Correlation of the conformational structure and catalytic activity of the highly thermostable xylanase of Thermomyces lanuginosus PC7S1T

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.