Improvement of the Catalytic Activity of nattokinase by Site-Saturation Mutagenesis of Glycine 131

Improvement of the Catalytic Activity of nattokinase by Site-Saturation Mutagenesis of Glycine 131 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 17 March 2025 V1 Latest version Share on Improvement of the Catalytic Activity of nattokinase by Site-Saturation Mutagenesis of Glycine 131 Authors : LI YUAN 0000-0002-4024-6174 , Wenhui Zhu , Dong Yang , Huiyang Jia , Aixia Ma , Kongfang Yu , Hong Wang , Ye Ma [email protected] , and Jinyao Li Authors Info & Affiliations https://doi.org/10.22541/au.174221477.73527147/v1 164 views 156 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Molecular modifications of nattokinase have been demonstrated to yield enzyme variants with enhanced catalytic performance, offering valuable insights into the structure-activity relationship of this enzyme. In the present study, PoPMuSiC 2.1, a web-based tool designed for predicting protein stability changes induced by mutations, was employed to investigate the catalytic properties of nattokinase. Through PoPMuSiC prediction and site-saturation mutagenesis, the G131A mutant, which exhibited improved catalytic activity, was identified. Comparative analysis of the G131A mutant and the wild type nattokinase revealed a 1.5-fold increase in enzyme activity and a 4.5-fold extension in half-life, effectively addressing the typical trade-off between enzyme activation and stability in protein engineering. Computational simulations further indicated that the G131A mutant adopts a more stable conformation, displays stronger interactions with the substrate, and features a more optimal substrate entry pocket. The fermentation process of G131A mutant was optimized and a yield of 623.43 U/mL was obtained. These findings offer a novel approach for studying the structure-activity relationship of nattokinase and could be extended to other serine proteases. 1. INTRODUCTION Nattokinase is an enzyme isolated from natto, a traditional Japanese food, which has the characteristics of low bleeding risk, high dose tolerance, no gene mutation and chromosome aberration induction[1, 2] . In addition to directly hydrolyze thrombus, nattokinase can also dissolve thrombus through a variety of pathways such as activation of prourokinase, stimulation of vascular endothelial cells to produce tissue plasminogen activator (t-PA), and degradation of inactivating plasminogen activator type I inhibitors[3]. Therefore, the research on the development of nattokinase health food and drug has become one of the hot spots in academic research. Nattokinase belongs to the subtilis protease family. The gene encoding nattokinase was 381 amino acids in length, including 29 amino acids of signal peptide sequence and 77 amino acids of propeptide. The molecular weight of the mature peptide was 27.7kDa[4]. As a disulfude-free single-chain polypeptide, nattokinase is characterized by a conserved catalytic triad (Asp 32 , His 64 , Ser 221 ) and a conserved substrate binding center (Ser 125 , Leu 126 , Gly 127 )[5]. The catalytic mechanism of nattokinase is similar to that of subtilis protease. Both nattokinase and subtilis protease hydrolyse the peptide chain by nucleophilic attack of Ser 221 on the hydroxyl carbon of the substrate peptide bond, while His 64 and Asp 32 act as proton acceptors for Ser 221 and stabilise the protonation state of His 64 , respectively[6]. The viability of enzyme industrial application hinges on thermostability, specific activity, and catalytic efficiency. The pursuit of heightened catalytic efficiency has increasingly garnered attention, with efforts focused on boosting enzyme activity or enhancing thermostability[7, 8]. Searching for enzymes from extremophiles is a viable approach to enhancing enzyme activity. Nonetheless, the process of isolating new enzymes is both time-consuming and labor-intensive. Protein engineering has emerged in recent years as a solution to these challenges, offering a means to significantly enhance enzyme properties[9]. In recent years, some researchers have improved the catalytic performance of nattokinase through protein engineering[10-12]. Liu et al. combined protein surface charge engineering, sequence alignment and a literature-based approach to successfully identify a Q59E mutant that enhances nattokinase enzyme activity[13]; Luo et al. engineered mutant M4 through alterations in the flexible region of nattokinase, this modification led to a notable 20.7-fold extension in half-life and a doubling of fibrinolytic activity[14]. As previously mentioned, the catalytic hydrolysis mechanism of nattokinase has been thoroughly elucidated. Moreover, various studies have explored methods to enhance both the fibrinolytic activity and stability of nattokinase. Despite these efforts, further improvements are required in both the fibrinolytic activity and stability of nattokinase to fully unlock its potential in functional foods and pharmaceuticals. The PoPMuSiC-2.1 web server predicts changes in thermodynamic stability due to single-site mutations within proteins[15]. It works by using a linear combination of statistical potentials with coefficients that depend on the solvent accessibility of the