In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10,000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S.carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment. Its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the Influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the peptidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coli beta-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S. carnosus. These results demonstrate that it is possible to immobilize normality soluble enzymes on the cell wall of S. carnosus-without radically altering their catalytic activity-by fusing them to a cell-wall-immobilization unit, consisting of a suitable cell-wall-spanning region and a standard cell-wall sorting signal.