Improving enzymatic properties of BlTDH from Bacillus licheniformis through site-directed mutagenesis

Abstract

L-threonine dehydrogenase (TDH) is a rate-limiting enzyme in the biosynthesis pathway of 2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine, which are widely used food additives. However, natural TDH enzymes suffer from low catalytic activity and poor environmental adaptability, limiting their industrial applications. This study hypothesized that strategic site-directed mutagenesis of conserved amino acid residues within the substrate-binding pocket and catalytic domain could affect both enzymatic activity and environmental stability of TDH. To test this hypothesis, BlTDH from Bacillus licheniformis was selected as the target enzyme, and structure-oriented alanine substitution mutagenesis was systematically applied to five conserved residues (T94, H95, N157, T293, and G294). The results showed that among the five mutants (T94A, H95A, N157A, T293A and G294A), N157A mutant had a specific enzyme activity of 120.47 ± 1.88 mU/mg, which was 2.1 times higher than that of the wild-type. In addition, the N157A mutant showed better temperature stability and pH adaptability. Structural analysis revealed that the side chain volume of N157A mutant decreased, thereby expanding substrate binding space and reducing steric hindrance, which was conducive to the catalytic reaction. These findings validated the original hypothesis, demonstrating that rational amino acid substitutions can significantly affect the catalytic performance of TDH. The superior N157A mutant presented immediate commercial viability for industrial-scale production of food-grade pyrazine additives. Meanwhile, the established structure-activity relationship provides an engineering framework for optimizing the application of relevant NAD⁺-dependent dehydrogenases in biotechnology.

Highlights

• •Based on structure-oriented mutagenesis, five mutants of BlTDH were obtained.
• •N157A, T94A and T293A mutants showed increased enzyme catalytic activity.
• •The N157A mutation had enhanced thermal stability and pH adaptability.

1. Introduction

2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP) have been widely discovered in traditional-fermented and heat-treated foods, including Baijiu, coffee, tea, nuts and roasted foods, and can be treated as raw materials for food, beverage, and tobacco flavor (Fayek et al., 2023; Liu & Quan, 2024). In food industries, 2,5-DMP and TMP have been used to improve the flavor of various foods such as bread, pudding, meat, soft drinks, milk, etc. (Guo et al., 2018; Yang et al., 2021). L-threonine dehydrogenase (TDH) acts as a key rate-limiting enzyme in the biosynthesis pathway of 2,5-DMP and TMP (Fig. 1), and its catalytic efficiency determines the conversion rate and yield of corresponding products (Zhang et al., 2019).

Fig. 1.

The biosynthesis pathway of 2,5-DMP and TMP.

TDH is a NAD⁺-dependent redox enzyme that can catalyze the oxidation of L-threonine to 2-amino-3-ketobutyric acid, accompanied by the reduction of NAD⁺ to NADH (Adjogatse et al., 2021). In recent years, with the rapid development of biotechnology and synthetic biology, TDH has received extensive attention due to its potential applications in fields such as the synthesis of pharmaceutical intermediates (Motoyama et al., 2021) and metabolic engineering (Liu et al., 2024). However, natural TDH faces many challenges in industrial applications, including insufficient catalytic efficiency, limited environmental adaptability and poor stability (Liu et al., 2023). The research on TDH mainly focuses on the separation and purification of enzymes, identification of basic characteristics and exploration of catalytic mechanisms. Earlier studies have shown that His90 and Cys38 mutations in Escherichia coli TDH substantially altered the catalytic efficiency (Johnson et al., 1998; Johnson & Dekker, 1998), laying the foundation for targeted mutagenesis approaches. Subsequently, the key catalytic residues of Pyrococcus horikoshii TDH was identified by crystal structure analysis (Higashi et al., 2008), and the activity of TDH in Thermococcus volcanium was enhanced by 4-fold by systematic mutagenesis(Desjardins et al., 2017).

