Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
Fungal Biol. 2014 May-Jun;118(5-6):507-15. doi: 10.1016/j.funbio.2014.04.002. Epub 2014 Apr 13.
Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a 'smooth' region and a 'hairy' region. The 'smooth' region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The 'hairy' region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40% the viscosity of pectin with a degree of esterification ≥85%. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8% esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.
木质纤维素是植物细胞壁的主要成分，也是可再生有机物质的重要来源。木质纤维素的成分之一是果胶。果胶由两个基本结构组成：“光滑”区和“毛状”区。“光滑”区（同型半乳糖醛酸聚糖）是由带有α-(1→4)键的半乳糖醛酸残基组成的线性聚合物，被甲基和乙酰基取代。“毛状”区更复杂，包含木半乳聚糖和鼠李半乳糖醛酸 I 和 II。降解果胶的酶（果胶酶）之一是果胶裂解酶（EC 4.2.2.10）。该酶作用于高度酯化的同型半乳糖醛酸聚糖，通过β消除作用催化甲氧基化半乳糖醛酸残基之间的α-(1→4)糖苷键断裂，形成 4,5-不饱和产物。在这项工作中，从青霉属紫色菌中测序了果胶裂解酶的基因和 cDNA，并在巴斯德毕赤酵母中表达了 cDNA。该基因长 1334bp，有三个内含子，编码 376 个氨基酸残基的蛋白质。重组酶被纯化至均一性并进行了表征。SDS-PAGE 测定果胶裂解酶的分子量为 45kDa。它对高度酯化的果胶具有活性，并使酯化度≥85%的果胶的粘度降低 40%。该酶对聚半乳糖醛酸和 8%酯化的柑橘果胶没有活性。重组酶的最适 pH 和温度分别为 6.0 和 50°C，在 50°C 下暴露 3 小时仍保持稳定。纯化的果胶裂解酶可用于生物技术应用，如食品工业，其中应避免果胶降解过程中甲醇的释放。
