PRIMARY STRUCTURE AND CHARACTERIZATION OF AN EXOPOLYGALACTURONASE FROM ASPERGILLUS-TUBINGENSIS

From the culture fluid of the hyphal fungus Aspergillus tubingensis, a n 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. T he enzyme has been characterized as an exopolygalacturonase [poly(1,4- alpha-D-galacturonide)ga lacturonohydrolase] that cleaves monomer unit s from the non-reducing end of the substrate molecule. K-m and V-max f or polygalacturonic acid hydrolysis were 3.2 mg ml(-1) and 3.1 mg ml(- 1) and 255 U mg(-1) and 262 U mg(-1) for the wild-type and recombinant enzymes, respectively, The kinetic data of exopolygalacturonase on ol igogalacturonates of different degree of polymerization (2-7) were int erpreted in terms of a subsite model to obtain more insight into catal ysis and substrate binding. On oligogalacturonates of different degree s of polymerization (2-7), the Michaelis constant (K-m) decreased with increasing chain length (n). The V-max value increased with chain len gth up to n = 4, then reached a plateau value. The enzyme was competit ively inhibited by galacturonic acid (K-i = 0.3 mM) as well as by redu ced digalacturonate (K-i = 0.4 mM). The exopolygalacturonase gene (pga X) was cloned by reverse genetics and shows only 13% overall amino aci d sequence identity with A. niger endopolygalacturonases. The exopolyg alacturonase is most related to plant polygalacturonases. Only four sm all stretches of amino acids are conserved between all known endogalac turonases and exopolygalacturonases. Expression of the pgaX gene is in ducible with galacturonic acid and is subject to catabolite repression . A fusion between the promoter of the A. niger glycolytic gene encodi ng pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no en dopolygalacturonases were produced.