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 fun ctions of the broad-host-range lactococcal plasmid pWVO1, the lactococ cal promoter P59, and the DNA sequences encoding the alpha-amylase sig nal sequence from Bacillus amyloliquefaciens and the mature part of th e alpha-galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant alph a-galactosidase and secreted the enzyme efficiently as judged by Weste rn blotting and activity assays. Expression levels of up to 4.3 mg ext racellular alpha-galactosidase g (dry weight) of biomass(-1) were achi eved in standard laboratory batch cultures. The alpha-galactosidase pr oduced by Lactococcus was active on the chromogenic substrate 5-bromo- 4-chloro-3-indolyl alpha-D-galactopyranoside, the trisaccharide raffin ose and on the galactomannan substrate, guar gum.