Naslov
Integrated approach for production of recombinant acetylacetone dioxygenase from Acinetobacter johnsonii

Autori
Straganz, Grit Daniela ; Slavica, Anita ; Hofer, Hannes ; Mandl, Ulrike ; Steiner, Walter ; Nidetzky, Bernd

Izvornik
Biocatalysis and Biotransformation (1024-2422) 23 (2005), 3-4; 261-269

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Dke1; non-heme metal-dependent dioxygenases; affinity purification; stability; Fe2+ cofactor

Sažetak
The C-C bond-cleaving acetylacetone dioxygenase Dke1 (EC 1.13.11.50) is a Fe2+-dependent enzyme from Acinetobacter johnsonii that activates oxygen to convert a range of  -dicarbonyl substrates into  -oxo-aldehyde and acid products. Previous methods of downstream processing yielded Dke1 with substoichiometric Fe2+ content. This paper reports the integration of enzyme production in E. coli and affinity chromatography to prepare recombinant Dke1 that is completely loaded with its metal cofactor. The specific activity of Dke1 in E. coli cell extracts could be increased up to 20-fold, compared to optimized enzyme production with the natural host. Introductionof an affinity-tag allowed the isolation of fully active Dke1 in a single purification step with high yield (70%). Mass spectrmetric analysis revealed at the level of >80% of sequence coverage that the isolated enzyme corresponded exactly to the predicted gene product. Tagged Dke1 is shown to have retained the functional properties of native Dke1.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

POVEZANOST RADA