Thermostable variants of pyranose 2-oxidase showing altered substrate selectivity for glucose and galactose.

Oliver Spadiut, Tien-Thanh Nguyen, Dietmar Haltrich

Index: J. Agric. Food Chem. 58(6) , 3465-71, (2010)

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Abstract

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.