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

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 fu...

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Bibliographic Details
Published in:Enzyme and microbial technology Vol. 38; no. 5; pp. 649 - 656
Main Authors: de Wet, B.J.M., van Zyl, W.H., Prior, B.A.
Format: Journal Article
Language:English
Published: Amsterdam Elsevier Inc 02-03-2006
Elsevier Science
Subjects:
Aureobasidium pullulans
Biological and medical sciences
Biotechnology
Enzyme
Fundamental and applied biological sciences. Psychology
Hemicellulose
Saccharomyces cerevisiae
Yeast-like fungus
α-Glucuronidase
Hemicellulose
Enzyme
α-Glucuronidase
Yeast-like fungus
Yeast
Gene expression
Fungi
Characterization
Aureobasidium pullulans
Glycosidases
Hydrolases
α-Glucosiduronase
Ascomycetes
Fungi Imperfecti
Recombinant protein
O-Glycosidases
Saccharomyces cerevisiae
Thallophyta
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Summary: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 K m 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.
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ISSN:0141-0229
1879-0909
DOI:10.1016/j.enzmictec.2005.07.018