Rational engineering of enzyme stability
Rational engineering of enzyme stability
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DOI：
10.1016/j.jbiotec.2004.03.026
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发表时间：
2004-09-30
影响因子：
4.1
通讯作者：
Vriend, G
中科院分区：
文献类型：
--
作者：
Eijsink, VGH;Bjork, A;Vriend, G
在过去的15年里，描述通过引入突变来稳定蛋白质的报道层出不穷。这些报告跨越了从T4溶菌酶和藤蔓酶等小酶的开创性理性设计工作到蛋白质设计和定向进化的时期。同时，天然超稳定蛋白的纯化和表征增加了我们对蛋白质稳定性的理解。在此过程中，人们提出了许多合理的蛋白质稳定策略，其中一些策略（例如通过引入脯氨酸或二硫桥来实现熵稳定）具有合理的成功率。另一方面，定向进化的比较研究和努力表明，有许多导致高稳定性的突变策略，其中一些不容易定义和合理化。该领域的最新发展包括人们越来越意识到蛋白质表面对稳定性的重要性，以及通常非常有限数量的突变可以大大提高稳定性的概念。另一个发展涉及到这样一个概念，即在高温下可逆和完全展开的小纯蛋白质的“实验室稳定性”与“工业稳定性”之间存在根本区别，后者通常由部分展开过程控制，然后是某种不可逆的失活过程（例如聚集）。只要对热失活的机理有足够的了解，就可以实现酶的成功和有效的合理稳定。(C) 2004 Elsevier b.v.版权所有
During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved. (C) 2004 Elsevier B.V All rights reserved.