AGUA, THE GENE ENCODING AN EXTRACELLULAR ALPHA-GLUCURONIDASE FROM ASPERGILLUS-TUBINGENSIS, IS SPECIFICALLY INDUCED ON XYLOSE AND NOT ON GLUCURONIC-ACID

An extracellular alpha-glucuronidase was purified and characterized fr om a commercial Aspergillus preparation and from culture filtrate of A spergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresi s 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 op timum for the enzyme is between 4.5 and 6.0, and the temperature optim um is 70 degrees C. The a-glucuronidase is active mainly on small subs tituted xylo-oligomers but is also able to release a small amount of 4 -O-methylglucuronic acid from birchwood xylan. The enzyme acts synergi stically with endoxylanases and beta-xylosidase in the hydrolysis of x ylan. The enzyme is N glycosylated and contains 14 putative N-glycosyl ation sites. The gene encoding this a-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 aci ds, containing a eukaryotic signal sequence of 20 amino acids. The mat ure protein has a predicted molecular mass of 91,790 Da and a calculat ed pi of 5.13. Multiple copies of the gene were introduced in A. tubin gensis, and expression was studied in a highly overproducing transform ant. The aguA gene was expressed on xylose, xylobiose, and xylan, simi larly to genes encoding endoxylanases, suggesting a coordinate regulat ion of expression of xylanases and a-glucuronidase. Glucuronic acid di d not induce the expression of aguA and also did not modulate the expr ession on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.