Thermostable variants of pyranose 2-oxidase showing altered substrate selectivity for glucose and galactose.
The homotetrameric flavoprotein pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications, among others as a biocatalyst for carbohydrate transformations toward higher-value products. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semirational enzyme engineering approach, namely, saturation mutagenesis of the amino acid His450 located at a pivotal point of the active site loop and subsequent screening of the libraries thus obtained for improved activity with the sugar substrate d-galactose. A variant with improved catalytic characteristics identified was H450G, which showed a significant, 3.6-fold decrease in K(M) together with a 1.4-fold increase in k(cat) for its substrate D-galactose and an overall improvement in the catalytic efficiency by a factor of 5. By combining H450G with other amino acid replacements, we obtained the P2Ox variants H450G/V546C and H450G/E542K/V546C, which can be of interest for applications in food industry due to their increased activity with D-galactose, high activity with D-glucose, and considerably increased stability for the latter variant. While the His-tagged recombinant wild-type enzyme strongly prefers D-glucose to D-galactose as its substrate, H450G/E542K/V546C converts both sugars, which are found in lactose hydrolysates, concomitantly, as was shown by laboratory-scale biotransformation experiments. The 2-keto sugars thus obtained can conveniently be reduced to the corresponding ketoses D-fructose and D-tagatose.
Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Oliver
O
Spadiut
Tien-Thanh
TT
Nguyen
Dietmar
D
Haltrich
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity; D055454:Trametes / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose; D005690:Galactose