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 nidulans 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 of the recombinant pectate lyase A resulted in the identification of two enzyme 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 specific activities. The N-glycosylation free pectate lyase A was further characterized with respect to product formation on polygalacturonic acid (alpha-1,4 linked D-galacturonic acid) and mode of action on oligogalacturonides of degree of polymerization 2-8. The bond cleavage frequencies for tetra-, penta-, and hexagalacturonides were studied as a function of [CaCl(2)]. The bond cleavage frequencies changed in a [CaCl(2)]-dependent way for penta- and hexagalacturonide. Kinetic studies using tetra- and hexagalacturonide revealed a strong sigmoidal [CaCl(2)]-dependent relation. The role of Ca(2+) ions in substrate binding is discussed.
Benen
J A
JA
Section Molecular Genetics of Industrial Microorganisms, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. jac.benen@algemeen.mgim.wau.nl
Kester
H C
HC
Parenicová
L
L
Visser
J
J
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics; D006031:Glycosylation; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase