Open Accessα‐Galactosidase A from Pseudomonas fluorescens subsp. cellulosa : cloning, high level expression and its role in galactomannan hydrolysis
Author(s) -
Halstead Jonathan R.,
Fransen Maikel P.,
Eberhart Ruth Y.,
Park Alison J.,
Gilbert Harry J.,
Hazlewood Geoffrey P.
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09382.x
Subject(s) - pseudomonas fluorescens , cloning (programming) , microbiology and biotechnology , galactomannan , biology , hydrolysis , biochemistry , chemistry , bacteria , genetics , computer science , polysaccharide , programming language
A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, constructed in λ ZAPII, was screened for α‐ 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 45 900, designated α‐galactosidase 27A (Aga27A). Aga27A exhibited extensive sequence identity with α‐galactosidases in glycoside hydrolase 27, and appeared to be a single domain protein. The recombinant α‐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 α‐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.