High-level production of bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3).
A fed-batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d-amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed-batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g . L(-1)). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U . g(-1) cell dry weight and 1154 U . L(-1) with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26-fold in the cost of the culture medium.
Martínez-Martínez
Irene
I
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30071 Murcia, Spain.
Kaiser
Christian
C
Rohde
Alexander
A
Ellert
Andree
A
García-Carmona
Francisco
F
Sanchez-Ferrer
Alvaro
A
Luttmann
Reiner
R
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D019149:Bioreactors / Q000382:microbiology; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D013379:Substrate Specificity
D003470:Culture Media; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase