Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis.
A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.
Overbeeke
N
N
Unilever Research Laboratorium, Vlaardingen, The Netherlands.
Termorshuizen
G H
GH
Giuseppin
M L
ML
Underwood
D R
DR
Verrips
C T
CT
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D015153:Blotting, Western; D007887:Fabaceae / Q000201:enzymology*; D005285:Fermentation; D005696:Galactosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D010957:Plasmids; D014169:Transformation, Bacterial; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism*
D005696:Galactosidases; D000519:alpha-Galactosidase