Chauvaux S, Beguin P, Aubert J P, Bhat K M, Gow L A, Wood T M, Bairoch A
Unité de Physiologie Cellulaire and URA 1300 CNRS, Départment des Biotechnologies, Institut Pasteur, Paris, France.
Biochem J. 1990 Jan 1;265(1):261-5. doi: 10.1042/bj2650261.
Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
由celD基因编码的嗜热栖热菌内切葡聚糖酶D（EC 3.2.1.4：EGD）被发现以2.03×10⁶ M⁻¹的缔合常数结合Ca²⁺。Ca²⁺使EGD对膨胀的微晶纤维素的活性提高了2倍。在Ca²⁺存在的情况下，该酶对对硝基苯基-β-D-纤维二糖苷和羧甲基纤维素的Kd降低了4倍。此外，Ca²⁺使该酶在75℃下的半衰期从13分钟增加到47分钟。由于celD的3'序列编码一个与几种Ca²⁺结合蛋白的Ca²⁺结合位点具有相似性的重复区域，构建了一个缺失克隆并用于纯化一种不再包含该重复区域的截短形式的酶。截短酶在活性、Ca²⁺结合动力学以及Ca²⁺对底物结合和热稳定性的影响方面与完整基因表达的EGD非常相似。因此，后述参数似乎不是通过重复的保守区域介导的。
