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Article Dans Une Revue Biotechnology Journal Année : 2009

Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design

Résumé

D-Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D-galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-Oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D-galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of kcat with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, kcat/KM is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D-galactose. Batch conversion experiments of D-galactose to 2-keto-D-galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis Menten kinetics.

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Dates et versions

hal-00477774 , version 1 (30-04-2010)

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Clara R Salaheddin, Oliver Spadiut, Roland Ludwig, Tien-Chye Tan, Christina Divne, et al.. Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design. Biotechnology Journal, 2009, 4 (4), pp.535-n/a. ⟨10.1002/biot.200800265⟩. ⟨hal-00477774⟩

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