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- Title
Purification, cloning, and properties of a-galactosidase fromSaccharopolyspora erythraeaand its use as a reporter system.
- Authors
Post, David A.; Luebke, Vicki E.
- Abstract
An a-galactosidase from the erythromycin-producing bacteriumSaccharopolyspora erythraeawas purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum,Km forp-nitrophenyl-a-d-glucopyranoside (pNPaG),Km for melibiose and theVmax are similar to those of other studied a-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 a-galactosidase enzymes. This fragment was used as a probe to clone the a-galactosidase gene, designatedmelA, from aS. erythraealambda phage chromosomal library.S. erythraeaappears to possess an unique a-galactosidase enzyme, encoded bymelA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product ofmelAas a reporter enzyme inS. erythraeahas been demonstrated. The a-galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl-a-d-galactosidase (X-a-gal) on agar media and pNPaG in liquid media.
- Publication
Applied Microbiology & Biotechnology, 2005, Vol 67, Issue 1, p91
- ISSN
0175-7598
- Publication type
Academic Journal
- DOI
10.1007/s00253-004-1764-6