Structure-oriented protein engineering provides a rational framework for predicting the functional consequences of specific mutations (Schwarte et al., 2017). It can avoid the resource consumption of large-scale random screening and provide a clear framework for understanding the molecular mechanism of mutations. Through structure-oriented site-directed mutagenesis, the activity of Lactobacillus brevis OTC protease was increased by 2–3 times (Fang et al., 2020) and the thermal stability of Geobacillus thermoglucosidasius FAE protein was improved (Yang et al., 2022). Due to the minimal steric requirements and neutral chemical properties of alanine, the alanine substitution strategy has distinct advantages over other mutagenesis methods and can systematically evaluate the contribution of individual residues without introducing promiscuous structural perturbations (Betts & Russell, 2003). Systematic alanine substitution analysis of pullulanase CBM68 identified critical binding residues and significantly enhanced its affinity for substrates through structure-oriented mutagenesis (Zeng et al., 2019). Additionally, the alanine substitution of larger residues in the MAS1 lipase substrate-binding pocket also prominently improved certain properties of the enzyme. The catalytic efficiencies of the obtained mutants H108A, F153A, and V233A were increased by 2.3 times, 2.1 times, and 1.4 times, respectively, while the thermal stability of F153A at 60 °C was increased by 5 times (Zhao et al., 2018). Furthermore, the iterative alanine scanning mutagenesis enabled L-amino dehydrogenase to exhibit the specificity and activity of aromatic ketones, which greatly expanded the substrate range of this enzyme (Mu et al., 2021).

Bacillus was reported as the main microorganism for producing 2,5-DMP and TMP (Dong et al., 2024). The TDH derived from B. licheniformis ATCC14580 had the enzyme activity of 99.1 ± 7.3 mU/mg (Yu et al., 2021). Therefore, improving the catalytic ability of Bacillus-derived TDH has become an urgent problem to be solved. Based on the structural conservation mode and catalytic mechanism analysis, we hypothesize that strategically replacing the key residue with alanine in the substrate-binding and catalytic regions will substantially enhance the catalytic efficiency and environmental robustness of BlTDH. In this study, five conserved residues of BlTDH were screened out as directed mutation sites (T94, H95, N157, T293, G294) through homology modeling and molecular docking analysis. The experimental design employed comprehensive kinetic characterization and stability assessment to establish structure-activity relationships and determine excellent mutants for industrial applications.

2. Materials and methods

2.1. Strains and reagents

B. licheniformis YC7 was previously isolated and identified from Daqu based on its high alkylpyrazine production capacity. E. coli BL21 (DE3) and E.coli DH5α competent cells were used for recombinant protein expression and plasmid construction, respectively.

BlTDH gene was cloned from B. licheniformis YC7 using its genomic DNA. The gene-specific primers contained NdeI and XhoI restriction sites, respectively (forward primer: 5’-CGCGGATCCATCTTGGACGGAATGAAAGCGCT-3′; reverse primer: 5’-CCGCTCGAGCTACGGTATCAGTACGACTTTGC-3′). The amplified BlTDH gene was subsequently ligated into the pET-28a expression vector via standard restriction enzyme digestion and T₄ DNA ligase-mediated ligation. The recombinant plasmid was transformed into E. coli DH5α competent cells. The positive transformants were identified by colony PCR and verified by DNA sequencing analysis, and preserved at −80 °C for long-term storage.

The plasmid mini purification kit, kanamycin, isopropyl-β-D-thiogalactoside (IPTG) and Ni-NTA-Sefinose™ resin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). All materials and reagents used in this study were analytical reagent grade or higher quality. DNA sequencing and primer synthesis were performed by TsingKe Biological Technology (Nanjing, China).

2.2. Design and construction of BlTDH mutants

The selection of BlTDH mutation sites was based on structural prediction and sequence conservation analysis. Three-dimensional (3D) structural modeling of BlTDH was performed using SWISS-MODEL (https://swissmodel.expasy.org/) homology modeling platform. The molecular docking simulation between BlTDH protein and substrate L-threonine was conducted using AutoDock software. The molecular docking results were visualized and analyzed using PyMOL software to identify potential key interaction sites based on binding affinity scores and intermolecular contact distances. To examine the evolutionary conservation of these sites, a multiple sequence alignment of TDH proteins from different species was performed using the online software ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/).

