Premium
Probing active‐site residues of pyranose 2‐oxidase from Trametes multicolor by semi‐rational protein design
Author(s) -
Salaheddin Clara,
Spadiut Oliver,
Ludwig Roland,
Tan TienChye,
Divne Christina,
Haltrich Dietmar,
Peterbauer Clemens
Publication year - 2009
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200800265
Subject(s) - pyranose , chemistry , galactose , substrate (aquarium) , enzyme kinetics , active site , stereochemistry , saturated mutagenesis , rational design , wild type , biochemistry , protein engineering , enzyme , mutant , biology , materials science , nanotechnology , ecology , gene
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 Tm P2Ox. 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 Tm P2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis‐Menten kinetics.