alpha-Galactosidase A from Pseudomonas fluorescens subsp cellulosa: cloning, high level expression and its role in galactomannan hydrolysis

A library of Pseudomonas fluorescens subsp. cellulosa genomic DNA, construc ted in lambda ZAPII, was screened for alpha -D-galactosidase activity. The DNA inserts from six galactosidase-positive clones were rescued into plasmi ds. Restriction digestion and Southern analysis revealed that each of the p lasmids contained a common DNA sequence. The sequence of the Pseudomonas DN A in one of the plasmids revealed a single open reading frame (aga27A) of 1 215 bp encoding a protein of M-r 45 900, designated alpha -galactosidase 27 A (Aga27A). Aga27A exhibited extensive sequence identity with alpha -galact osidases in glycoside hydrolase 27, and appeared to be a single domain prot ein. The recombinant alpha -galactosidase was expressed at high levels in E scherichia coli and the biophysical properties and substrate specificity of the enzyme were evaluated. The data showed that Aga27A was a mesophilic ne utral 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 M anA followed by AgaA enhanced product release. The positive effects of sequ ential hydrolysis are discussed. (C) 2000 Federation of European Microbiolo gical Societies. Published by Elsevier Science B.V. All rights reserved.