Site-directed mutagenesis of BlTDH was performed using PCR-based methodology (Schwartz et al., 1991). The pET-28a-BlTDH plasmid was used as the PCR template, and the mutation primers were shown in Table S1. The PCR cycling program was carried out as follows: 94 °C for 3 min, 94 °C for 90 s, 56 °C for 60 s, 72 °C for 10 min (35 cycles) and 72 °C for 15 min. The reaction product was digested with DpnI to remove the template, and then transformed into competent cells of E. coli DH5α. Positive transformants were identified by PCR, and plasmids were extracted from for DNA sequencing analysis to confirm the mutation sites.

2.3. Protein expression and purification

The successful mutants were transformed into E.coli BL21 (DE3) and inoculated into LB medium containing kanamycin (50 μg/mL). When OD₆₀₀ reached 0.6–0.8, IPTG was added until its final concentration was 1 mmol/L to induce protein expression. After shaking and culturing at 25 °C for 14 h, bacteria cells were collected by centrifugation and resuspended with pre-cooled PBS buffer (pH 7.5). After sonication, the cell extract was centrifuged at 12,000g for 20 min at 4 °C to collect the supernatant. The target protein was purified by nickel column affinity chromatography. The PBS buffer containing 10–500 mmol/L imidazole was used for gradient elution. The purified proteins were analyzed by SDS-PAGE, and the protein concentration was determined by using NanoDrop (Thermo Fisher Scientific, Wilmington, USA).

2.4. Enzyme assay of BlTDH and its variants

Enzymatic activity was determined by monitoring the formation of NADH at 340 nm using a UV-1800 spectrophotometer (Aoe Instruments (Shanghai) Co., Ltd., China) based on the established spectrophotometric method (Zhang et al., 2019). The standard reaction system (with a total volume of 3 mL) was as follows: 2.4 mL of 50 mmol/L Tris-HCl buffer (pH 8.0), 0.1 mL of 20 mmol/L NAD⁺, 0.3 mL of 100 mmol/L L-threonine and 0.2 mL of appropriately diluted enzyme solution. After preheating in a water bath at 37 °C for 1 min, the OD₃₄₀ absorbance was continuously monitored for 2 min. One unit of enzyme activity represents the amount of enzyme that catalyzes the generation of 1 μmol NADH per minute. Specific enzyme activity is defined as the enzyme activity units per milligram of total protein (U/mg protein).

2.5. Determination of optimum temperature and pH of BlTDH and its variants

After adding the substrate to the enzyme solution, the reaction was immediately conducted under the conditions of 30, 40, 50, 60, 70 and 80 °C to measure the enzyme activity and determine the optimal temperature. The reactions were performed in a temperature-controlled water bath with an accuracy of ±0.1 °C to ensure accurate temperature maintenance. The relative enzyme activity was calculated by taking the highest activity as 100 %.

To determine the optimal pH, the enzyme activity was measured in different pH buffers (pH 3.0–11.0, with an interval of 1.0). The buffer solutions (50 mmol/L) used in the experiment were as follows: citric acid-phosphate buffer solution with pH 3.0–6.0, Tris-HCl buffer solution with pH 7.0–9.0, carbonate-bicarbonate buffer solution with pH 10.0–11.0.

2.6. Thermal and pH stability of BlTDH and its mutants

The thermal and pH stability of BlTDH and its mutants were determined according to Liu et al. (2023). For thermal stability determination, the enzyme solution was incubated at 20, 30, 40, 50 and 60 °C for 20 min, respectively. After thermal treatment, samples were immediately cooled to 4 °C for 5 min. The residual enzyme activity was determined under the optimal reaction temperature condition. The relative residual activity was calculated with the activity of control samples as 100 %. For pH stability determination, the enzyme solution was placed in different pH buffers (pH 3.0–9.0) at 30 °C for 1.5 h, and the residual enzyme activity was determined under the optimal condition. The relative residual activity was calculated with the initial activity as 100 %.

