Molecular cloning and expression in Pichia pastoris of a Irpex lacteus exo-beta-(1-3)-galactanase gene

A gene encoding exo-β-(1→3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-β-(1→3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed β-(1→3)-galactooligosaccharides, as did other exo-β-(1→3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as β-(1→3)-galactan. Product analysis revealed that the enzyme released β-(1→6)-galactobiose, β-(1→6)-galactotriose, and α-L-arabinofuranosyl-(1→3)-β-galactosyl-(1→6)-galactose together with Gal from β-(1→3)-galactans attached with and without β-(1→6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-β-(1→3)-galactanase from I. lacteus efficiently hydrolyzes β-(1→3)-galactan main chains of AGPs by bypassing β-(1→6)-galactosyl side chains.