mutated residue. In particular, PoPMuSiC demonstrates robust predictive capabilities, with a correlation coefficient of 0.8 between predicted and measured stability changes in cross-validation, with outliers accounting for only 10%[15]. PoPMuSiC has been applied to the modification of a variety of enzymes, such as κ-carrageenase[16], cellulase[17], dextranase[18] and chitosanase[7]. In this study, nattokinase from Bacillus mojavensis LY-06 (AprY) was cloned and expressed in Escherichia coli BL21 (DE3), and the recombinant nattokinase (rAprY) was characterized. To further improve the enzyme properties, one amino acid mutation sites (G131A) with improved catalytic performance were rationally selected using the PoPMuSiC algorithm. This study aims to deliver an enzyme molecule optimized for the industrial-scale production and application of nattokinase. 2. MATERIALS AND METHODS 2.1 Plasmids, Strains, and cultivation conditions The recombinant plasmid employed was pET-28a-AprY, resistant to kanamycin, harboring a complete nattokinase peptide (inclusive of signal peptide, propeptide, and mature peptide), derived from Bacillus mojavensis LY-06. Escherichia coli BL21 (DE3) served as the expression host for the recombination AprY (rAprY). Plasmid amplification and overexpression occurred in Luria-Bertani (LB) medium, comprising 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl, adjusted to pH 7.0. Induction of recombinant protein expression was initiated using Isopropyl-beta-D thiogalactopyranoside (IPTG). 2.2 Site-directed mutagenesis and production of nattokinase variants Primers synthesised by Shanghai Sangon Biotech Co., Ltd. facilitated amplification of the target sequences. Using the plasmid pET-28a-AprY as a template, variants were constructed by site-directed mutagenesis. The AprY gene mutations were introduced by PCR using the X-up forward primer, X-down reverse primer and TransStart FastPfu Fly PCR SuperMix (Beijing TransGen Biotech Co., Ltd., China) ( Table S1 and Table S2 ). The PCR products were then treated with DMT enzyme before transformation into Escherichia coli DH5α competent cells. The transformed cells were then cultured on LB plates supplemented with kanamycin, and the resulting colonies were sequenced by Shanghai Sangon Biotech Co. (Shanghai, China)[19]. 2.3 Bacterial expression and purification of rAprY and its variants The methodology for expression and purification of rAprY and its variants was adapted from previous work by Li et al[20]. Briefly, Escherichia coli BL21 (DE3) cells containing the pET-28a-AprY expression vector and its variant counterparts were cultured in LB medium supplemented with 50 μg/mL kanamycin at 37 ℃. When an OD 600 of 0.7-0.8 was reached, overexpression was induced by adding IPTG to a final concentration of 0.1 mM. Cells were then cultured at 16°C for a further 20 hours before being harvested by centrifugation. The bacterial pellet was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 5% glycerol, pH 7.4) and subjected to sonication to disrupt cell membranes, followed by centrifugation to remove debris. The resulting supernatant was further purified at 0-4°C by column chromatography using a Ni-NTA column (Invitrogen, Carlsbad, CA, USA). After achieving homogeneity in the purification, the protein concentration was determined using the Bradford protein assay reagent kit (Beyotime, Shanghai, China). 2.4 Assay of thrombolytic activity The enzymatic properties and kinetic parameters of rAprY and its variants were evaluated using the tetrapeptide substrate method[21]. Activity of purified nattokinase was determined by measuring the increase in p-nitroaniline (p-NA; Sigma, Louis, MO) released from the chromogenic substrate suc-AAPF-pNA. Reactions were carried out in a 96-well plate at 25 ℃, with 200 μL of PBS buffer (8 mM Na 2 HPO 4 , 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.0-7.2) containing the appropriate enzyme concentration. Upon addition of suc-AAPF-pNA at a final concentration of 0.25 mM, reactions were monitored continuously at 405 nm for 3 minutes using the SpectraMax® iD5 multimode microplate reader (MolecularDevices, San Jose, CA, USA). One unit of enzyme activity was defined as the amount that catalyzes the production of 1 μmol p-NA per minute under assay conditions. To assess thrombolytic activity and thermostability of each rAprY variant’s crude enzyme, the fibrin plate method was employed[22]. Transparent circle areas on the fibrin plate were measured after 18 hours of incubation at 37 ℃, with urokinase serving as a control standard for both rAprY and its variants. 