Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture.
The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F' episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl beta-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I.
Sakamoto
S
S
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Terada
I
I
Lee
Y C
YC
Uehara
K
K
Matsuzawa
H
H
Iijima
M
M
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D013824:Thermus / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
D003470:Culture Media; D004269:DNA, Bacterial; D007544:Isopropyl Thiogalactoside; D012697:Serine Endopeptidases; C055102:aqualysin I