Characterization of the Aureobasidium pullulans α-glucuronidase expressed in Saccharomyces cerevisiae

The α-glucuronidase gene (aguA) of Aureobasidium pullulans NRRL Y-2311-1 was amplified by PCR and sequenced. Based on its deduced amino acid sequence, AguA was found to be a member of family 67 of the glycoside hydrolases. It shares greater than 60% identity and between 34% and 42% identity with fungal and with bacterial α-glucuronidases, respectively. The open reading frame lacks introns and encodes a polypeptide of 836 amino acids that contains a putative signal peptide of 15 amino acids resulting in a mature protein with a calculated molecular mass of 91.0 kDa. A construct of the aguA gene encoding an additional C-terminal hexahistidine tag was cloned on an episomal plasmid under control of the ADH2 promoter and terminator and expressed in Saccharomyces cerevisiae Y294. The heterologous α-glucuronidase was purified to homogeneity by Ni-chelation affinity chromatography, and displayed an electrophoretic mobility of 157 kDa on SDS–PAGE. Maximal activity was measured at 65 °C and at pH 5 and pH 6. The enzyme had Km values in the millimolar range for the series of substrates from aldobiouronic acid to aldopentaouronic acid, but was unable to hydrolyze an internally substituted aldopentaouronic acid.