2.5 Biochemical characterization of rAprY and its variants Optimal temperature determination involved subjecting the pure enzyme solution to various temperature ranges (20℃-70℃), followed by assessing enzyme activity using the tetrapeptide substrate method. For optimal pH determination, the pure enzyme solution underwent exposure to different pH buffers (ranging from pH 3 to pH 11), with enzyme activity assessed via the tetrapeptide-substrate method. Citric acid maintained pH levels within the range of 3.0-5.0, while disodium hydrogen phosphate and sodium dihydrogen phosphate buffered pH levels from 6.0 to 8.0, and sodium carbonate was utilized for pH values between 9.0 and 11.0. Evaluation of pH stability and temperature stability involved incubating the wild type nattokinase and mutant pure enzyme solutions at various pH levels or temperatures in buffer solutions at 4°C for half hours, followed by measuring residual enzyme activity. The highest enzyme activity recorded was set at 100%. Half-life determination entailed subjecting the wild type nattokinase and mutant pure enzyme solutions to 55°C for durations ranging from 10 to 60 minutes, respectively, with subsequent determination of the half-life (t 1/2 ) for both the wild type nattokinase and mutant enzymes. Activity of untreated enzymes exposed to high temperatures was set as the baseline at 100%. 2.6 Determination of the kinetic parameters The kinetic parameters for rAprY and its variants were assessed using suc-AAPF-pNA across concentrations spanning from 0.05 to 1 mM in PBS buffer (comprising 8 mM Na 2 HPO4, 136 mM NaCl, 2 mM KH 2 PO4, 2.6 mM KCl, pH 7.0-7.2) at 25 ℃. Employing the Michaelis-Menten method facilitated the determination of K m and k cat values, with GraphPad Prism 8 software from San Diego, CA, utilized for analysis. Results presented represent the averages obtained from three distinct experiments. 2.7 Bioinformatics analysis The protein sequences of rAprY and its variants underwent structural modeling through the AlfaFold3 workstation, which produced precise models (https://golgi.sandbox.google.com, accessed on 12 May 2024). Subsequent analysis of these models utilized the PROCHECK module within SAVES 6.0 (https://saves.mbi.ucla.edu/, accessed on 12 May 2024) to generate Ramachandran plots and assess model reliability[23]. DoGSiteScorer online software (https://proteins.plus/) was used to calculate the volume and surface area of the active center of nattokinase and its mutants[24]. The PoPMuSiC-2.1 algorithm, accessible at http://dezyme.com/ on 12 May 2024, was utilized to identify potential amino acid substitutions impacting the enzyme properties of nattokinase[15]. Subsequently, site-directed mutagenesis was employed to introduce the corresponding mutations into the nattokinase enzyme. Molecular docking of ligands (suc-AAPF-pNA) with AprY and its variants was performed using the AutoDock 4.2.6 program[25]. The protein models underwent molecular dynamic simulation (MD) utilizing the YASARA (Yet Another Scientific Artificial Reality Application) software[26]. To enhance solute stability, the hydrogen-bonding network was optimized[27]. Predictions for pKa were conducted to adjust the protonation states of protein residues at pH 8.0. Physiological concentrations of NaCl ions (0.9%) were achieved, with an excess of either Na + or Cl - to neutralize the cell. Following steepest-descent and simulated-annealing minimizations to resolve clashes, the simulation proceeded for 100 ns, employing the AMBER14 force field for the solute and TIP3P for water. Van der Waals forces were capped at a cutoff of 10 Å, while long-range electrostatics were computed using the particle-mesh Ewald method with a distance of 1.0 nm[28, 29]. Motion equations were integrated using a previously described algorithm[27]. Bonded interactions were computed with a multiple timestep of 1.25 fs, and non-bonded interactions with a multiple timestep of 2.5 fs. The simulation operated at a temperature of 328.15 K/298.15 K and a pressure of 1 atm in the NPT ensemble. The initial 50 ns were considered equilibration time and were excluded from further analysis. Various programs were employed for analysis, including md_run for simulation execution, md_analyze for evaluating parameters such as radius of gyration (Rg), potential energy, solvent accessible surface area (SASA), protein secondary structure content, and number of hydrogen bonds, and md_analyzeres for calculating the root mean square fluctuation (RMSF) value. Visualization of the enzyme structure was accomplished using PymoL software. Steered molecular dynamics (SMD), employed to elucidate the responses of biomolecules to external mechanical manipulations at the atomic level, mirrors the principles of atomic force microscopy (AFM)[30]. Integration of motion equations utilized a multiple timestep of 1.25 fs for bonded interactions and 2.5 fs for non-bonded interactions at a temperature of 298K and a pressure of 1 atm (NPT ensemble), following algorithms detailed previously. Following a 3 ps equilibration period, a minimum acceleration of 2000 pm/ps² was imposed on all ligand atoms concurrently with the non-bonded forces (every 2.5 fs). Given the ligand mass of 624.24 Dalton and the equation F=m×a, this yielded a pulling force of [2000×624.24×0.00166] picoNewton. The pulling direction was defined as the vector connecting the centers of mass of the receptor and ligand, continually adjusted to accommodate rotations of the complex. The maximum distance between the centers of mass of the receptor and ligand was regularly updated, with a 500 pm/ps² increase in acceleration if it remained constant for 400 simulation steps. Acceleration was scaled down by a factor of 1-(1-4000/MaxDisSpeed)² as soon as the maximum distance increased with a ’MaxDisSpeed’ exceeding 4000 m/s, albeit not below the initial minimum. This validation occurred every 20 simulation steps. Simulation termination ensued upon the ligand reaching a 15 Å displacement. The peak pulling force and total work performed were computed to assess their correlation with binding strength. 