通过定点突变增强纳豆激酶的热稳定性

Enhancing Thermostability of Nattokinase by Site-directed Mutagenesis

摘要:

血栓导致的心脑血管疾病是当前危害人类健康、导致死亡率最高疾病之一，因此近年来溶栓药物及具有溶栓功能保健食品的研发进展得十分迅速。纳豆激酶因其特有的高效溶血栓作用，成为国内外科研热点之一。本研究针对目前纳豆激酶稳定性的问题，以纳豆激酶的晶体结构和序列比对为基础，通过定点突变构建组纳豆激酶突变体，对可能影响纳豆激酶热稳定性的氨基酸进行研究。在构建的5个突变体中，P14L、N76D两个突变体的热稳定性有明显提高，在65 ℃下的半衰期由20 min分别提高为30 min和50 min。同源建模和分子动力学研究结果表明，这两个氨基酸位点是通过不同机理对纳豆激酶催化活性以及热稳定性产生的重要作用。本研究可为纳豆激酶的基因工程改造以及其生产和应用提供理论基础和技术支持。

Abstract:

Cardiovascular and cerebrovascular diseases caused by thrombi are often life-threatening, contributing to the highest mortality rate. This has fuelled rapid development of thrombolytic drugs and functional foods with thrombolytic activity in the past few years. Due to its strong thrombolytic effect, nattokinase (NK) has become the focus of international interest. The aim of this study was to enhance the thermostability of NK. Based on its sequence alignments and crystal structure, NK mutants were prepared by site-directed mutagenesis and the amino acids that might affect NK thermostability were studied. Among the five mutants generated in this study, P14L and N76D showed significantly increased thermostability and their half-life at 65 ℃ improved from 20 min (wild type) to 30 and 50 min, respectively. Homology modeling and molecular dynamics analysis showed that two amino acid sites influenced thermostability and catalytic activity of NK through different mechanisms. This study provides a theoretical and technical basis for the genetic reconstruction, production, and application of NK.