Excretion into the culture medium of a Bacillus beta-glucanase after overproduction in Escherichia coli.
The beta-glucanase gene (bgl) from Bacillus amyloliquefaciens was expressed in E. coli CSH 55 under the control of the PR promoter of phage lambda that is repressed by the thermosensitive repressor C1857. Production of beta-glucanase was drastically stimulated by a temperature shift to 42 degrees C. This overexpression of the bgl gene (about 20% of the total cellular protein) led to an almost complete excretion of the otherwise periplasmic protein into the extracellular medium, beta-glucanase accounted for more than 50% of the extracellular proteins. Col E 1 related plasmid (pEG 1) are amplified in E. coli relA strains in response to an amino acid limitation leading to a 10-fold increase in the activity of plasmid encoded genes. In this work we intended to maximize the expression of the bgl gene by a concerted action of a plasmid amplification and temperature induction. Surprisingly we could not increase the beta-glucanase production above the level reached by plasmid amplification or temperature induction alone. The reasons for this unexpected result will be discussed. Under all conditions tested the expression of the bgl gene was much lower in the E. coli relA strain NF 162 than in E. coli CSH 55; the low beta-glucanase production was accompanied by a reduced excretion rate of the enzyme.
Riethdorf
S
S
Sektion Biologie, Ernst-Moritz-Arndt-Universität, Greifswald.
Ulrich
A
A
Völker
U
U
Hecker
M
M
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D005936:Glucans / Q000378:metabolism; D005959:Glucosidases / Q000096:biosynthesis*; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D005936:Glucans; D011994:Recombinant Proteins; D005959:Glucosidases; D001617:beta-Glucosidase