基于序列和结构分析的酶热稳定性改造策略*

功能酶被广泛应用于食品、化工、医药等领域,但却容易受高温环境限制,导致催化效率降低。以分子改造为目的的蛋白质工程技术是解决这一问题的关键环节,其能够对酶结构和功能进行改造,获得热稳定性好的工业酶。传统的定向进化方法只能依靠随机突变进行人工筛选,具有效率低、针对性差等缺点;理性设计作为酶热稳定性改造的主要方法,可借助各种计算机程序和软件预测潜在突变位点,但其要求对酶的催化机制、热稳定性机制有深入了解。对于大多数天然酶而言,酶的序列和晶体结构是最容易获取的信息,也是预测功能的重要基础。从酶的序列和晶体结构入手,重点介绍了共识突变、基于序列偏好性的突变、截短柔性区域、优化分子内相互作用力、刚化催化活性区域及计算机辅助筛选柔性位点等常用策略,这些策略具有筛选效率高、改造准确性高、实用性强等优点。结合多种酶的热稳定性改造案例进行分析,旨在为不同酶的改造策略选择提供有效参考,同时也为工业酶的耐热性研究提供理论支持。

Modification Strategy of Enzyme Thermal Stability Based on Sequence and Structure Analysis

Functional enzymes have been widely used in the fields of food, chemicals, medicine, etc. However, high temperature reduces the catalytic efficiency of enzymes. The protein engineering technology can be used as a key link to modify enzymes’ structure and function, and to obtain industrial enzymes with thermostability. Traditional directed evolution methods can only rely on random mutations for manual screening with low efficiency. Rational design, as the main method of thermostability modification, can be used to predict potential mutation sites with various computer programs and software, but it requires deep understanding of the catalytic and thermal stability mechanism. For most natural enzymes, it is easy to obtain sequence and crystal structure, and is also an important basis for predicting function. This paper focuses on the modification strategies as follows: common mutation, the mutation based on amino acid preference, trunking of flexible regions, the optimization of intramolecular interactions, the modification of catalytic active regions and computer-aided design with the sequence and crystal structure analysis. These strategies have the advantages of high screening efficiency and modification accuracy, and strong practicability. And it also analyzes the thermostability modification cases of different enzymes, aiming to provide an effective reference for the selection of modification strategies, and also give theoretical support for the heat resistance research of industrial enzymes.