High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.
By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.
Austrian Centre of Industrial Biotechnology, Petersgasse 14, Graz, 8010, Austria.
Sandra
S
Abad
Jozef
J
Nahalka
Margit
M
Winkler
Gabriele
G
Bergler
Robert
R
Speight
Anton
A
Glieder
Bernd
B
Nidetzky
D019840:2-Propanol; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics; D008954:Models, Biological; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics
D003605:D-Amino-Acid Oxidase; D019840:2-Propanol