Hcm. Kester et al., PRIMARY STRUCTURE AND CHARACTERIZATION OF AN EXOPOLYGALACTURONASE FROM ASPERGILLUS-TUBINGENSIS, European journal of biochemistry, 240(3), 1996, pp. 738-746
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.