Celestino S Maria C, Maria de Freitas S, Javier Medrano F, Valle de Sousa M, Filho E Ximenes Ferreira
Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, CEP 70910-900, Brazil.
J Biotechnol. 2006 May 3;123(1):33-42. doi: 10.1016/j.jbiotec.2005.10.024. Epub 2005 Dec 7.
An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.
通过超滤以及凝胶过滤和离子交换色谱法相结合的方法，从嗜热真菌纳尼阿顶孢霉的液态培养物中纯化出一种细胞外果胶酶（PECI），使其达到表观均一性。通过SDS - PAGE和质谱法测定，PECI的分子量分别为35,500 Da和30,749 Da。它在60℃和pH 8.0时活性更高，在50℃时表现出高稳定性，半衰期为7天。然而，在60℃和70℃时，PECI的稳定性要低得多，半衰期分别约为20分钟和3分钟。还通过荧光光谱和圆二色光谱研究了纯化的PECI的热稳定性。荧光显示在45℃至70℃之间观察到解折叠转变区域。在70℃时发现稳定性大幅下降。在pH值介于5.0和9.0之间进行的圆二色性测量显示转变温度（T(m)）范围为50 - 55℃。对这些结果的热力学分析表明EPGI是一种热稳定蛋白，在pH 8.0和9.0时分别表现出最大稳定性（ΔG(25)）为22.65和19.19 kcal/mol。对柑橘类水果果胶的表观K(m)值为4.22 mgml(-1)。PECI未表现出可检测到的果胶甲酯酶、内切多聚半乳糖醛酸酶、甘露聚糖酶、木聚糖酶和纤维素酶活性。然而，它表现出外切多聚半乳糖醛酸酶和果胶裂解酶活性。在纯PECI中检测到碳水化合物的存在。它被l - 色氨酸、DEPC、DTT、DTNB、DTP、l - 半胱氨酸和β - 巯基乙醇激活，并被NBS、Fe(2+)、Cu(2+)、Zn(2+)、Mn(2+)、Al(3+)和Ca(2+)抑制。该酶与来自野油菜黄单胞菌和地衣芽孢杆菌的果胶裂解酶具有同源性。