2.7. Statistical analysis

All experiments were performed with three technical replicates and repeated in three independent experiments. Data was presented as mean ± standard deviation. One-way ANOVA was used to analyze the significance differences of the enzyme activity.

3. Results

3.1. Selection of targeted mutation sites

In this study, the 3D structural model of BlTDH protein was constructed using SWISS-MODEL (Fig. 2A). As shown in the Ramachandran conformation diagram, the quality of the model was evaluated (Fig. 2B). The result showed that 92.5 % of the amino acid residues were located in the most favorable region, 7.5 % in the permissive region, and no residues in the marginal or the forbidden region, confirming the high quality reliability of the constructed model. The substrate L-threonine was molecularly docked with BlTDH protein using AutoDock software. The molecular docking results were analyzed using PyMOL software, and it was found that L-threonine mainly interacted with THR-94 (T94), HIS-95 (H95), ASN-157 (N157), THR-293 (T293) and GLY-294 (G294) sites of the BlTDH protein (Fig. 2C). T94 was located near the NAD⁺ binding pocket, which was a key region connecting the NAD⁺ binding domain and the catalytic domain. N157 was located within the active pocket and directly participated in the binding process of the substrate. H95 formed key hydrogen bonds with the substrate, while T293 and G294 were located in the catalytic domain. A two-dimensional (2D) interaction diagram was established, detailing various interaction patterns between L-threonine and the surrounding amino acid residues (Fig. 2D). It was showed that T94 interacted with the side chain donor-acceptor formed by the substrate, and H68 interacted with the aromatic hydrogen of the substrate. The above results provided a reliable structural basis for the selection of sites for site-directed mutation.

Fig. 2.

Three-dimensional structure model of BlTDH and substrate docking analysis. (A) 3D structure model of BlTDH. (B) Laplace conformation diagram. (C) Molecular docking 3D diagram. (D) Molecular docking 2D interaction diagram.

To understand the conserved regions of BlTDH protein, multiple sequence alignments were performed between BlTDH and other MDR-TDH proteins from P. furiosus, T. kodakaraensis, and B. amyloliquefaciens (Fig. 3). Notably, the key interaction sites identified by molecular docking were precisely located in some conserved regions, further verifying the importance of these sites in the substrate recognition and catalysis. Based on the dual validation of structural simulation and sequence analysis, the T94, H95, N157, T293 and G294 sites of BlTDH protein were ultimately selected as the targets of site-directed mutations.

Fig. 3.

Multiple sequence alignment of TDH proteins.

3.2. Construction and purification of BlTDH mutants

The mutations were introduced via site-directed mutagenesis based on PCR. The results of sequencing analysis confirmed that all target sites were successfully mutated to alanine (Fig. 4A). Accordingly, the original threonine (ACG) was replaced by alanine (GCG) in T94A mutant. Histidine (CAC) was replaced by alanine (GCG) in H95A mutant. The N157A, T293A and G294A mutants also showed the corresponding codon changes, respectively. Subsequently, we conducted heterologous expression and purification of BlTDH gene and its four mutants. The purified target proteins were analyzed using 12 % SDS-PAGE (Fig. 4B). Both the wild-type and each mutant had clear protein bands (about 40 kDa), which was consistent with the theoretical molecular weight of BlTDH.

Fig. 4.

Sequencing verification of BlTDH mutants (A) and SDS-PAGE analysis of wild-type BlTDH and its mutants (B).

3.3. Enzyme activity analysis of BlTDH and its mutants

To reveal the effect of different mutation sites on the catalytic function of BlTDH, the enzyme activity of BlTDH and its mutants were measured (Fig. 5). The results showed that the enzymatic activities of the T94A, N157A and T293A mutants were significantly increased. Among them, the enzyme activity of the N157A mutant was the highest, reaching 120.47 mU/mg, which was nearly 2.1 times higher than that of the wild type. This indicated that the mutations at these three sites had a positive impact on the catalytic efficiency of BlTDH. In contrast, the mutations at the H95 and G294 sites markedly reduced the enzyme activity, suggesting that these two sites played a key role in maintaining the catalytic function of BlTDH. It can be seen that the different sites of BlTDH protein play distinct roles in its catalytic function, which provides an important basis for understanding the relationship between protein structure and function.