2.8 Optimization of the fermentation process of G131A production In order to investigate the effect of different single-factor conditions on nattokinase production, the enzyme activity of G131A crude enzyme, measured by the fibrin plate method, was employed as an examination index for single-factor optimisation experiments[10]. The optimal results of each round of optimisation were then accumulated and incorporated into the subsequent round. The LB medium was utilised for the optimisation of enzyme production conditions, with the factors examined encompassing fermentation time (14, 16, 18, 20, and 22h), fermentation temperature (14, 16, 18, 20, and 22°C), bottling volume (5, 10, 15, 20, 25%), initial pH (5, 6, 7, 8, and 9), and inoculum volume (0.5, 1.0, 1.5, 2.0, and 2.5%). 3. RESULTS AND DISCUSSIONS 3.1 Identification of candidate sites for catalytic performance enhancement of nattokinase Folding free energy, an important parameter of protein thermodynamics, is a general measure of the thermostability of a protein, with smaller folding free energy indicating higher protein stability[31]. PoPMuSiC (http://dezyme.com) is an online prediction software that can predict the effect of each mutant amino acid on the protein unfolding free energy. This is achieved by calculating the solvent accessibility and linear combination coefficient of the mutant amino acid, thereby identifying amino acids that play a key role in protein stability[15]. A significant body of research has demonstrated the efficacy of utilising PoPMuSiC to predict the folding free energy change at each amino acid site, thereby enhancing the stability or activity of the enzyme. For instance, Guo et al. [7]utilised PoPMuSiC to predict the free energy change of a single point mutation of BsCsn46A in Bacillus subtilis , and identified mutants P121N, P121C and P121V, whose activities increased by 1.69, 1.97 and 2.15 times, respectively. In a similar manner, Zhu et al.[32] utilised PoPMuSiC to predict an increased thermostability of arylsulfatase K253H. Guo Jing et al.[9] utilised PoPMuSiC to predict the free energy changes of tyrosinase from Streptomyces kathirae SC-1 and identified mutants Arg95Tyr and Gly123Trp. The half-lives of these mutants were found to be 1.35-fold and 1.82-fold higher than those of the wild type, respectively. Furthermore, Liao et al.[33] constructed 14 mutants of α-l-rhamnosidase from Aspergillus niger JMU-TS528 based on the folding free energy changes predicted by PoPMuSiC, six of which exhibited higher thermostability than the wild type. These findings demonstrate that the calculation and prediction of the effect of amino acid mutations on protein stability by PoPMuSiC is an effective method to enhance protein stability and enzyme activity. The protein structure of wild type nattokinase was submitted to the PoPMuSiC algorithm tool for calculation. The PoPMuSiC algorithm was utilised to demonstrate all possible point mutations in the wild type nattokinase, according to the folding free energy difference between the wild type nattokinase and mutant enzymes ( Figure 1 ). We selected amino acids with a folding free energy < -6 kJ /mol as candidate sites (D41、N123、G131、G178、S207、N243). In order to ascertain the effect of candidate sites on the catalytic performance of nattokinase, alanine scans were performed on each candidate site and their crude enzyme activity and stability was determined by fibrin plate assay ( Figure 2 ). The results demonstrated that the majority of the mutants exhibited reduced activity, with N123A, N243A and G178A mutants demonstrating complete loss of activity and stability. Furthermore, D41A and S207A mutants exhibited a decline in activity, suggesting that these sites are indispensable for the catalytic efficacy of the enzyme. Consequently, modification of these sites is deleterious to the activation of nattokinase catalytic performance[19]. It is noteworthy that the G131A mutant showed 1.5-fold and 4.5-fold increased activity and thermostability in comparison to the wild type nattokinase. In a previous study, a nattokinase mutant G131S was screened, exhibiting a 1.2-fold increase in thermostability at 55 °C[34]. This finding suggests that modifying amino acid residue 131 may enhance the catalytic performance of nattokinase. Subsequently, amino acid residues at position 131 were saturated, and the crude enzyme activity and thermostability of the mutants was determined using the fibrin plate assay ( Figure 3 ). The results obtained demonstrated that approximately half of the mutants exhibited either maintenance or enhancement of nattokinase activity. However, only the G131A and G131M mutants demonstrated a significant increase in thermostability, with an 4.5-fold and 1.23-fold increase, respectively, in comparison with the wild-type nattokinase. Previous studies have shown there is an activity-stability trade-off phenomenon for enzymes. This phenomenon can be observed during enzyme engineering modifications, where the increase in enzyme activity is often accompanied by a decrease in stability[35]. The G131A and G131M enzymes obtained in this study achieved simultaneous improvement of fibrinolytic activity and thermostability, and overcame the activity-stability trade-off phenomenon. This indicates the application value of the strategy employed in the engineering of nattokinase. 3.2 Purification and characterization of G131A To determine the effects of the five screened mutants on fibrinolytic activity and stability, rAprY as well as G131A mutant were purified and characterized ( Figure S1 ). Wild type nattokinase and G131A were successfully purified, and their enzymatic properties were detected by using the tetrapeptide substrate method. Studies on the optimal pH and pH stability of rAprY and its mutants showed that the optimal pH of G131A mutant was unchanged, but the acid stability of G131A was slight decreased at pH 10 ( Figure 4A-B ). Studies have shown that substitution of key amino acids with different charge properties in the catalytic centre of the enzyme can influence the optimal pH of the enzyme by affecting the pKa value of the active centre, and that changing the polarity of amino acid residues on the surface of the enzyme can also influence the optimal pH of the enzyme by changing the microenvironment profile[19]. The G131A mutant did not alter the charged state of amino acid residue 131, which may be the reason why its optimal pH and stability did not change significantly. We further investigated the optimal reaction temperature and thermostability of wild type nattokinase and G131A mutant. The results showed that the optimal reaction temperature of G131A mutant did not change ( Figure 4C ). Notably, although the optimal reaction temperature of G131A mutant was not significantly different from that of the wild type, its thermostability was enhanced at 55℃ ( Figure 4D ). Notably, the wild type showed less than 50% residual activity after incubation at 55℃ for 20 min, whereas the G131A mutant showed more than 70% of the corresponding residual enzyme activity, showing superior stability. When half-lives were measured, G131A had the best halflife, which were 4.5-fold higher than the wild type. The negative correlation between enzyme stability and function, known as the stability-activity trade-off, has long limited engineering to robust enzyme access, therefore, we compared the specific enzyme activities of G131A and wild-type nattokinase. The specific enzyme activity of the G131A mutant was 1.5-fold higher than that of the wild type, indicating that the G131A mutant can break the limitation of the activity stability trade-off in enzyme engineering ( Table 1 ). The enzyme kinetic parameters of rAprY and G131A mutant were also determined ( Table 1 ). The K m values of G131A mutant were 52.6% lower than those of the wild type, respectively, indicating an increased affinity of G131A mutant for the substrate. In proteins, perturbations of amino acids will propagate unevenly throughout the structure: a residue that is more strongly coupled to a neighbour will be more affected by the perturbation of the neighbor[34]. The k cat values of G131A mutant was increased, probably due to the distal effect of residue 131 on the active site. Our previous prediction of AprY using the Ohm online workstation showed a strong interresidue allosteric correlation of the terminal sequence with the active center, confirming our speculation[36]. 3.3 Understanding the mechanism for enhanced catalytic performance of the G131A mutant. To further elucidate the potential effects of two terminal mutants of nattokinase on the catalytic performance of nattokinase, we modelled wild type nattokinase and G131A, using Alfafold3 online team building. The reliability of the established protein model was determined by generating Ramachandran plots using the SAVES online workstation ( Figure S2 ). Molecular dynamics simulations were also performed at 298K for 100 ns. The root mean square deviation (RMSD) is the sum of all atomic deviations between the conformation at a given time and the target conformation[8]. It is an urgent measure of system stability. At the beginning of the MD simulation, wild type nattokinase and G131A mutant showed relatively large atomic vibrations. However, as the simulation progressed, the RMSD value of the whole protein stabilised (Within 0.3 Å), indicating that the potential and total energy of the system had reached a steady state. Therefore, all MD data were analysed using the data obtained after 50 ns ( Figure 5A ). From the perspective of the overall structure, G131A exhibits a more stable conformation with A lower RMSD (1.67 Å) compared to wild-type nattokinase (1.93 Å) in the presence of constant Rg ( Figure 5A-B ). The SASA value can be used to describe the solubility of a protein and its ability to interact with other molecules[10]. We found that the SASA of the G131A mutant was reduced, which is consistent with the fact that the number of hydrogen bonds formed by the G131A mutant was reduced, suggesting that the G131A mutant has a weaker interaction with the surrounding solvent than the wild-type nattokinase, thereby increasing the stability of the protein structure ( Figure 5C and Table 2 ). It is worth noting that the total