In this study, a novel pectate lyase (ApPel1) was identified and characterized from Aspergillus parasiticus. The ApPel1 hydrolysed oligogalacturonides (OGs) effectively and produced 4,5-unsaturated OGs from low-methoxyl (LM) pectin, with DP 2 to DP 5 as the major products. Furthermore, the multiple sequence alignments, structure model and phylogenetic analyses of the ApPel1 indicated that its catalytic active sites were highly conserved with other pectin lyases (PLs) and the Ca2+ binding amino acid residues are different compared with pectate lyases (Pels). N187D, N191D and N187D/N191D mutants were constructed to test for both Ca2+ binding properties and the effects on catalytic ability. The three mutations sharply decreased the activity of ApPel1 and Ca2+ tolerance, indicating that the Ca2+ binding amino acid residues are different from the other Pels. Based on the sequence and structure comparison between PLs and Pels, and mutation analysis, the ApPel1 may be direct evolution from PLs. Thus, this enzyme has potential for use in producing unsaturated OGs for biological activity study, and contributes to an improved understanding of the evolutionary relationships between PLs and Pels.
Keywords: Evolutionary analysis; Pectate lyase; Site-directed mutation; Unsaturated oligogalacturonides.
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