Bioprocess development to improve foreign protein production from recombinant Streptomyces.
Bioprocessing strategies to improve production of the heterologous protein parathion hydrolase from recombinant Streptomyces lividans were investigated. Initial limitations to increased production were overcome by using large amounts of nutrients and feeding these nutrients throughout the fermentation. Batch addition of such large amounts of nutrients resulted in byproduct acid accumulation. Our data suggest that byproducts resulted from incomplete utilization of peptide medium ingredients and not from an overflow of glucose catabolism. Over extended fed-batch operation, oxygen transfer became limiting and these limitations were overcome by sparging oxygen-enriched gas. When cultivation was continued past about 90 h, we observed that despite nutrient feeding and oxygen enrichment enzyme activities no longer increased. Our results show that during such late cultivation periods the rates of enzyme synthesis and deactivation became balanced. If synthesis is prevented, either by a nutritional limitation or by the addition of the protein synthesis inhibitor chloramphenicol, enzyme activities were observed to decrease. Since deactivation rate constants in these experiments were similar to those observed in cell-free studies, and because extracellular protease activities were not detected in our fermentation, it appears that deactivation results from the inherent instability of the parathion hydrolase enzyme.
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21229.
N
N
DelaCruz
G F
GF
Payne
J M
JM
Smith
S J
SJ
Coppella
D043303:Aryldialkylphosphatase; D002701:Chloramphenicol / Q000494:pharmacology; D004795:Enzyme Stability; D005285:Fermentation; D005798:Genes, Bacterial; D010100:Oxygen / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D002701:Chloramphenicol; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase; D010100:Oxygen