Polizeli M de L, Jorge J A, Terenzi H F
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
J Gen Microbiol. 1991 Aug;137(8):1815-23. doi: 10.1099/00221287-137-8-1815.
The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].
利用高产突变体exo - 1在粗糙脉孢菌中研究了果胶酶的产生，该突变体合成和分泌的酶比野生型多五到六倍。聚半乳糖醛酸酶、果胶裂解酶和果胶酸裂解酶由果胶诱导产生，且这种诱导作用受葡萄糖抑制。半乳糖诱导聚半乳糖醛酸酶的效率比果胶高四倍；相比之下，裂解酶的活性不受半乳糖影响。半乳糖对聚半乳糖醛酸酶的诱导作用不受葡萄糖抑制。通过离子交换色谱法分离细胞外果胶酶。果胶酸和果胶裂解酶洗脱成三个主要部分，均含有这两种活性；聚半乳糖醛酸酶以单一的对称峰洗脱，显然不含其他蛋白质污染物，纯化了56倍。纯化的聚半乳糖醛酸酶是一种单体糖蛋白（碳水化合物含量为38%），表观分子量分别为36.6 - 37.0 kDa（分别通过Sephadex G - 100和尿素 - SDS - PAGE测定）。该酶主要水解聚果胶酸。果胶也能被水解，但水解速率仅为聚果胶酸的7%。聚果胶酸水解的Km和Vmax分别为5.0 mg/ml和357 μmol min-1（mg蛋白）-1。最适温度和pH分别为45℃和6.0。纯化的聚半乳糖醛酸酶使聚果胶酸钠溶液的粘度降低了50%，还原糖基团增加了7%。初始反应时间的水解产物由寡聚半乳糖醛酸组成，未检测到单体。因此，纯化的粗糙脉孢菌酶被归类为内切聚半乳糖醛酸酶[聚（1,4 - α - D - 半乳糖醛酸）聚糖水解酶；EC 3.2.1.15]。
