Characterization of Aspergillus niger pectate lyase A

The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99.3) was cloned from a chromosomal lambda (EMBL4) library using the Aspergillus nidu lans pectate lyase encoding gene [Dean, R. A., and Timberlake, W. E. (1989) Plant Cell 1, 275-284] as a probe. The plyA gene was overexpressed using a promoter fusion with the A, niger pyruvate kinase promoter. Purification o f the recombinant pectate lyase A resulted in the identification of two enz yme forms of which one appeared to be N-glycosylated and the other appeared to be free of N-glycosylation. The two enzyme forms showed identical speci fic activities. The N-glycosylation free pectate lyase A was further charac terized with respect to product formation on polygalacturonic acid (alpha - 1,4 linked D-galacturonic acid) and mode of action on oligogalacturonides o f degree of polymerization 2-8. The bond cleavage frequencies for tetra-, p enta-, and hexagalacturonides were studied as a function of [CaCl2]. The bo nd cleavage frequencies changed in a [CaCl2]-dependent way for penta- and h exagalacturonide. Kinetic studies using tetra- and hexagalacturonide reveal ed a strong sigmoidal [CaCl2]-dependent relation. The role of Ca2+ ions in substrate binding is discussed.