Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase

Abstract

Food-derived nattokinase has strong thrombolytic activity and few side effects. In the field of medicine, nattokinase has been developed as an adjuvant drug for the treatment of thrombosis, and nattokinase-rich beverages and health foods have also shown great potential in the field of food development. At present, the poor thermostability of nattokinase limits its industrial production and application. In this study, we used several thermostability-prediction algorithms to predict nattokinase from Bacillus mojavensis LY-06 (AprY), and screened two variants S33T and T174V with increased thermostability and fibrinolytic activity. The t1/2 of S33T and T174V were 8.87-fold and 2.51-fold those of the wild type AprY, respectively, and their enzyme activities were also increased (1.17-fold and 1.28-fold, respectively). Although the thermostability of N218L was increased by 2.7 times, the fibrinolytic activity of N218L was only 73.3% of that of wild type AprY. The multiple-point mutation results showed that S33T-N218L and S33T-T174V-N218L variants lost their activity, and the T174V-N218L variant did not show any significant change in catalytic performance, while S33T-T174V increased its thermostability and activity by 21.3% and 24.8%, respectively. Although the S33T-T174V variant did not show the additive effect of thermostability, it combined the excellent transient thermostability of S33T with the better thrombolytic activity of T174V. Bioinformatics analysis showed that the overall structure of S33T and T174V variants tended to be stable, while the structure of S33T-T174V variant was more flexible. Local structure analysis showed that the increased rigidity of the active center region (positions 64–75) and the key loop region (positions 129–130, 155–163, 187–192, 237–241, and 268–270) determined the increased thermostability of all variants. In addition, the enhanced flexibility of S33T-T174V variant in the Ca1 binding region (positions 1–4, 75–82) and the peripheral region of the catalytic pocket (positions 210–216) may account for the inability to superpose its thermostability. However, the increased flexibility in the active central region (positions 210–216) and the interaction region with Ca1 binding site (positions 1–4, 210–216) of the S33T-T174V variant may contribute to the decreased thermostability of the S33T-T174V variant compared with the corresponding single-point mutation. We explored the effective strategy to enhance the thermostability of nattokinase, and the resulting variants have potential industrial production and application.

Enhanced Thermostability of Nattokinase by Computation-Based Rational Redesign of Flexible Regions

“…Multiple algorithms have been developed to predict changes in protein stability caused by mutations. The stability effect of each candidate mutant is evaluated using ΔΔ G , which represents the mutation-induced free energy change. , To calculate ΔΔ G for all possible point mutations, we used the thermostability prediction software CUPSAT to perform virtual saturation mutations of amino acid residues in loops 10 and loop12. To decrease the mutation library, we selected the variant with the most significant ΔΔ G reduction in residues predicted to be negative in loops 10 and 12 as the candidate site for thermostability improvement.…”

“…The fibrin plate method was employed to determine the thrombolytic activity and thermostability of each rAprY variant’s crude enzyme . Thrombolytic activity was assessed by measuring the transparent circle area on the fibrin plate after 18 h of incubation at 37 °C, with urokinase as a control standard, for both rAprY and its variants.…”

“…The most significant obstacle hindering the enzyme’s industrial application is its poor thermostability. Nattokinase typically operates optimally between 40 and 60 °C, making it susceptible to high-temperature denaturation during industrial production . At present, the research on structural modification of nattokinase mainly focuses on improving its activity, acid stability, and oxidative stability.…”

“…It utilizes computer simulations and bioinformatics technology to obtain data on the structure, free energy, interaction entropy, interaction enthalpy, and other related properties of protein molecules through algorithms such as quantum mechanics, molecular docking, and molecular dynamics (MD) simulations. , This allows the establishment of small mutation libraries with a high accuracy. Liu et al combined protein surface charge engineering, sequence alignment, and a literature-based approach to successfully identify a mutant Q59E that enhances nattokinase enzyme activity; Li et al created a mutant K12D with improved fibrinolytic activity and mutants S33T, R45E, and T174 V with enhanced stability using computer-aided prediction and semirational design strategies; , and Luo et al generated mutant M4 by modifying the flexible region of nattokinase, resulting in a 20.7-fold increase in half-life and a 2-fold increase in fibrinolytic activity . However, in some cases, these strategies may not be effective due to the limited availability of relevant studies on protein sequence–structure–function relationships.…”