Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis.
The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens alpha-amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.
Research Laboratories, Alko Ltd., Helsinki, Finland.
M
M
Paloheimo
D
D
Haglund
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S
Aho
M
M
Korhola
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D000516:alpha-Amylases / Q000235:genetics
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D000516:alpha-Amylases