New secretory strategies for Kluyveromyces lactis {beta}-galactosidase

We examined several strategies for the secretion of Kluyveromyceslactis ß-galactosidase into the culture medium, inorder to facilitate the downstream processing and purificationof this intracellular enzyme of great industrial interest. Weconstructed plasmids by fusing the LAC4 gene or engineered variantsto the secretion signal of the K.lactis killer toxin or to thesecretion signal of the Saccharomyces cerevisiae -factor. Withthese plasmids we transformed strains of the yeasts K.lactisand S.cerevisiae, respectively and tested ß-galactosidaseextracellular activity in different culture media. We achievedpartial secretion of ß-galactosidase in the culturemedium since the high molecular weight and oligomeric natureof the enzyme, among other factors, preclude full secretion.The percentage of secretion was improved by directed mutagenesisof the N-terminus of the protein. We developed several deletionmutants which helped us to propose structure–functionrelationships by comparison with the available data on the homologousEscherichia coli ß-galactosidase. The influence ofthe culture conditions on heterologous ß-galactosidasesecretion was also studied.