Extracellular production of cloned alpha-amylase by Escherichia coli.
Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.
Suominen
I
I
Department of Biochemistry, University of Turku, Finland.
Karp
M
M
Lähde
M
M
Kopio
A
A
Glumoff
T
T
Meyer
P
P
Mäntsälä
P
P
D001692:Biological Transport; D002462:Cell Membrane / Q000502:physiology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000502:physiology*; D005110:Extracellular Space / Q000201:enzymology; D005786:Gene Expression Regulation; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D009113:Muramidase / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D000516:alpha-Amylases; D009113:Muramidase; D008274:Magnesium