Fig. 5.

Comparison of specific enzyme activity of BlTDH and its mutants. The vertical bars with different lower-case letters are significantly different from each other at p < 0.05 (one-way ANOVA).

3.4. Optimum temperature and pH of BlTDH and its mutants

To determine the optimal conditions for TDH to exert catalytic function, the enzyme activities of BlTDH and its mutants under different temperature ranges (30–80 °C) and various pH values (3−11) were determined (Fig. 6). As shown in Fig. 6A, the relative activity of wild-type BlTDH was highest at 60 °C. Meanwhile, the H95A and G294A mutants remained consistent with the optimal temperature of the wild type. However, the optimal temperature for the T94A, N157A and T293A mutants dropped to 50 °C. It was worth noting that the N157A mutant exhibited higher relative activity at lower temperature (40 °C), which is of great value for industrial applications of this enzyme under mild temperature conditions. The adaptability analysis of pH showed that the relative activities of wild-type BlTDH and its mutants were the same and reached the maximum value at pH 8.0 (Fig. 6B). At the same time, we found that the N157A mutant had higher enzyme activity than the wild-type and other mutants under acidic conditions of pH 3.0–5.0 and alkaline conditions of pH 8.0–10.0, indicating that this mutant had a wide pH adaptability.

Fig. 6.

Optimum temperature (A) and pH (B) of BlTDH and its mutants.

3.5. Thermal and pH stability of BlTDH and its mutants

To analyze the stability of these enzymes, we determined the residual enzyme activities of the wild-type and its mutants after treatment at different temperatures and pH values (Fig. 7). The results displayed that the N157A mutant exhibited significantly higher relative activity than the wild-type BlTDH and other mutants, especially at 40–60 °C (Fig. 7A). Especially, when the treatment temperature was 60 °C, the residual enzyme activity of the N157A mutant was about 50 % of its initial enzyme activity, while the wild-type BlTDH retained only about 30 %. Nevertheless, the residual relative activities of the H95A, T293A, and T94A mutants were significantly lower than that of the wild-type, especially at 30–60 °C. Among them, the H95A mutant was the most vulnerable, retaining only about 10 % of its initial relative activity at 60 °C (Fig. 7A). The remaining enzyme activities of the wild-type and its mutants showed the highest values respectively at pH 7.0 (Fig. 7B).Obviously, the pH stability of the H95A, T293A and G294A mutants was generally worse than that of the wild-type. However, the N157A mutant retained a relatively high residual enzyme activity, especially maintaining relative activity of approximately 40 % and 55 % at pH 4.0 and pH 9.0, respectively, which was much higher than that of the wild-type under the corresponding conditions (Fig. 7B). These results suggested that the mutation at the N157 site signally enhanced the temperature and pH stability of the corresponding mutant.

Fig. 7.

Thermal (A) and pH (B) stability of BlTDH and its mutants.

3.6. Structural analysis of the five mutants

To elucidate the molecular mechanism of enzyme activity changes after site mutation, PyMOL software was used to analyze and visualize the changes in protein structure before and after mutation (Fig. 8 and Fig. 9). The mutation at T94 caused threonine, which originally had a hydroxyl side chain, to be replaced by alanine, which had a methyl side chain (Fig. 8A, B). The mutation at N157 led to the substitution of asparagine, which originally had a large polar amide side chain, with alanine, which had a relatively small methyl side chain (Fig. 8C, D). The mutation of T293A removed the hydroxyl side chain, altering the local hydrogen bond network and charge distribution (Fig. 8E, F). The H95A mutation resulted in the replacement of histidine with alanine, the removal of the key imidazole side chain involved in substrate recognition and proton transfer, and the disruption of the original catalytic network and hydrogen bond system (Fig. 9A, B). The mutation site was located in the catalytic domain of BlTDH. The G294A mutation replaced the highly flexible glycine, which originally had no side chains, with alanine containing a methyl side chain (Fig. 9C, Fig. 9D).