potential energy of G131A is lower than that of wild-type nattokinase, and the Coulomb potential energy is the major contributor, which also supports the increased rigidity of the internal structure of the G131A mutant ( Figure 5D and Table 2 ). Secondary structure analysis of wild-type nattokinase and the G131A mutant showed that the proportion of α-helix in G131A was slightly increased while that of β-sheet was decreased, which may be responsible for the change in protein rigidity ( Table 2 ). The catalytic activity of proteins is more related to changes in the active centre or local structure than to the global structure[20]. During the decomposition of the total potential energy, the bond formation potential energy and the bond angle potential energy of G131A increased, and we speculated that the regions within the protein related to catalytic activity may change, which in turn affects the catalytic performance of the G131A mutant ( Table 2 ). RMSF is a measure of the range of position variation of each atom in the simulated molecular structure. RMSF has the capacity to reflect the motion of each atom in the system. When the RMSF value of an atom is large, it can be deduced that the position of the atom changes significantly and is more flexible. This may indicate structural instability or local movement[37]. Analysis of the RMSF of wild-type and G131A mutant showed that the RMSF of G131A was increased at positions 103-105 (loop) and 210-216 (β-sheet), but decreased at positions 40-43 (loop) and 217-219 (loop) ( Figure 6A ). The drastic change in RMSF at positions 210-219 in the G131A mutant resulted in a shift of the corresponding β-sheet structure to random coiling, which may be the main reason for its catalytic performance[38] ( Figure 6B ). Further analysis of the binding network at positions 210-219 of the G131A mutant revealed that the original hydrogen bond network between positions 210-216 and the surrounding amino acid residues disappeared completely (including three hydrogen bonds between L209 and G212 and T213, and two hydrogen bonds between S207 and G215 and A216). However, the hydrogen bond network corresponding to regions 217 to 219 with increased rigidity is largely preserved ( Figure 6C ). The loop containing positions 103-105 is located above the catalytic centre. Our previous study confirmed that the flexible changes in this region are related to the activity and stability of the enzyme. Under low-temperature (298-300 K) simulation conditions, S78T and Y217K with increased stability but decreased activity decreased their RMSF around position 100[13], while K12D and N109D with increased activity but decreased stability increased their RMSF around position 100[19]. Luo et al. designed M4 with increased stability and activity under high temperature conditions with increased 400K RMSF at positions 95-105[14]. In this study, the RMSF of the G131A mutant was increased at position 100 at 298K, which was basically in agreement with the previously reported results, confirming that the flexible change in this region of G131A could to some extent overcome the trade-off between stability and activity, i.e. it could improve thermostability while retaining better catalytic activity. Amino acid residues 40-43 are located in the Ca1 binding domain, and Ca ² ⁺ has been shown to be associated with the stability of nattokinase. Therefore, stiffening this domain may help to overcome the trade-off between stability and activity in the modification of nattokinase[3]. To analyse the effect of the G131A mutant on the active site, we first calculated the changes in the binding network of the tetrapeptide substrate to the active site of the enzyme. The results showed that the number of hydrogen bonds formed between G131A and tetrapeptide substrate (0.998 Å on average) was lower than that of wild type nattokinase (1.306 Å on average), which was consistent with the change in K m value of G131A and wild type nattokinase, indicating that the increase in enzyme catalytic rate ( k cat ) was the key factor determining the improvement in catalytic efficiency of G131A. The results of steered molecular dynamics further support the above conclusion: we performed molecular docking using the tetrapeptidic substrate suc-AAPF-pNA as a substrate and applied a multimolecule dynamic approach to pull the substrate out of the protein bindingpocket of each mutant; We then used steered molecular dynamics to pull the docked substrate onto the enzyme’s substrate binding centre 15 Å and analysed the changes in the interaction between the substrate and the enzyme during the stretching process. The results showed that the work of complete dissociation of the ligand from wild type nattokinase was 2669.15 kJ/mol, whereas the corresponding work of the G131A mutant was 2897.76 kJ/mol ( Figure S3 ). Furthermore, The analysis of the stretching process showed that the key residues (Ser221, His64 and their surrounding regions) of the active centre of the G131A mutant had more stable and longlasting hydrogen bonds to the substrate. In addition, G131A also showed a more stable hydrogen bond interaction in the region around amino acid position 100 ( Figure 7A ). The increased volume of the enzyme active centre has been demonstrated to facilitate the faster entry of the substrate into the active centre, thereby enabling its participation in hydrolysis[39]. Further analysis of the average conformation of the enzyme molecular dynamics simulation showed that the cavity volume of the active centre of the G131A mutant was 717.63 Å 3 , which was larger than that of the wild type nattokinase (699.71 Å 3 ). This suggests that the rapid transfer of substrate and product in the active centre of the G131A mutant may be one of the reasons for the improved catalytic efficiency of the G131A mutant ( Figure 7B ). 