Molecular Cloning and Expression in Pichia pastoris of a Irpex lacteus Exo-β-(1 → 3)-galactanase Gene

A gene encoding exo-beta-(1 -> 3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(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 beta-(1 -> 3)-galactooligosaccharides, as did other exo-beta-(1 -> 3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1 -> 3)-galactan. Product analysis revealed that the enzyme released beta-(1 -> 6)-galactobiose, beta-(1 -> 6)-galactotriose, and alpha-L-arabinofuranosyl beta-(1 -> 3)-beta-galactosyl-beta-(1 -> 6)-galactose together with Gal from beta-(1 -> 3)-galactans attached with and without beta-(1 -> 6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1 -> 3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1 -> 3)galactan main chains of AGPs by bypassing beta-(1 -> 6)-galactosyl side chains.