Purification, cloning, and properties of alpha-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 (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.
Post
David A
DA
Abbott Laboratories, Fermentation Microbiology Research and Development, Building NCF3, 1400 Sheridan Road, North Chicago, IL 60064-6264, USA. david.post@abbott.com
Luebke
Vicki E
VE
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002863:Chromogenic Compounds / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005690:Galactose / Q000378:metabolism; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016930:Saccharopolyspora / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D004269:DNA, Bacterial; D005960:Glucosides; C019502:4-nitrophenyl alpha-glucoside; D008553:Melibiose; D000519:alpha-Galactosidase; D005690:Galactose