Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis

Abstract

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly( 1,4-a-~-ga~acturonide)galacturonohydrolasel that cleaves monomer units from the non-reducing end of the substrate molecule.K,,, and V,,,, for polygalacturonic acid hydrolysis were 3.2 mg mlV' and 3.1 mg ml-' and 255 U mg I and 262U mg-' for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2 -7), the Michaelis constant (K,,,) decreased with increasing chain length (n). The V,;,, value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid ( K , = 0.3 mM) as well as by reduced digalacturonate (K, = 0.4 mM).The exopolygalacturonase gene (pgax) was cloned by reverse genetics and shows only 13 % overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pguX-coding region was used to achieve high level production of exopol ygalacturonase under conditions where no endopolygalacturonases were produced.

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C

“…A. E. Benen, unpublished results). Then, the cleavage mixture was adjusted to pH 4.2 with acetic acid and incubated for 48 h at 30 8C with 1 mg of exopolygalacturonase isolated from Aspergillus tubingensis [13] to hydrolyse the saturated oligogalacturonates to GalpA. The remaining mixture of GalpA and D4,5-unsaturated oligomers, with degree of polymerization 3±10, was converted into the acidic form by the subsequent addition of three times 40 g of 5.1 meq´g ±1 Dowex 50W-X8 (H + ) (Bio-Rad).…”

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

“…The K m value of 2.4 mg/mL has been reported for an endo-PG from A. niger (Parenicova et al 1998). An exo-PG from A. tubingensis has K m value of 3.2 mg/mL (Kester et al 1996). The K m values of most of the microbial PGs are in the range of 0.1-5.0 (Polizeli et al 1991;Zhang et al 1999;Niture et al 2001;Singh and Rao 2002;Thakur et al 2010a, b;Yadav et al 2012;Martins et al 2013;Chen et al 2014).…”

Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates

“…Oligogalacturonates of defined length [degree of polymerization (DP) 2-6] were prepared as described previously [11]. The oligomers (50 mg) were dissolved in 5 ml of methanol\0.02 M HCl.…”