aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid

Abstract

An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

FIG. 1

SDS-PAGE of the different steps in the purification of α-glucuronidase. Lane 1, low-molecular-mass standard proteins; lane 2, starting material; lane 3, 45% ammonium sulfate precipitate; lane 4, Phenyl Sepharose FF pool; lane 5, Q-Sepharose pool; lane 6, Superdex 200 PG pool; lane 7, Mono Q pool; lane 8, Butyl Sepharose pool; lane 9, Poros 10 HQ pool.

FIG. 2

Restriction map of the insert containing the aguA gene (arrow) which is present in the functional construct pIM3212.

FIG. 3

Nucleotide sequence of aguA and derived amino acid sequence. The signal peptide (lowercase letters), putative (boldface roman letters) and confirmed (boldface italics) N-glycosylation sites, and the determined amino acid sequences (underlined) are indicated.

FIG. 4

Alignment of the amino acid sequences of α-glucuronidase from A. tubingensis, T. reesei, and T. maritima. Identical amino acids are depicted below the amino acid sequences.

FIG. 5

Northern analysis of the induction of aguA on different carbon sources. The top panel was probed with the internal 2-kb SalI fragment of aguA, and the bottom panel was probed with a 700-bp EcoRI fragment from the A. niger 18S ribosomal DNA and served as a loading control. Lane 1, mycelium from the preculture on fructose; other lanes, mycelium transferred to the following carbon sources: lane 2, 1% glucose; lane 3, 1% fructose; lane 4, 1% xylose; lane 5, 1% arabinose; lane 6, 1% glycerol; lane 7, 1% glucuronic acid; lane 8, 0.2% xylobiose; lane 9, 0.5% birchwood xylan; lane 10, 1% xylose–0.2% glucose; lane 11, 1% xylose–1% glucose; lane 12, 1% xylose–2% glucose; lane 13, 0.5% birchwood xylan–1% glucose; lane 14, 1% glucose–1% glucuronic acid; lane 15, 1% fructose–1% glucuronic acid; lane 16, 1% xylose–1% glucuronic acid; lane 17, 1% arabinose–1% glucuronic acid; and lane 18, 1% glycerol–1% glucuronic acid.