IN-VIVO IMMOBILIZATION OF ENZYMATICALLY ACTIVE POLYPEPTIDES ON THE CELL-SURFACE OF STAPHYLOCOCCUS-CARNOSUS

Many surface proteins of Gram-positive bacteria are covalently anchore d to the cell wall by a ubiquitous mechanism, involving a specific, C- terminal sorting signal, To achieve cell-wall immobilization of a norm ally secreted enzyme in vivo, we constructed a hybrid protein consisti ng of Staphylococcus hyicus lipase and the C-terminal region of Staphy lococcus aureus fibronectin binding protein B (FnBPB). This region com prised the authentic cell-wall-spanning region and cell-wall sorting s ignal of FnBPB. Expression of the hybrid protein in Staphylococcus car nosus resulted in efficient cell-wall anchoring of enzymatically activ e lipase, The cell-wall-immobilized lipase (approximately 10 000 molec ules per cell) retained more than 80% of the specific activity, compar ed to the C-terminally unmodified S. hyicus lipase secreted by S. carn osus cells, After releasing the hybrid protein from the cell wall by l ysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB p er se was fully compatible with folding of the lipase to an active con formation, To study the influence of the distance between the cell-wal l sorting signal and the C-terminus of the lipase on the activity of t he immobilized lipase, the length of this spacer region was varied, Re duction of the spacer length gradually reduced the activity of the sur face-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating t hat 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 lipa se was replaced by another enzyme, the Escherichia coil beta-lactamase , the resulting hybrid was also efficiently anchored in an active conf ormation to the cell wall of S. carnosus, These results demonstrate th at it is possible to Immobilize normally soluble enzymes on the cell w all of S. carnosus - without radically altering their catalytic activi ty - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cell-wall-spanning region and a standard cell-wall sorting signal.