Purification, characterization and gene analysis of a new α-glucosidase from shiraia sp SUPER-H168

A new alpha-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified alpha-glucosidase were 4.5 and 60 A degrees C, respectively, using p-nitrophenyl-alpha-glucopyranoside (alpha-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe2+, Cu2+, and Ag+ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K (m), V (max), and k (cat)/K (m) of the alpha-glucosidase were 0.52 mM, 3.76 U mg(-1), and 1.3 x 10(4) L s(-1) mol(-1), respectively. K (m) with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The alpha-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The alpha-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 alpha-glucosidase is an ascomycetes alpha-glucosidase. This is the first report of alpha-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and alpha-pNPG, transglycosylation and conversion activity of maltose into trehalose.