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

Abstract

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 (pNPalphaG), 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 pNPalphaG in liquid media.

Potential of Cometabolic Transformation of Polysaccharides and Lignin in Lignocellulose by Soil Actinobacteria

“…Although cellulose decomposition was found to be more rapid in decomposing plant litter than that of hemicelluloses, the utilisation of xylose and arabinose-containing hemicelluloses was relatively fast [51]. Although the production of 1,4-β-mannosidase, 1,4-β-galactosidase and 1,4-α-arabinosidase by individual taxa of Actinobacteria was reported previously [54]–[56], we show that they belong to a set of enzymes that are simultaneously produced by saprotrophic taxa during their growth on lignocellulose. The enzyme 1,4-β-glucuronidase, which is involved in the degradation of pectins, was not produced by the tested strains.…”

Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt–Protease Tolerance and Transglycosylation Activity

“…To date, the number of GH 27 α-galactosidases from bacteria is far more than that from eukaryota (). However, we found that only three GH 27 α-galactosidases from bacteria have been characterized. − …”

Galactomannan Degrading Enzymes from the Mannan Utilization Gene Cluster of Alkaliphilic Bacillus sp. N16-5 and Their Synergy on Galactomannan Degradation

“…As the development of sequencing technology continues, more and more homologues of GH27 α-galactosidases have been found from bacterial genomes, which are much more than those from eukaryote genomes (). However, only a few of them have been characterized so far, including α-galactosidases from Pseudomonas fluorescens, Saccharopolyspora erythraea, Clostridium josui, and Pontibacter sp. ,− Among those enzymes, AgaA from Pseudomonas fluorescens is the only one whose synergistic interaction with mannanase has been studied . It has been reported that the yield of galactose and reducing sugar increased when AgaA acted on galactomannan in combination with the GH 26 family β-mannanase.…”