methylglucoside; D014994:Xylose; D002237:Carbohydrate Dehydrogenases; C489823:poxB protein, Phlebiopsis gigantea; D005947:Glucose; D013013:Sorbose; D005690:Galactose