PGAE ENCODES A 4TH MEMBER OF THE ENDOPOLYGALACTURONASE GENE FAMILY FROM ASPERGILLUS-NIGER

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.