Characterization of pectate lyase A from Aspergillus niger

J.A.E. Benen, L. Parenicova, H.C.M. Kester, J. Visser

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Abstract

The Aspergillus niger plyA gene encoding pectate lyase A (EC 4.2.99.3) was cloned from a chromosomal 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 (-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 [CaCl2]. The bond cleavage frequencies changed in a [CaCl2]-dependent way for penta- and hexagalacturonide. Kinetic studies using tetra- and hexagalacturonide revealed a strong sigmoidal [CaCl2]-dependent relation. The role of Ca2 ions in substrate binding is discussed
Original languageEnglish
Pages (from-to)15563-15569
JournalBiochemistry
Volume39
DOIs
Publication statusPublished - 2001

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