Biotechnology and Bioengineering: Volume 116, Number 10, October 2019

Funding information Priority Academic Program Development of Jiangsu Higher Education Institutions; 111 Project, Grant/Award Number: No. 111‐2‐06; National Natural Science Foundation of China, Grant/Award Number: 3140078; National First‐Class Discipline Program of Light Industry Technology and Engineering, Grant/ Award Number: LITE2018‐04; Fundamental Research Funds for the Central Universities, Grant/Award Number: JUSRP51713B; National Key R&D Program of China, Grant/ Award Number: 2016YFE0127400 Abstract 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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