Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency |
| |
Authors: | Sabine Bastian Matthias J. Rekowski Klaus Witte Dorothée M. Heckmann-Pohl Friedrich Giffhorn |
| |
Affiliation: | (1) Lehrstuhl für Angewandte Mikrobiologie, Universität des Saarlandes, Postfach 15 11 50, Saarbrücken, 66041, Germany |
| |
Abstract: | 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 (kcat/Km) for d-xylose and l-sorbose. P2OxB1 was provided with a C-terminal His6-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 kcat/Km for d-glucose (5.3-fold), methyl- -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.This revised version was published online in February 2005 with corrections to Table 2. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|