Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A.
In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage lambda pR promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42 degrees C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved.
Engel
H
H
Department of Biochemistry, BIOSON Research Institute, University of Groningen, The Netherlands.
van Leeuwen
A
A
Dijkstra
A
A
Keck
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial / Q000502:physiology; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*
D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D009238:N-Acetylmuramoyl-L-alanine Amidase