 Hi, I'm Alessandro Ferrari, one of the authors of the paper entitled Expanding the Substitute Scope of Kite-Oligo-Saccharide Oxidase from Fusarium Graminiarum by Structure-Inspired Metagenesis. The flaboprotein Kite-Oligo-Saccharide Oxidase from now on called Kite is a carbohydrate oxidase with a peculiarity. It's the only of its kind capable of oxidizing an acetylated sugars. In fact, it can oxidize the constitutive element of chitin, anacetylglucosamine, and its oligomers. The oxidation takes place at the C1 position, resulting in the formation of the corresponding lactone which subsequently hydrolyzes to the respective aldonic acid. In a previous study, a point mutation in the active site, Q268R, changed the substrate specificity of Kite from Kite-Oligo-Saccharide to Glucol-Oligo-Saccharide. In this study, by comparison with two other known carbohydrate oxidase, namely the Glucol-Oligo-Saccharide oxidase from Acremonium Strictum from now on called Gox, and the lactose oxidase from Microdochium Nibale from now on called Laot, Kite was redesigned in order to accept other carbohydrates as substrates. By superimposition of the model structure of Kite-O with the two other known carbohydrate oxidase, we took a close look at the active site at the substrate binding pocket, and eight residues were identified which were different between Kite-O, Gox, and Laot. So we designed eleven mutations in order to either change the residue in Kite-O with the residue type found either in Laot or Gox, or change the physical properties of the selected amino acid, for example from a methionine to a tyrosine. After screening with 23 different carbohydrates, two mutants G270E and S410R turned out to be interesting. In fact, they improved the catalytic efficiency towards oxidation of cellobios, lactose and maltose two times compared to the wild type. Then we decided to combine these mutations together with the previously characterized one Q268R, generating three different combinations, G270E with S410R, Q268R with S410R, and the triple mutant, which included the combination of all the three together. Well the results were way beyond our expectations, the double mutant G270E S410R improved the catalytic efficiency of Enacetyl Glucosamine by 16 times compared to the wild type, generating the best Enacetyl Glucosamine oxidase so far reported in the literature. And the results were even more surprising for the triple mutant. The oxidation of cellobios improved by 200 times compared to the wild type, and the oxidation of the lactose improved even more than 500 times. As you can see, by rational design of mutation, the certain scope of Kaito was successfully changed. We generated variants with superior activity towards different carbohydrates compared to the wild type. You can read in more detail in our paper in Biotechnology and Bioengineering, and we can read how we achieved this and the reasons that might be behind these drastic improvements. For any questions, comments or requests, please feel free to contact Professor Marco Freyhe at the mail address below. Thank you for your attention, and we hope you will enjoy our paper.