Cloning and Expression of a Thermostable α-Galactosidase from the Thermophilic Fungus Talaromyces emersonii in the Methylotrophic Yeast Pichia pastoris

• Published in: Journal of microbiology and biotechnology Vol. 20; no. 12; pp. 1653 - 1663
• Main Authors: Janika Simila, Anita Gernig, Patrick Murray, Sara Fernandes, Maria G. Tuohy
• Format: Journal Article
• Language: Korean
• Published: 한국미생물생명공학회 30.12.2010
• Subjects: overexpression; Pichia pastoris; Talaromyces emersonii; thermostability; α-Galactosidase
• ISSN: 1017-7825

The first gene (α-gall) encoding an extracellular α-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The α-gall 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-gall gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant α-Gall was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using His-binding 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-Gall followed Michaelis-Menten kinetics (Vmax of 240.3 μM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km(obs) of 0.57 mM, Ki 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-Gall is of particular interest for possible biotechnological applications involving the processing of plant materials.