L. Parenicova et al., PGAE ENCODES A 4TH MEMBER OF THE ENDOPOLYGALACTURONASE GENE FAMILY FROM ASPERGILLUS-NIGER, European journal of biochemistry, 251(1-2), 1998, pp. 72-80
In the present study, the molecular and basic biochemical characteriza
tion of endopolygalacturonase E, the fourth Aspergillus niger N400 end
opolygalacturonase, is reported. The entire endopolygalacturonase E ge
ne consists of 1293 bp interrupted by three short introns (50, 50, and
59 bp, respectively) as concluded from the cDNA sequence. The deduced
amino acid sequence comprises 378 residues that include 39 N-terminal
amino acids of the prepropeptide. The calculated M-r and pI of the ma
ture protein are 35 584 and 3.6, respectively. Compared with other end
upolygalacturonases from A. niger N400, the mature protein endopolygal
acturonase E has the highest sequence identity with endopolygalacturon
ase C (77.6%) followed by endopolygalacturonase I (57.6%) and endopoly
galacturonase II (54.3%). For overproduction of endopolygalacturonase
E, an A. niger multicopy strain was used that was transformed with a p
romoter gene fusion construct that directs expression from the glycoly
tic A. niger pyruvate kinase promoter. The enzyme was purified and cha
racterized as an endopolygalacturonase based on product analysis after
polygalacturonate hydrolysis and on bond cleavage frequencies of olig
ogalacturonates of different degree of polymerisation (n = 2-7). The p
H optimum was 3.8. The K-m and V-max for polygalacturonate hydrolysis
were 2.5 +/- 0.4 mg.ml(-1) and 1.3 +/- 0.2 mu kat.mg(-1), respectively
. A subsite map was calculated by the combination of the methods of Su
ganuma et al. [Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (19
78) J. Biochem. (Tokyo) 84, 293-316] and Nitta et al. [Nitta, Y., Mizu
shima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567-576
]. This indicated that the enzyme was composed of at least five subsit
es.