Parry N J, Beever D E, Owen E, Nerinckx W, Claeyssens M, Van Beeumen J, Bhat M K
Food Materials Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
Arch Biochem Biophys. 2002 Aug 15;404(2):243-53. doi: 10.1016/s0003-9861(02)00301-6.
A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.
从橙色嗜热子囊菌中纯化至同质的一种主要细胞外内切葡聚糖酶，其相对分子质量为34 kDa，等电点为3.7，在70 - 80℃和pH 4.0 - 4.4时活性最佳。在50℃下，pH 2.8 - 6.8时它可稳定存在48 h，在pH 5.0和2.8时，分别在高达70℃时仍能保持其二级结构和折叠构象。N端的33个氨基酸序列与14种微生物内切葡聚糖酶具有相当的同源性，其中有8个氨基酸高度保守（第10 - 17位），且在第8、22、23和32位分别为甘氨酸、脯氨酸、甘氨酸和脯氨酸。该酶富含天冬氨酸（15%）和谷氨酸（10%），碳水化合物含量为2.7%。内切葡聚糖酶的多克隆抗体与其自身抗原以及橙色嗜热子囊菌的其他纯化纤维素酶发生交叉反应。该内切葡聚糖酶对聚合底物具有特异性，对羧甲基纤维素活性最高，其次是大麦β - 葡聚糖和地衣多糖。它优先切割Glc(n)和MeUmbGlc(n)的内部糖苷键，并拥有一个具有五个亚位点的扩展底物结合位点。数据表明，橙色嗜热子囊菌的内切葡聚糖酶是糖苷水解酶家族5的成员。
