Expression and characterization of an endo-β-1,6-galactanase from <i>Arabidopsis thaliana</i>

To date, only fungal and bacterial endo-β-1,6-galactanases from glycoside hydrolase subfamilies GH5_16 and GH30_5 have been characterized. β-1,6-galactan chains are primarily structural components of type II arabinogalactans present in plant and algal cell walls. The ATIYA1 gene, originating from Arabidopsis thaliana, was cloned and expressed in Komagataella phaffii. The precursor protein consists of an N-terminal signal peptide, a glycosyl hydrolase domain, and a C-terminal ricin B-like module. The recombinant enzyme was able to hydrolyze β-1,6-linked galactan chains. Optimal conditions for enzymatic activity were observed at pH 5 and 30°C. The ATIYA1 gene encodes the first endo-β-1,6-galactanase identified from plants and represents the first characterized member within the GH5_11 subfamily.