Fig. 8.

Structural analysis of T94A, N157A and T293A mutation sites.

Fig. 9.

Structural analysis of H95A and G294A mutation sites.

To further explore the reasons for the increase of enzyme activity, thermal and pH stability of the N157A mutant, the molecular docking analysis between the mutant and the substrate L-threonine was performed by AutoDock software (Fig. 10). It was found that L-threonine formed more stable multiple hydrogen bonds with the key amino acid residues ARG-59, LYS-61 and VAL-120 of the N157A mutant, with bond lengths of 3.1 A, 1.7 A, 2.0 A, 2.6 A and 2.2 A respectively.

Fig. 10.

Molecular docking of N157A mutant with substrate L-threonine.

4. Discussion

In this study, the change of catalytic efficiency of BlTDH from Bacillus licheniformis was realized through site-directed mutagenesis. The temperature stability and pH adaptability of the corresponding mutants were significantly altered. Meanwhile, the molecular structures of the mutants were analyzed to reveal the reasons for the different catalytic efficiencies between the mutants and the wild-type.

Studies on Streptomyces klenkii phospholipase D demonstrated that the transformation from polar amino acids to non-polar amino acids could promote the interaction between the enzyme and the substrate, and reduce the energy barrier for the substrate to enter the active center (Hu et al., 2024). In this study, the N157A mutant also involved the substitution of polar amino acids (asparagine) with non-polar amino acids (alanine) (Fig. 4A), which resulted in a significant increase in the enzyme activity (Fig. 5). It was speculated that this mutation (N157A) optimized the overall folding energy pattern of the enzyme, thereby enhancing its adaptability to extreme environments (Gianni et al., 2021). In addition, this structural change increased the area of the substrate binding pocket and reduced the spatial obstruction (Hu et al., 2023), improving the substrate proximity and binding efficiency. Compared to the wild-type, the N157A mutant formed a more accessible substrate-binding structure (Fig. 8C, D). As a result, the mutation of N157A not only improved the enzyme activity, but also enhanced the enzyme adaptability to different temperature and pH environments (Fig. 7), providing important clues for the construction of highly efficient TDH variants adapted to multiple industrial conditions. In contrast, the wild-type had relatively bulky side chain, which occupied portions of the substrate binding space and increased the interaction distance between L-threonine and key residues (Fig. 2C). However, the N157A mutant-substrate complex exhibited superior stability and a more favorable conformation (Fig. 10), further explaining the significantly improved catalytic efficiency and environmental adaptability of the N157A mutant at the molecular level.

The catalytic efficiency of the T293A mutant was slightly improved (Fig. 5), which was analogous to the findings on the T136A mutant reported by Mu et al. (2021). In their study, molecular docking analysis revealed that the removal of the hydroxyl side chain in the T136A mutant expanded the volume of the substrate binding pocket. Specifically, this substitution optimized the charge distribution around the active center and reduced the steric hindrance effect of the substrate entering the active center. Therefore, this led to a decrease in the energy barrier for substrate binding and an increase in the affinity and catalytic efficiency between the enzyme and the substrate (Mu et al., 2021). The modest increase of enzyme activity was also found in the T94A mutant (Fig. 5). The T94A mutation eliminated the hydrogen bond restriction formed by the hydroxyl side chain, thereby reducing the rigidity of the binding pocket and decreasing the steric hindrance when the substrate bound to NAD⁺. This might be the main reason for the improved catalytic efficiency of the T94A mutant (Sinha et al., 2020). In addition, the increased enzyme activity of the T94A mutant might be due to the reduced constraint of the local hydrogen bond network after the mutation, which could enhance the conformational flexibility of the enzyme and contribute to the catalytic reaction (Wang et al., 2017).

