+ Site Statistics
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

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

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

Biotechnology Journal 4(4): 535-543

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.

(PDF emailed within 0-6 h: $19.90)

Accession: 055189787

Download citation: RISBibTeXText

PMID: 19370721

DOI: 10.1002/biot.200800265

Related references

Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design. Febs Journal 276(3): 776-792, 2009

Impact of pyranose oxidase from Trametes multicolor, glucose oxidase from Aspergillus niger and hydrogen peroxide on protein agglomeration in wheat flour gluten-starch separation. Food Chemistry 148: 235-239, 2014

Engineering of pyranose 2-oxidase: improvement for biofuel cell and food applications through semi-rational protein design. Journal of Biotechnology 139(3): 250-257, 2009

Kinetic mechanism of pyranose 2-oxidase from trametes multicolor. Biochemistry 48(19): 4170-4180, 2009

Production of a novel pyranose 2-oxidase by basidiomycete Trametes multicolor. Applied Biochemistry and Biotechnology 70-72: 237-248, 1998

Biochemical characteristics of Trametes multicolor pyranose oxidase and Aspergillus niger glucose oxidase and implications for their functionality in wheat flour dough. Food Chemistry 131(4): 1485-1492, 2012

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor. Applied and Environmental Microbiology 67(8): 3636-3644, 2001

The bread dough stability improving effect of pyranose oxidase from trametes multicolor and glucose oxidase from Aspergillus niger: unraveling the molecular mechanism. Journal of Agricultural and Food Chemistry 61(32): 7848-7854, 2014

Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor. Biocatalysis and Biotransformation 26(1-2): 120-127, 2008

Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor. Acta Crystallographica. Section D, Biological Crystallography 60(Pt 1): 197-199, 2003

Conformational heterogeneity in pyranose 2-oxidase from Trametes multicolor revealed by ultrafast fluorescence dynamics. Journal of Photochemistry and Photobiology A: Chemistry 234: 44-48, 2012

Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor. Analytical Biochemistry 314(2): 235-242, 2003

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications. Biotechnology Journal 4(4): 525-534, 2009

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover. Plos One 11(2): E0148108, 2016

Enzymatic formation of dicarbonyl sugars: C-2 oxidation of 1->6 disaccharides gentiobiose, isomaltose and melibiose by pyranose 2-oxidase from Trametes multicolor. Journal of Carbohydrate Chemistry 18(8): 999-1007, 1999