Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite-directed mutagenesis.

Authors:
Zhongmei Liu
Zhongmei Liu
Jiangnan University
China
Han Zhao
Han Zhao
Provincial Hospital Affiliated to Shandong University
China
Laichuang Han
Laichuang Han
From the Life Science College
Wenjing Cui
Wenjing Cui
Jiangnan University
China
Li Zhou
Li Zhou
The Third Affiliated Hospital of Sun Yat-sen University
Guangzhou Shi | China
ZheMin Zhou
ZheMin Zhou
Nankai University
China

Biotechnol Bioeng 2019 Apr 1. Epub 2019 Apr 1.

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.

Nattokinase (NK) is a serine protease of the subtilisin family; as a potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

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Source
http://dx.doi.org/10.1002/bit.26983DOI Listing
April 2019
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