Applied Microbiology and Biotechnology, Vol.44, No.1-2, 75-80, 1995
Production of Secreted Guar Alpha-Galactosidase by Lactococcus-Lactis
A plant alpha-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 pWVO1, the lactococcal promoter P59, and the DNA sequences encoding the alpha-amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant alpha-galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular alpha-galactosidase g (dry weight) of biomass(-1) were achieved in standard laboratory batch cultures. The alpha-galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl alpha-D-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum.
Keywords:CYAMOPSIS-TETRAGONOLOBA GUAR;BACILLUS-SUBTILIS;HANSENULA-POLYMORPHA;STREPTOCOCCUS-LACTIS;NUCLEOTIDE-SEQUENCE;SUBSP LACTIS;PLASMID DNA;EXPRESSION;CLONING;GENE