The adverse effects observed in the H95A and G294A mutants provided us different mechanistic insights (Fig. 5). The H95A mutation disrupted the original catalytic network and hydrogen bond system, consequently leading to a substantial decline in activity. This observation was consistent with the research on E. coli TDH (Johnson & Dekker, 1998). Their results demonstrated that His-90 served as a key residue for precisely targeting NAD⁺ cofactors and substrates during catalysis, and also facilitated the necessary proton transfer steps (Johnson & Dekker, 1998). Therefore, the H95A mutation was likely to disrupt these crucial interactions, thereby resulting in a significant reduction in the catalytic efficiency. The G294A mutation introduced a methyl side chain, which could limit the conformational flexibility required by the enzyme in the catalytic process (Zhang et al., 2025). Previous investigations have shown that glycine-to-alanine mutations can significantly reduce enzyme activity by disrupting spatial flexibility, confirming that minor structural modifications near the active site can substantially affect substrate specificity and catalytic efficiency (Seah et al., 1995).

Interestingly, we found that after the site mutation, the optimal temperature of the three mutants (T94A, N157A and T293A) decreased to 50 °C, while the optimal temperature of the wild-type was 60 °C (Fig. 6A). Beneficial mutations (e.g., T94A, N157A and T293A) reduced steric constraints or polar interactions (e.g., removal of hydroxyl/amide groups), increasing the local flexibility of active-site regions (Richard, 2019). These structural changes enhanced conformational dynamics at moderate temperatures, accelerating substrate binding/catalysis. However, at high temperatures, excessive flexibility compromised the integrity of the structure, reducing the optimal thermal perfomance (Miller, 2017). The optimal temperature for the other two mutants with reduced enzyme activity also remained at 60 °C (Fig. 6A). Detrimental mutations (e.g., H95A, G294A) disrupted the interactions necessary for catalysis (e.g., H95A cancels proton transfer) or introduced rigidity (e.g., G294A adds methyl sterics). These perturbations impaired the function of the enzyme at different temperatures, but had less of an impact on the optimal thermal performance, as their primary defect was catalytic rather than stability-related (Chiuri et al., 2009).

The comprehensive characterization of BlTDH mutants provides significant insights into structure-function relationships and demonstrates substantial improvements in catalytic performance through rational protein engineering. The N157A mutant demonstrated significant practical value for industrial applications, with 2.1-fold (Fig. 5) enhanced activity and improved environmental adaptability (broader pH tolerance and thermal stability) (Fig. 7) enabling cost-effective production of food-grade pyrazine additives. The enhanced catalytic efficiency directly reduced enzyme requirements and production costs for manufacturing 2,5-DMP and TMP used in food, beverage, and flavor industries. Further specialized research in the future may include the optimization of food matrices, sensory evaluation and regulatory compliance assessment.

5. Conclusion

In this study, we systematically evaluated the effects of five conserved sites (T94, H95, N157, T293 and G294) on the catalytic function of BlTDH by using a structure-oriented site-directed mutation strategy. Among the five mutants, the N157A mutant was the most outstanding, with about 2.1 times higher enzyme activity than the wild-type, and showed a wider pH adaptation range and higher thermal stability. Additionally, structural analysis revealed that the N157A mutation expanded the substrate-binding space, reduced steric hindrance, and optimized the substrate-enzyme interaction network by reducing the side chain volume. These findings clarify the role of specific amino acid residues in substrate recognition, binding and catalytic transformation, providing new molecular insights into the catalytic mechanism of BlTDH. The N157A mutant provides commercial viability for industrial pyrazine production and enhances economic feasibility. This also lays a scientific research foundation for the development of cost-effective food additives through biotechnology processes.

CRediT authorship contribution statement

Xun Liu: Writing – review & editing, Writing – original draft, Software, Funding acquisition, Conceptualization. Hongyi Gu: Writing – original draft, Visualization, Software, Methodology. Han Li: Resources, Methodology, Data curation. Shuanglian Chen: Visualization, Software. Zhen Tang: Formal analysis, Data curation. Wenli Quan: Writing – review & editing, Supervision, Project administration, Conceptualization.

Funding

This work was supported by the Natural Science Foundation of Sichuan Province of China (No. 2022NSFSC1782) and Sichuan Province Scientific Research Foundation for the Returned Overseas Chinese Scholars (2022).

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.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.