Bezus Brenda, Esquivel Juan Carlos Contreras, Cavalitto Sebastián, Cavello Ivana
Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI, UNLP, CCT-La PlataCONICET), Calle 47 y 115, B1900ASH, La Plata, Provincia de Buenos Aires, Argentina.
Laboratory of Applied Glycobiotechnology, Academic Group of Food Science and Technology, School of Chemistry, Universidad Autonoma de Coahuila, Unidad Saltillo, 2528, Saltillo, Coahuila, Mexico.
Appl Biochem Biotechnol. 2025 Feb;197(2):1090-1111. doi: 10.1007/s12010-024-05069-0. Epub 2024 Oct 1.
The aim of the present work was to partially purify and characterize an Antarctic polygalacturonase and to determine the enzyme's potential in pectin extraction and vegetal maceration at 20 °C. Polygalacturonase was purified by chromatography to obtain an enzymatic preparation of specific activity 30.3 U.mg. Optimal conditions for the polygalacturonase activity were 45 °C and pH 5.0-6.0, and the activation energy for the reaction was 41.8 kJ.mol. Of the enzyme activity, 100% was retained after 3 h at 40 °C. The enzyme was remarkably stable for an hour over a wide range of pH (2.0-12.0). Polygalacturonase activity was slightly reduced in the presence of Ca, Fe, K, Mn, and Zn, whereas Hg reduced the activity by 60%, suggesting a thiol-dependent catalysis. The apparent molecular weight of the enzyme was 33 kDa. The kinetic constants evaluated against polygalacturonic acid were 0.17 mg.ml (K), 480 s (K), and 7.9 µmol.mg.min (V). The enzyme was active against different pectic substrates. Thin-layer chromatography revealed an endo-mechanism of action. Polygalacturonase digested lime pomace to aid the extraction of high-methoxylated pectin at 20 °C and increased the vegetal maceration of Capsicum annuum by 24% over the control values.
本研究的目的是对一种南极多聚半乳糖醛酸酶进行部分纯化和特性鉴定，并测定该酶在20℃下用于果胶提取和植物浸渍的潜力。通过色谱法对多聚半乳糖醛酸酶进行纯化，得到比活性为30.3 U.mg的酶制剂。多聚半乳糖醛酸酶活性的最佳条件是45℃和pH 5.0 - 6.0，反应的活化能为41.8 kJ.mol。在40℃下3小时后，酶活性保留100%。该酶在较宽的pH范围（2.0 - 12.0）内1小时内都非常稳定。在Ca、Fe、K、Mn和Zn存在的情况下，多聚半乳糖醛酸酶活性略有降低，而Hg使活性降低60%，表明是硫醇依赖性催化。该酶的表观分子量为33 kDa。以聚半乳糖醛酸为底物评估的动力学常数为0.17 mg.ml（Km）、480 s（kcat）和7.9 μmol.mg.min（Vmax）。该酶对不同的果胶底物具有活性。薄层色谱显示其作用机制为内切型。多聚半乳糖醛酸酶在20℃下消化酸橙果渣以辅助提取高甲氧基果胶，并且使辣椒的植物浸渍比对照值增加了24%。
