Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth.
The Bacillus subtilis levanase structural gene sacC was expressed under the regulated control of sacR, the inducible levansucrase leader region, in a degU32(Hy) strain. In this genetic context, exocellular levanase is overproduced (0.5% of total protein) during the exponential phase of growth upon induction by sucrose at 37 degrees C and pH 7. No precursor form that comprised a signal peptide was detected in pulse-chase experiments. The subsequent release of the cell-associated processed protein is a slow event (t(1,2) = 80+/-10 s). The unfolding-folding transition of pure levanase monitored in vitro by the resistance to proteolysis was achieved within the same time range (t(1/2) = 50 s) under the same conditions of pH and temperature. Calcium ions, which modulate the rate and the yield of refolding, have a low affinity for the protein. Comparison of these results with those obtained previously with levansucrase and alpha-amylase overproduced in the same genetic and physiological context suggests that the precursor processing is more efficient in levanase and alpha-amylase than in levansucrase. This discrepancy could lie in information borne by the signal peptide sequence of these exoproteins. However, the rate of the ultimate stage of release of these three proteins, which includes the passage through the cell wall, is correlated with the rate of folding and appears to be independent of their molecular size.
Leloup
Laurence
L
Petit-Glatron
Marie-Françoise
MF
Chambert
Régis
R
Le Saux
Jérome
J
D001412:Bacillus subtilis / Q000201:enzymology / Q000254:growth & development*; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C021512:levanase; D002118:Calcium