蛋白质的温度适应性：在一种冷适应α淀粉酶中构建类似中温酶的活性和稳定性

Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase.

作者信息

D'Amico Salvino, Gerday Charles, Feller Georges

机构信息

Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000, Liege-Sart Tilman, Belgium.

出版信息

J Mol Biol. 2003 Oct 3;332(5):981-8. doi: 10.1016/j.jmb.2003.07.014.

DOI:10.1016/j.jmb.2003.07.014

PMID:14499602

Abstract

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature.

摘要

产生了嗜冷α-淀粉酶的两个多重突变体，带有五个突变（每个突变引入了猪胰α-淀粉酶中存在的额外弱相互作用），有或没有一个特定于温血动物的额外二硫键。与单突变相比，两个多重突变体均表现出因协同效应而导致的稳定性和活性的大幅改变。新引入的弱相互作用和二硫键赋予了类似嗜温菌的稳定性参数，如熔点t(m)、量热焓ΔH(cal)的增加以及对热失活的保护作用增强，同时还表现为解折叠的协同性和可逆性降低。此外，催化反应的动力学和热力学活化参数均向猪酶的值靠近。这项研究证实了弱相互作用在调节酶的稳定性和活性之间的平衡以适应环境温度方面的核心作用。