The influence of protein folding on late stages of the secretion of alpha-amylases from Bacillus subtilis.
A derivative of the alpha-amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme. This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane. When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca2+ ions for in vitro folding. When the concentration of Ca2+ in the growth medium was increased, so too did the relative yield of AmyLQS50.5. We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B. subtilis.
School of Microbiological, Immunological and Virological Sciences, The Medical School, University of Newcastle upon Tyne, UK.
K
K
Stephenson
N M
NM
Carter
C R
CR
Harwood
M F
MF
Petit-Glatron
R
R
Chambert
D001407:Bacillus / Q000201:enzymology*; D001412:Bacillus subtilis / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D003470:Culture Media; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases; D002122:Calcium Chloride; D002118:Calcium