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.

中文翻译：

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通过鉴别底物降解分析比较表征曲霉果胶裂解酶

真菌基因组通常包含几个基因拷贝，这些基因编码具有相似活性的碳水化合物活性酶。拷贝通常具有轻微的序列变异性，并且有人提出多基因性代表酶的不同反应最佳版本。副本是否代表酶的底物攻击能力的差异很少被考虑。曲霉属物种的基因组编码几种果胶裂解酶 (EC 4.2.2.10)，它们都属于 CAZy 数据库中的多糖裂解酶亚家族 PL1_4。这些酶在序列同一性和系统发育方面不同，并且在它们的同源模型中在活性位点附近表现出结构差异。这些酶通过消除 α-(1, 4) 高半乳糖醛酸中的糖苷键，优先选择甲基酯化的半乳糖醛酸残基之间的键。本研究检查了由相同曲霉属物种编码的四种不同的果胶裂解酶（PelB、PelC、PelD 和 PelF）。（即 A. luchuensis），并进一步比较了来自两种相关曲霉属的两种 PelA 果胶裂解酶。(A. aculeatus 和 A.tubingensis)。我们报告了酶对苹果果胶、柑橘果胶和甜菜果胶的作用的系统发育、酶动力学和酶降解谱。所有果胶裂解酶对苹果果胶的反应率最高[甲氧基化度（DM）69%，乙酰化度（DAc）2%]，对甜菜果胶反应率最低（DM 56%，DAc 19%）。pH 5–5.5 下的活性比较产生以下排名：PelB > PelA > PelD > PelF > PelC。通过液相色谱与质谱 (LC-MS) 检测分析反应过程中同型半乳糖醛酸寡聚体产物谱的演变。该分析揭示了产物谱的细微差异，表明酶之间存在不同的底物降解偏好，特别是在乙酰取代方面。因此，LC-MS 产品谱分析揭示了多基因似乎为真菌提供了额外的底物降解多功能性。此外，该产品分析代表了一种在功能上比较果胶降解酶的新方法，这有助于解释碳水化合物活性酶不同副本的结构-功能关系和反应特性。