Evidence for importance of the Staphylococcus hyicus lipase pro-peptide in lipase secretion, stability and activity.
To investigate the function of the pro-peptide (PP) region of the Staphylococcus hyicus exolipase, restriction sites were created in the lipase gene to facilitate the construction of deletions in this region. Lipase gene expression was carried out in Staphylococcus carnosus. In the presence of the entire PP region, the 86-kDa pro-lipase was efficiently exported, had high lipolytic activity, and hardly any degradation products were seen in Western blot analysis. In addition to the 86-kDa pro-lipase, the membrane fraction contained a 106-kDa immunoreactive form. If the PP was completely or partially deleted, signal peptide processing, lipase secretion, lipase activity and/or lipase stability were impaired. The results obtained with lipase PP deletion mutants indicate that the PP region may have two functional domains. The N-terminal region of the lipase PP appears to be more important for lipase activity and the C-terminal portion for lipase secretion and proteolytic stability. In the presence of only the C-terminal part of the PP lipase, secretion was hardly affected. However, the activity of the extracellular lipase was markedly reduced. If only a small portion of the C-terminal part of the PP was present, lipase secretion was again markedly reduced and no lipase activity was detectable. In the presence of the N-terminal half of the PP region, lipase secretion was affected to a lesser extent. However, the resulting 60-kDa form, which showed comparably good specific lipase activity, suffered severe proteolytic degradation.
Demleitner
G
G
Universität Tübingen, FRG.
Götz
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics*; D017353:Gene Deletion; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics
D004792:Enzyme Precursors; D008049:Lipase