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Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design.
KTH, School of Biotechnology (BIO), Glycoscience.
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2009 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 4, no 4, 535-543 p.Article in journal (Refereed) Published
Abstract [en]

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 k(cat) with both D-glucose and D-galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k(cat)/K(M) 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.

Place, publisher, year, edition, pages
2009. Vol. 4, no 4, 535-543 p.
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-92622DOI: 10.1002/biot.200800265PubMedID: 19370721ScopusID: 2-s2.0-65549111724OAI: diva2:514052
QC 20120410Available from: 2012-04-04 Created: 2012-04-04 Last updated: 2012-04-10Bibliographically approved

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Salaheddin, ClaraSpadiut, OliverTan, Tien-ChyeDivne, Christina
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