血红密孔菌外切多聚半乳糖醛酸酶的纯化与特性分析
Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus.
作者信息
Quiroga Emma N, Sgariglia Melina A, Molina César F, Sampietro Diego A, Soberón José R, Vattuone Marta A
机构信息
Cátedra de Fitoquímica, Instituto de Estudios Vegetales Dr. A.R. Sampietro, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, (4000) San Miguel de Tucumán, Argentina.
出版信息
Mycol Res. 2009 Dec;113(Pt 12):1404-10. doi: 10.1016/j.mycres.2009.09.007. Epub 2009 Sep 23.
The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.
本研究描述了血红密孔菌在柑橘果胶上生长时产生的一种新型胞外多聚半乳糖醛酸酶PGase I的纯化及特性。与蔗糖和乳糖相比，该底物能提高酶的产量。通过薄层层析（TLC）和间苯二酚 - 硫酸染色测定，PGase I是一种胞外酶，其主要水解产物为半乳糖醛酸。它水解二半乳糖醛酸的能力表明PGase I是一种外切多聚半乳糖醛酸酶。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）显示PGase I是一种N - 糖基化单体。该酶分子量为42kDa，最适pH为4.8，在pH 3.8至8.0之间稳定。观察到最适温度为50 - 60摄氏度，在80摄氏度时仍保留一些酶活性。其活化能为5.352卡/摩尔（-1）。PGase I对聚半乳糖醛酸（PGA）的亲和力高于柑橘果胶（Km分别为0.55±0.02和0.72±0.02mg/ml）。因此，PGase I是一种外切多聚半乳糖醛酸酶，酶学委员会编号为3.2.1.82。
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