Directed evolution of galactose oxidase

The fungal enzyme galactose oxidase is an extracellular enzyme that is produced by various Fusarium spp. Its unique active centre consists of one copper ion and a cysteine covalently linked to a tyrosine carrying a radical. The enzyme catalyses the oxidation of primary alcohols, e.g. galactose, to the corresponding aldehyds, e.g. galactose 6-aldehyde without reacting with other reducing groups. In traditional organic chemistry this reaction can only be performed using highly toxic chemicals like chrome (VI) salts. Despite its broad substrate specificity (including sugar alcohols, aliphatic and aromatic alcohols) when it comes to carbohydrates galactose oxidase accept only galactose and closely related sugars as substrates. This narrows the usable range of application for galactose oxidase. It is the objective of this proposal to broaden the substrate specificity of galactose oxidase especially improving the activity against glucose. Two different approaches will be used. The first approach is to use saturation mutagenesis to alter the amino acids in and around the active centre. The produced mutants will be screened for improved activity. In the second approach the whole galactose oxidase gene will be mutated using error prone PCR. In a simple and fast membrane based pre-screening active clones will be selected and analysed in detail. This directed evolution will be repeated several times. The best clones from both approaches will be combined via gene shuffling. The most interesting clones will be characterised in detail. Batch conversion of promising substrates will be performed in medium scale and the so produced aldehyds will be used by our Italian partner (S. Riva) for chemical and chemo-enzymatic synthesis. Together with our first Swedish partner (L. Gorton) we will use the variants for mediator free 3rd generation biosensor to detect carbohydrates like glucose or fructose in high concentrations. By solving the crystal structure of these mutants by our second Swedish partner (C. Divne) and comparing them with the structure of the wild type enzyme, we hope to increase our knowledge about the reaction mechanism of this unique enzyme.

Galactose oxidase (GalOx) is an enzyme produced by a variety of fungi. GalOx has found a number of interesting applications, for example is it used for the determination of galactose in blood and in clinical analytics for the histological detection of cancer cells. In industrial biotechnology it can serve as a biocatalyst for the specific oxidation of sugars. Only oxygen is needed for this reaction. For the traditional chemical syntheses of these oxidized sugars highly toxic and carcinogenic substances like chrom (VI) salts are used. As an example one can give the reaction with galactose, with is found as a component in the milk sugar lactose. Galactose can be specifically oxidized by GalOx to galactose 6-aldehyde, which in turn can be converted to immunoactive galactoseamines. Galactose 6- aldehyde can also be used in food technology to produce hydrogels and emulsifier. GalOx is, however, very selective in its substrate specificity, accepting only galactose and closely related sugars. This narrows the usable range of application for this enzyme in biotechnology. It was the objective of this proposal to broaden the substrate specificity of GalOx, especially improving the activity against glucose (grape-sugar). For this purpose GalOx from three different fungi strains were isolated, characterised in detail, and amino acids presumed to play an important role in the reaction mechanism were identified and modified. The so produced enzyme variants were analysed for their potential for biotechnology applications. In the frame of this project we also studied a closely related enzyme, glyoxal oxidase, in detail. This enzyme has a very similar structure compared with GalOx but a completely different substrate spectrum. We isolated this enzyme from different sources, produced it and compared it to GalOx.