Younesi Fereshteh S, Pazhang Mohammad, Najavand Saeed, Rahimizadeh Parastou, Akbarian Mohsen, Mohammadian Mehdi, Khajeh Khosro
Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran.
Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
Mol Biotechnol. 2016 Jan;58(1):12-21. doi: 10.1007/s12033-015-9900-3.
Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.
嗜酸热脂环酸芽孢杆菌的内切葡聚糖酶Cel9A（AaCel9A）是一种含有537个残基的单体酶。该酶在催化结构域的N端有一个免疫球蛋白样结构域。在本研究中，通过将缺失免疫球蛋白样结构域的截短酶（AaCel9AΔN）与野生型酶进行比较，研究了免疫球蛋白样结构域对AaCel9A活性、稳定性和结构刚性的作用以及钙对酶活性和稳定性的影响。我们的结果表明，免疫球蛋白样结构域的缺失使截短酶的催化效率提高了三倍，而酶的米氏常数没有任何显著变化。此外，通过缺失免疫球蛋白样结构域，活性的最适pH从6.5变为7.5，最适温度从65℃变为60℃。热稳定性和荧光猝灭结果表明，截短酶的稳定性和刚性高于野生型酶。与AaCel9AΔN相比，钙同样提高了酶的催化效率（高达40%），并显著提高了AaCel9A的稳定性。这表明免疫球蛋白样结构域在野生型酶中对钙提高酶稳定性起到了作用。
