Abstract
Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes’ action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5–5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes.
Highlights
Pectin lyases (EC 4.2.2.10) catalyze cleavage of the α-(1,4) glycosidic linkages between methyl-esterified galacturonic acid (GalA) units in the homogalacturonan backbone of pectin through a β-elimination reaction (Figure 1)
Differences in pH optima (Supplementary Table S1) and expression inducers may partially explain the large gene families observed for several different classes of pectinolytic enzymes (Martens-Uzunova and Schaap, 2009; Andersen et al, 2012), but there is a need for rigorous biochemical characterization of the gene products including specificity toward pectins of different origin and in particular determination of product profiles in order to fully understand this multigenecity
The genomes of the A. tubingensis and A. luchuensis strains from which pectin lyases were sourced in the current work contain pelE genes (UniProt entries A0A100I604 and G7XZW9, respectively), but no reports on PelE expression or activity of the pectin lyases from these strains exist
Summary
Pectin lyases (EC 4.2.2.10) catalyze cleavage of the α-(1,4) glycosidic linkages between methyl-esterified galacturonic acid (GalA) units in the homogalacturonan backbone of pectin through a β-elimination reaction (Figure 1). Pectin lyases prefer linkages between methyl-esterified (methoxylated) GalA units, and catalyze bond cleavage at sites where only the GalA moiety in the +1 subsite is methoxylated, albeit with lower specific activity (Mutenda et al, 2002; Van Alebeek et al, 2002). Most commercial pectinases originate from filamentous fungi, notably several derive from the Aspergillus section Nigri such as Aspergillus niger and A. aculeatus (Sandri et al, 2011) These fungi are specialized in secreting a broad spectrum of enzymes, including pectin-degrading enzymes, which can degrade and utilize the surrounding biomass (Benoit et al, 2012; de Vries et al, 2017; Kowalczyk et al, 2017)
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