PMID:15801782
| Citation |
Kasche, V, Ignatova, Z, Märkl, H, Plate, W, Punckt, N, Schmidt, D, Wiegandt, K and Ernst, B Ca2+ is a cofactor required for membrane transport and maturation and is a yield-determining factor in high cell density penicillin amidase production. Biotechnol. Prog. 21:432-8 |
|---|---|
| Abstract |
Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed. |
| Links |
PubMed Online version:10.1021/bp049636a |
| Keywords |
Alcaligenes faecalis/enzymology; Alcaligenes faecalis/growth & development; Bioreactors; Calcium/metabolism; Cell Membrane/metabolism; Penicillin Amidase/biosynthesis; Penicillin Amidase/metabolism; Water Pollutants, Chemical/isolation & purification |
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