A plant α-galactosidase gene was inserted in the expression vector pGKV259. The resulting plasmid pGAL2 consisted of the replication functions of the broad-host-range lactococcal plasmid pWV01, the lactococcal promoter P59, and the DNA sequences encoding the α-amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the α-galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant α-galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular α-galactosidase g (dry weight) of biomass-1 were achieved in standard laboratory batch cultures. The α-galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl α-d-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum.
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology