Cloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris

The first gene (α-gal1) encoding an extracellular α-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The α-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal α-galactosidases belonging to glycosyl hydrolase family 27. The α-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant α-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70°C, pH 4.5, and lost no activity over 10 days at 50°C. α-Gal1 followed Michaelis-Menten kinetics (V_max of 240.3 μM/min/mg, K_m of 0.294 mM) and was inhibited competitively by galactose (K_m ^obs of 0.57 mM, K_i of 2.77 mM). The recombinant T. emersonii α-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the α-galactosidic linkage. Owing to its substrate preference and noteworthy stability, α-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.