New secretory strategies for Kluyveromyces lactis β-galactosidase

Abstract

We examined several strategies for the secretion of Kluyveromyces lactis beta-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed plasmids by fusing the LAC4 gene or engineered variants to the secretion signal of the K.lactis killer toxin or to the secretion signal of the Saccharomyces cerevisiae alpha-factor. With these plasmids we transformed strains of the yeasts K.lactis and S.cerevisiae, respectively and tested beta-galactosidase extracellular activity in different culture media. We achieved partial secretion of beta-galactosidase in the culture medium since the high molecular weight and oligomeric nature of the enzyme, among other factors, preclude full secretion. The percentage of secretion was improved by directed mutagenesis of the N-terminus of the protein. We developed several deletion mutants which helped us to propose structure-function relationships by comparison with the available data on the homologous Escherichia coli beta-galactosidase. The influence of the culture conditions on heterologous beta-galactosidase secretion was also studied.

Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase

“…Such mechanism has not been reported for the KL-b-Gal and, even when we have observed that it is important for protein activity (Becerra et al, 2001), no function has been attributed to this region yet. Domain 1 is very similar in all three proteins.…”

Metabolic engineering ofSaccharomyces cerevisiaefor lactose/whey fermentation

“…69,70 Recombinant S. cerevisiae strains that are able of secreting K. lactis β-galactosidase have also been constructed. 71,72 These approaches were used with the aim of developing a system for K. lactis β-galactosidase production and not for lactose bioconversion to ethanol. More recently, and with the same aim, a hybrid protein between K. lactis and A. niger β-galactosidase was constructed that increased the yield of the recombinant protein released to the growth medium.…”

Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

“…Therefore, the corresponding constructions were made by PCR amplification of the selected domains, restriction and ligation. Since previous work had demonstrated that, in K. lactis , mutant β-galactosidases with large deletions in the N-terminal region were inactive [3] we designed two hybrid proteins between K. lactis and A. niger β-galactosidases interchanging the C-terminal region. Constructions were made in the pSPGK1 plasmid [26] and were called pSPGK1-LAC4-LACA- Bam HI and pSPGK1-LAC4-LACA- Kpn I.…”

“…In contrast, yeast β-galactosidase optimum pH is near neutral, consequently making it suitable for saccharifying milk and sweet whey. However, the production and industrial use of this intracellular enzyme are problematic due to the high cost associated with its extraction from the cells and to the low yields obtained as a result of its instability [3]. …”