Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Penicillin amidases (PAs) from E. coli and A. faecalisare periplasmic enzymes that contain one tightly bound Ca2+ 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 PAEC) and Sec‐ (A. faecalis PAAF) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca2+ binding is required for the membrane transport and maturation of the pro‐enzyme to active enzyme. Pro‐enzyme without Ca2+ was targeted to the membrane but not translocated. Influence of Ca2+ in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca2+ in the feed the synthesis of the pre‐pro‐enzyme was hardly influenced. At optimal Ca2+ content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PAEC) and 2.3 g/L (PAAF) or 4% (PAEC) and 8% (PAAF) of the cell dry weight. The corresponding specific activities are 1700 U (PAEC) and 14000 U (PAAF) 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 Ca2+ or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.