3.4 Fermentation process optimisation to enhance the yield of G131A mutants The heterologous expression of nattokinase in Escherichia coli is characterised by low expression levels and susceptibility to inclusion body formation[20]. Consequently, a sequential optimisation of the fermentation conditions (bottling volume, inoculum volume, induction temperature, pH, induction time, IPTG concentration) of the G131A mutant in Escherichia coli was conducted. A single-factor experiment was conducted to determine the optimal volume of bacterial solution to be bottled. The results indicated that a volume of 20% of the total bottling volume resulted in optimal expression, with a value of 569.17 U/mL ( Figure 8A ). In view of these findings, a subsequent investigation was undertaken to examine the influence of inoculum quantity and induction temperature on the expression of the G131A mutant. The results demonstrated that the impact of inoculum quantity on the expression of the G131A mutant was relatively minor in comparison to that of the induction temperature. The expression of the G131A mutant at a concentration of 576.29/mL was achieved simultaneously under the conditions of a 2% inoculum quantity and an induction temperature of 20°C ( Figure 8B-C ). The pH of the fermentation broth has been demonstrated to exert a substantial influence on the metabolism and enzyme expression of Escherichia coli[40]. The G131A mutant expression was further elevated to 593.29 U/mL when the pH was 8 ( Figure 8D ). The induction time was identified as a key determinant of soluble protein expression. Our single factor experiment, which varied the induction time, demonstrated that 20 h represented the optimal induction time for the G131A mutant, with an expression level of 615.14 U/mL ( Figure 8E ). Induction times either above or below this optimal point resulted in a substantial decrease in expression. Finally, the impact of IPTG concentration on protein expression was investigated. The G131A mutant exhibited the highest expression at an IPTG addition of 0.7 mM/ml, reaching 623.43 U/mL ( Figure 8F ). 4. CONCLUSION The molecular modification of nattokinase has been shown to yield enzyme molecules with enhanced catalytic performance. Furthermore, this process facilitates the elucidation of the structure-activity relationship of nattokinase. In this study, PoPMuSiC 2.1, a web server for the estimation of protein stability changes upon mutation and sequence optimality, was utilised to screen the catalytic properties of nattokinase. The mutant G131A, which exhibited enhanced catalytic performance, was identified through this process. A comparison of the mutant G131A with the wild-type nattokinase revealed a 1.5-fold increase in activity and a 4.5-fold increase in half-life, respectively, thereby overcoming the activation-stability trade-off that is typically observed in enzyme engineering. Computer simulations revealed that G131A has a more stable conformation, stronger substrate interaction and more suitable substrate entry pocket. This study provides a new research strategy for the structure-activity relationship study of nattokinase and even basic serine proteases. CREDIT AUTHORSHIP CONTRIBUTION STATEMENT Yuan Li and Jinyao Li: conceptualization; Yuan Li and Wenhui Zhu: methodology; Yuan Li and Wenhui Zhu: software; Yuan Li and Dong Yang: validation; Yuan Li, Aixia Ma and Huiyang Jia: formal analysis, investigation, data curation, and visualization; Yuan Li and Jinyao Li: resources; Yuan Li, Ye Ma, Kongfang Yu, and Hong Wang: writing—original draft preparation; Yuan Li: writing—review and editing; Jinyao Li and Yuan Li: supervision; Yuan Li: project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript. ACKNOWLEDGMENTS This research was funded by the Science and Technology Department of Xinjiang Uygur Autonomous Region, grant number 2022D01C96. DECLARATION OF COMPETING INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. REFERENCES 40. 