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. 2025 Mar 4;24(1):51.
doi: 10.1186/s12934-025-02681-5.

Enhanced thermostability of nattokinase by rational design of disulfide bond

Kongfang Yu #  1 , Liangqi Chen #  1   2 , Yaolei Tang  3 , Aixia Ma  1   2 , Wenhui Zhu  1 , Hong Wang  1 , Xiyu Tang  1   2 , Yuan Li  4   5 , Jinyao Li  6   7
Affiliations

Enhanced thermostability of nattokinase by rational design of disulfide bond

Kongfang Yu et al. Microb Cell Fact. .

Abstract

Nattokinase, the thrombolytically active substance in the health food natto, nevertheless, its lower thermostability restricts its use in food and pharmaceutical applications. In this study, two heat-resistant variants of nattokinase, designated 50-109 (M1) and 15-271 (M2), were successfully obtained by introducing a disulfide bonding strategy. Their half-lives at 55℃ were found to be 2.50-fold and 5.17-fold higher, respectively, than that of the wild type. Furthermore, the specific enzyme activities of the variants, M1 and M2, were also increased by 2.37 and 1.66-fold, respectively. Meanwhile, the combination of two mutants increased the thermostability of nattokinase by 8.0-fold. Bioinformatics analyses indicated that the enhanced thermostability of the M1 and M2 variants was due to the increased rigidity and structural contraction of the overall structure. Finally, the fermentation process of mutant M1 was optimized to increase the expression of nattokinase. Study provides substantial molecular and theoretical support for the industrial production and application of nattokinase.

Keywords: Bioinformatics design; Disulfide bond; Enzyme synthesis; Nattokinase; Thermostability.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Enzymatic properties of rAprY and its variants with single-point mutations. (A) Optimum pH; (B) pH stability; (C) Optimum temperature; (D) Half-life period
Fig. 2
Fig. 2
Nattokinase activity (A) and thermostability (B) measured using the fibrin plate assay.Samples marked with the plus sign on fibrin plates were those that had been heat-treated (55℃ for 30 min)
Fig. 3
Fig. 3
MD simulation results of AprY and its variants at 328.15 K for 100ns: (A) RMSD; (B) SASA; (C) Rg; (D) RMSF
Fig. 4
Fig. 4
(A) Changes in the secondary structure corresponding to each amino acid residue during MD at 298.15 K; (B) Changes in substrate interaction with wild-type rAprY, M1 and M2 variants during steered dynamics simulations
Fig. 5
Fig. 5
(A) Enzyme activity and Gray-scale scanning analysis of supernatants from different hosts; (B) Effects of different induction time, pH, bottling volume, induction temperature and inoculum volume on the production of nattokinase
See this image and copyright information in PMC

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