Purification, cloning, and properties of β-galactosidase from Saccharopolyspora erythraea and its use as a reporter system

An alpha-galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K-m for p-nitrophenyl-alpha-D-glucopyranoside (pNP alpha G), K-m for melibiose and the V-max are similar to those of other studied alpha-galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related alpha-galactosidase enzymes. This fragment was used as a probe to clone the alpha-galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique alpha-galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The alpha-galactosidase uses the substrates 5-bromo- 4-chloro-3-indoyl-alpha-D-galactosidase (X-alpha-gal) on agar media and pNP alpha G in liquid media.