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Ca 2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield‐Determining Factor in High Cell Density Penicillin Amidase Production
Author(s) -
Kasche Volker,
Ignatova Zoya,
Märkl Herbert,
Plate Wilhelm,
Punckt Nils,
Schmidt Dorothea,
Wiegandt Katharina,
Ernst Burkhard
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049636a
Subject(s) - periplasmic space , enzyme , biochemistry , penicillin amidase , cofactor , lysis , chemistry , amidase , enzyme assay , membrane , biology , escherichia coli , immobilized enzyme , gene
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.