Pectin methyl esterase (PME) activity was detected by coupled assays using the pectate lyase family 1 enzyme from Aspergillus niger (AnPL1, Megazyme: E-PCLYAN), an enzyme only capable of cleaving unmethylated galacturonan. Assays of 200 µL contained 0.035% pectin (various types supplied from Megazyme, K-PECID) or 1% poly-methylgalacturonan (Biosynth) in 100-mM potassium phosphate buffer at pH 7, 225 U of AnPL1 (Megazyme), and were supplemented with or without 0.5 nmol of PvCE15 or 1:100 of the PME NovoShape from Aspergillus aculeatus (University of Reading, UK). Pectate lyase product formation was observed by measuring the absorbance at 235 nm using a UV-Star UV transparent 96-well microplate (Greiner Bio-One) as described previously (46 ).
Seveso A., Mazurkewich S., Banerjee S., Poulsen J.C., Lo Leggio L, & Larsbrink J. (2024). Polysaccharide utilization loci from Bacteroidota encode CE15 enzymes with possible roles in cleaving pectin-lignin bonds. Applied and Environmental Microbiology, 90(1), e01768-23.
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