Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells.
Gram-positive bacteria code for one or more enzymes termed sortases which catalyze the covalent anchoring of substrate proteins on their cell wall. They recognize an amino acid sequence designated sorting motif, present close to the C-terminal end of the substrate proteins, cleave within this motif and catalyze anchoring of the polypeptide chain to the peptide crossbridge linking the peptidoglycan strands in a transpeptidation reaction. Bacillus subtilis has been reported to code for two different sortases but the sorting sequences recognized by them are yet unknown. To be able to immobilize proteins on the surface of B. subtilis cells, we introduced the srtA gene coding for sortase A of Listeria monocytogenes with the known sorting motif (LPXTG) into B. subtilis. L. monocytogenes and B. subtilis share the same peptide crossbridge. Next, we fused the coding region of an alpha-amylase gene to the C-terminal region of Staphylococcus aureus fibronectin binding protein B containing the sorting motif. Covalent linkage could be proven by treatment of the cells with lysozyme and by immunofluorescence microscopy. Up to 240,000 molecules of alpha-amylase could be immobilized per cell, 24 times more than previously reported for other bacterial species. To study the influence of the distance between the sorting motif and the C-terminus of alpha-amylase on the activity of the enzyme, the length of the spacer was varied. It turned out that the highest activity was measured with a spacer length of 123 amino acid residues.
Nguyen
Hoang Duc
HD
Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany.
Schumann
Wolfgang
W
D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008089:Listeria monocytogenes / Q000166:cytology / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D000516:alpha-Amylases; D003546:Cysteine Endopeptidases