title: Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis.
abstract: 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.
authors.affiliation: Unilever Research Laboratorium, Vlaardingen, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Overbeeke
authors.forename: G H
authors.initials: GH
authors.lastname: Termorshuizen
authors.forename: M L
authors.initials: ML
authors.lastname: Giuseppin
authors.forename: D R
authors.initials: DR
authors.lastname: Underwood
authors.forename: C T
authors.initials: CT
authors.lastname: Verrips
mesh_terms: 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*
chemical_list: D005696:Galactosidases; D000519:alpha-Galactosidase