alpha-Galactosidase A from Pseudomonas fluorescens subsp. cellulosa: cloning, high level expression and its role in galactomannan hydrolysis.
FEMS Microbiol Lett
; 192(2): 197-203, 2000 Nov 15.
Article
en En
| MEDLINE
| ID: mdl-11064195
A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, constructed in lambda ZAPII, was screened for alpha-D-galactosidase activity. The DNA inserts from six galactosidase-positive clones were rescued into plasmids. Restriction digestion and Southern analysis revealed that each of the plasmids contained a common DNA sequence. The sequence of the Pseudomonas DNA in one of the plasmids revealed a single open reading frame (aga27A) of 1215 bp encoding a protein of M(r) 45900, designated alpha-galactosidase 27A (Aga27A). Aga27A exhibited extensive sequence identity with alpha-galactosidases in glycoside hydrolase 27, and appeared to be a single domain protein. The recombinant alpha-galactosidase was expressed at high levels in Escherichia coli and the biophysical properties and substrate specificity of the enzyme were evaluated. The data showed that Aga27A was a mesophilic neutral acting non-specific alpha-galactosidase. Both P. fluorescens subsp. cellulosa mannanase A (ManA) and Aga27A hydrolyse the polymeric substrate, carob galactomannan. Sequential hydrolysis with AgaA followed by ManA, or ManA followed by AgaA enhanced product release. The positive effects of sequential hydrolysis are discussed.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Pseudomonas fluorescens
/
Alfa-Galactosidasa
/
Mananos
Idioma:
En
Revista:
FEMS Microbiol Lett
Año:
2000
Tipo del documento:
Article
Pais de publicación:
Reino Unido