Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept.
Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing the widespread adoption of biocatalysts for industrial synthesis. A crucial distinction of many redox enzymes used to synthesize chiral compounds is the need for cofactors (e.g., NAD(P)(H)) for function. Because of the prohibitively high prices of nicotinamide cofactors, a robust cofactor-regenerating enzyme is required for the economical synthesis of fine chemicals by biocatalysis. Here we test the structure-guided consensus for the generation of a thermostable glucose dehydrogenase (GDH). The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46 %. Of the 24 variants, seven were located at the subunit interface-known to influence GDH stability-and six were more stable (86 % success). The best variants feature a half-life of approximately 3.5 days at 65 degrees C, in contrast to approximately 20 min at 25 degrees C for the wild type, thus enhancing stability 10(6)-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologues from Bacillus thuringiensis and Bacillus licheniformis. The thermal stability as measured by half-life and CD(222 nm) of the GDH variants was increased, as expected. The resulting stability changes provide further support for the view that these residues are critical for stability of GDHs and reinforce the success of the consensus approach for identifying stabilizing mutations.
Vázquez-Figueroa
Eduardo
E
School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, 315 Ferst Drive, Atlanta, GA 30332-0363, USA.
Chaparro-Riggers
Javier
J
Bommarius
Andreas S
AS
D001419:Bacteria / Q000201:enzymology; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000235:genetics; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D009154:Mutation; D013329:Structure-Activity Relationship; D013696:Temperature
D042843:Glucose 1-Dehydrogenase