Bronnenmeier K, Rücknagel K P, Staudenbauer W L
Institute for Microbiology, Technical University Munich, Federal Republic of Germany.
Eur J Biochem. 1991 Sep 1;200(2):379-85. doi: 10.1111/j.1432-1033.1991.tb16195.x.
Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N-terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75 degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70 degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non-reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non-reducing end of the cellulose chains.
阿魏纤维素酶II从粪堆梭菌的培养上清液中纯化至同质。由于阿魏纤维素酶II的选择性滞留，通过在Superose 12上进行快速蛋白质液相色谱（FPLC）凝胶过滤，实现了与主要纤维素分解酶活性（阿魏纤维素酶I）的完全分离。该酶的表观分子量为87 kDa，等电点为3.9。N端氨基酸的测定表明，阿魏纤维素酶II不是阿魏纤维素酶I的蛋白水解加工产物。阿魏纤维素酶II在pH 5至6之间观察到最大活性。在Ca2+存在下，该酶具有高度的热稳定性，最适温度约为75℃。在70℃下，阿魏纤维素以线性速率水解三天。阿魏纤维素酶II对未取代的纤维素、纤维四糖和更大的纤维糊精有活性。它对羧甲基纤维素和大麦β-葡聚糖没有活性。与其他细菌外切葡聚糖酶不同，阿魏纤维素酶II不水解芳基-β-D-纤维二糖苷。阿魏纤维素以大约4:1的摩尔比降解为纤维二糖和纤维三糖。以酸膨胀的阿魏纤维素为底物时，纤维四糖也作为中间产物形成，并进一步裂解为纤维二糖。还原型纤维糊精的降解模式与预期的作用于链的非还原端的纤维二糖水解酶不同；纤维五醇降解为纤维二糖醇和纤维三糖，而纤维六醇最初裂解为纤维二糖醇和纤维四糖。综合这些发现表明，阿魏纤维素酶II代表了一种新型的外切葡聚糖酶（纤维糊精水解酶），它根据底物的可及性从纤维素链的非还原端释放纤维四糖、纤维三糖或纤维二糖。
