Enhancing Thermostability of Nattokinase by Site-directed Mutagenesis
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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.
