Abstract

Thermomyces lanuginosus PC7S1T xylanase was purified after four chromatographic steps involving columns of ion exchange and molecular filtration. The purified xylanase revealed the molecular mass to be 21.3 kDa, high specificity for beechwood xylan and its main hydrolysis products were xylobiose and xylotriose. Xylanase exhibited increased activity at pH 6.5 and a temperature of 75 °C and was stable-over a wide range of pHs (100 hours) and temperatures (3 hours at 75 °C). Circular dichroism spectrum deconvolution showed a β-structure-rich secondary structure with a melting temperature of 73 °C. In addition, its secondary structure was preserved, even at extreme pHs and temperatures up to 70 °C in the presence of MnCl₂ (1 mM), DTT (5 mM) and high concentrations of guanidine (6 M). On the other hand, changes in the tertiary structure of the enzyme, which was monitored using intrinsic and/or extrinsic fluorescence (ANS probe), in the presence of MnCl₂, DTT and extreme values of pH may be related to variations in enzyme activity. Therefore, T. lanuginosus PC7S1T xylanase exhibited resistance to high temperatures, observed through its ability to remain active and demonstrate a high-degree of rigidity and resistance to denaturation; very extreme conditions were necessary in order to change the enzymes’ secondary and/or tertiary structures.

摘要

Thermomyces lanuginosus PC7S1T 木聚糖酶在涉及离子交换柱和分子过滤的四个层析步骤后得到纯化。纯化后的木聚糖酶分子量为21.3 kDa，对山毛榉木聚糖具有高度特异性，其主要水解产物为木二糖和木三糖。木聚糖酶在 pH 6.5 和 75 °C 的温度下表现出更高的活性，并且在广泛的 pH（100 小时）和温度（75 °C 下 3 小时）范围内稳定。圆二色谱解卷积显示富含 β 结构的二级结构，熔点为 73 °C。此外，即使在极端 pH 值和高达 70 °C 的温度下存在 MnCl _{2 ，其二级结构也得以保留}(1 mM)、DTT (5 mM) 和高浓度胍 (6 M)。另一方面，在存在 MnCl ₂、DTT 和极端 pH 值的情况下，使用内在和/或外在荧光（ANS 探针）监测酶的三级结构的变化可能与酶活性的变化有关. 因此，T. lanuginosus PC7S1T 木聚糖酶表现出耐高温性，通过其保持活性的能力观察到，并表现出高度的刚性和抗变性；为了改变酶的二级和/或三级结构，需要非常极端的条件。