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The residual activity is plotted as a percentage of the initial activity. Figure 3. The activity (A) and thermostability (B) of wild-type rAprY and a saturating mutation at amino acid residue 131 were determined by fibrin plate assay. The thermostability of each enzyme was determined by measuring residual activity after incubating the enzymes at 55℃ for 30 min. The residual activity is plotted as a percentage of the initial activity. Figure 4 .Enzymatic properties of rAprY and its mutant with single-point mutations. (A) The optimal pH of rAprY and single-point mutants was measured using different pH conditions; (B) The pH stability of rAprY and its mutant was determined by measuring residual activity after the enzyme was incubated in various pH conditions for 4h at 4℃; (C) The optimum temperature of rAprY and single-point mutants; (D) The thermostability of each enzyme was determined by measuring residual activity after incubating the enzymes at 55℃ for 0-100 min. The residual activity is plotted as a percentage of the initial activity. Figure 5. The root mean square deviation (RMSD) (A), Radius of Gyration (Rg) (B), solvent-accessible surface area (SASA) (C) and total potential energy (D) of rAprY and G131A mutant. Figure.6 RMSF (A), secondary structure changes (B) and local bonding changes at region 210-219 (C) for wild type nattokinase and G131A at MD at 298K. Figure 7. Mechanism analysis of the enhanced fibrinolytic activity of G131A mutant. (A)Changes in substrate interaction with wild-type rAprY and G131A mutant during steered dynamics simulations; (B) Volume and surface area determination of the catalytic center of wild-type rAprY and G131A mutant. Figure 8. Effects of different bottling quantity (A), inocuium size (B), fermentation temperature (C), pH (D), fermentation time (E), and induction concentration of IPTG (F) on the production of G131A mutant. Fig. 1: Figure 1. Folding free energy changes of wild-type nattokinase. The helix structure is shown in red. The sheet structure is shown in green. The turn structure is shown in pink. The coli structure is shown in light green. The bugle structure is shown in blue. Fig. 2: Figure 2. Alanine scan results of wild-type rAprY and each mutant crude enzyme were determined by fibrin plate assay for activity. (A) Enzyme activity; (B) thermostability. The thermostability of each enzyme was determined by measuring residual activity after incubating the enzymes at 55℃ for 30 min. The residual activity is plotted as a percentage of the initial activity. Fig. 3: Figure 3. The activity (A) and thermostability (B) of wild-type rAprY and a saturating mutation at amino acid residue 131 were determined by fibrin plate assay. The thermostability of each enzyme was determined by measuring residual activity after incubating the enzymes at 55℃ for 30 min. The residual activity is plotted as a percentage of the initial activity. Fig. 4: Figure 4. Enzymatic properties of rAprY and its mutant with single-point mutations. (A) The optimal pH of rAprY and single-point mutants was measured using different pH conditions; (B) The pH stability of rAprY and its mutant was determined by measuring residual activity after the enzyme was incubated in various pH conditions for 4h at 4℃; (C) The optimum temperature of rAprY and single-point mutants; (D) The thermostability of each enzyme was determined by measuring residual activity after incubating the enzymes at 55℃ for 0-100 min. The residual activity is plotted as a percentage of the initial activity. Fig. 5: Figure 5 .The root mean square deviation (RMSD) (A), Radius of Gyration (Rg) (B), solvent-accessible surface area (SASA) (C) and total potential energy (D) of rAprY and G131A mutant. Fig. 6: Figure 6. RMSF (A), secondary structure changes (B) and local bonding changes at region 210-219 (C) for wild type nattokinase and G131A at MD at 298K. Fig. 7: Figure 7 .Mechanism analysis of the enhanced fibrinolytic activity of G131A mutant. (A)Changes in substrate interaction with wild-type rAprY and G131A mutant during steered dynamics simulations; (B) Volume and surface area determination of the catalytic center of wild-type rAprY and G131A mutant. Fig. 8: Figure 8 .Effects of different bottling quantity (A), inocuium size (B), fermentation temperature (C), pH (D), fermentation time (E), and induction concentration of IPTG (F) on the production of G131A mutant. Table 1: Table 1. Specific activities and kinetic parameters of wild-type rAprY and G131A mutant. Table 2: Table 2. MD simulation results of wild type nattokinase nattokinase (AprY) and G131A mutant at 298K for 100 ns. Information & Authors Information Version history V1 Version 1 17 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords catalytic performance fermentation process molecular dynamics nattokinase site-saturation mutagenesis Authors Affiliations LI YUAN 0000-0002-4024-6174 Xinjiang University View all articles by this author Wenhui Zhu Xinjiang University View all articles by this author Dong Yang Xinjiang University View all articles by this author Huiyang Jia Xinjiang University View all articles by this author Aixia Ma Xinjiang University View all articles by this author Kongfang Yu Xinjiang University View all articles by this author Hong Wang Xinjiang University View all articles by this author Ye Ma [email protected] Xinjiang University View all articles by this author Jinyao Li Xinjiang University View all articles by this author Metrics & Citations Metrics Article Usage 164 views 156 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation LI YUAN, Wenhui Zhu, Dong Yang, et al. Improvement of the Catalytic Activity of nattokinase by Site-Saturation Mutagenesis of Glycine 131. Authorea . 17 March 2025. DOI: https://doi.org/10.22541/au.174221477.73527147/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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