The CGTase gene derived from Bacillus sp. G1 (NCBI number: P31746) was obtained from the National Center for Biotechnology Information.¹ Escherichia coli DH5α [F⁻, φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(r_K⁻, m_K⁺), phoA, supE44, λ⁻, thi-1, gyrA96, relA1] was used for plasmid construction, and BL21 (DE3) {F⁻ ompT hsdS (r_B⁻ m_B⁻) dcm⁺ Tet^r galλ (DE3) endA Hte [argU proL Cam^r] [argU ileY leuW Strep/Spec^r]} was applied for gene expression. All plasmids used to express recombinant protein were derived from pET28a.

Bacillus sp. G1 CGTase (BsCGT) (Goh et al., 2012b) structure was obtained by comparison with the structure of B. thermophilus CGTase (PDB ID: 1CYG) (Song et al., 2023), which share 62.15% sequence similarity. Homology modeling using the SWISS-MODEL online service generated a three-dimensional structural model of BsCGT (Figure 1A1) which was employed in docking analyses. The structural integrity of the BsCGT model was assessed using the SAVES server. ²

This study employed a one-step PCR method for targeted mutagenesis of plasmids. Specific primers for the CGTase gene from Bacillus sp. G1 facilitated targeted mutations, as detailed in Supplementary Table S1 (rows 1–27). The construction of thermostable mutant was initially carried out by importing the 3D structural model of CGTase was imported into PyMOL for structural analysis and image visualization. Discovery Studio was utilized to dock CGTase and calcium ions, identify amino acid residues near the binding site, conduct virtual mutations on selected residues, and calculate the energy change associated with these mutations near the binding site (Li et al., 2023). Mutations were performed on the plasmid using the one-step PCR method outlined. With the template containing the single mutation N33K, Primer sequences for these mutations are detailed in Supplementary Table S1 (rows 28–36). Following DpnI digestion to remove the template, the product was transformed into E. coli DH5α. The transformed cells were selected on Luria-Bertani (LB)-kanamycin (Kan) agar, cultured in LB-Kan liquid, and plasmid DNA was extracted using the TIANGEN kit for identification and sequencing. The mutant plasmids were then chemically transformed into E. coli BL21(DE3) (Figure 1B). This study analyzed enzyme expression at different fermentation temperatures.

The engineered mutant strains were stored at −80°C in glycerol and revived on LB-Kan plates overnight at 37°C. Selected colonies were grown in LB-Kan broth at 37°C, 200 rpm for 8–10 h. These starter cultures were then inoculated into TB-Kan broth at a 5% rate and incubated until OD600 reached 1.5–2.0. IPTG was added to induce expression, and the cultures were transferred to 25°C for 12 h of fermentation.

The mutant CGTase and wild-type CGTase were purified using Ni-NTA His Bind Resin (7SEA Biotech). The enzyme concentration was measured using the microplate method with a BCA kit (Boster Biological Technology).

The disproportionation activity of CGTase was evaluated using the method described by van der Veen et al. (2000). The assay mixture included 12 mM 4-nitrophenyl-α-D-maltoheptaose-4,6-O-ethylidene (EPS) and 10 mM maltose, both obtained from Shanghai Yuanye Bio-Technology Co., Ltd. The procedure was initiated with a pre-incubation at 55°C for 10 min, followed by the addition of appropriately diluted CGTase for another 10 min at the same temperature. Afterwards, the reaction was incubated with 100 μL of α-glucosidase (10 U mL⁻¹) (EC 3.2.1.20 from Shanghai Yuanye Bio-Technology Co., Ltd) at 37°C for 1 h. The reaction was terminated by adding 100 μL of 1 M sodium carbonate, and absorbance was measured at 401 nm. Enzyme activity, expressed in units per milliliter (U mL⁻¹), is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of EPS per minute (van der Veen et al., 2001).

Maltose solutions at concentrations of 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, 10, 20, 50, and 100 mM were prepared and preheated. The enzyme activity at these varying substrate concentrations was assessed under optimal reaction conditions: pH 6.0 and a temperature of 55°C, using the method described in Section 2.5. The resulting data were analyzed using GraphPad Prism software for curve fitting, from which kinetic constants were derived.

For the synthesis of trehalose, the purified fusion enzymes, Sase-CcDoc and CtDoc-Hase, along with the scaffold protein ScafCCR, were initially assembled into a bi-enzyme complex system. Specifically, they were mixed at an equimolar ratio and underwent a reaction in a constant-temperature water bath at 37°C for 2 h (Wang et al., 2023). Subsequently, 200 g L⁻¹ maltodextrin (with a glucose equivalent of 9) was employed as the substrate. The assembled double enzyme was added, in conjunction with 5 U g⁻¹ Pullulanase, and the reaction lasted for 48 h at 60°C, with regular sampling. Moreover, CGTAses were introduced and samples were collected at regular intervals (Chen et al., 2020). To inactivate the enzymes, the sample was boiled for 10 min following the reaction, followed by the addition of amylase, and then shaken in a water bath at 60°C for 6 h. Eventually, the mixture was boiled for another 10 min to ensure the complete deactivation of the enzymes.

After the reaction was completed, the resulting solution was centrifuged at 20,000 × g for 10 min to separate the supernatant, which was then appropriately diluted. The trehalose content in the conversion solution was quantified using high-performance liquid chromatography (HPLC). This was performed on a Shimadzu HPLC system (LC-10ATV) equipped with an Inertsil NH₂ chromatography column (4.6 mm × 250 mm, 5 μm, GL Sciences (Shanghai) Co., Ltd). The analysis conditions included a mobile phase of acetonitrile to water in a 4:1 ratio, a flow rate of 1.0 mL/min, and a column temperature of 40°C (Trakarnpaiboon and Champreda, 2022).

Each experiment included three replicates. Results are presented as the average value ± standard deviation (SD). Statistical evaluations were performed using one-way analysis of variance (ANOVA). The Origin 9.4 software platform (OriginLab Corporation, United States) was utilized for statistical analysis. Post-hoc comparisons of means were conducted using Tukey’s Honest Significant Difference (HSD) test to identify statistically significant differences between groups.

Evaluation of the Ramachandran plot of the BsCGT model (Figure 1A2) obtained from homologous modeling, indicated that the model was credible, since 86.6% of residues fell within the most favored region, 99.7% in allowed regions and only 0.3% in disallowed regions. To identify mutations that enhance substrate affinity, this enzyme model was docked with maltose, and the amino acids near the substrate binding site were identified according to the docking results. An alanine scan is then performed to determine which amino acids may change affinity when mutated. Virtual saturation mutations of these amino acids were subsequently performed, and the results showed that mutations at Asn33, Tyr119, Tyr122, Leu216, His255, Glu258, Pro394, and Glu566 may be conducive to improving affinity (Table 1).

Enzyme expression was tested under different fermentation temperatures, revealing that cultivation at 25°C yielded optimal enzyme activity (Supplementary Figure S1). Thus enzymatic activity was evaluated for several single-point mutants, identifying mutations with advantageous characteristics. These mutations underwent further mutagenesis and activity results are shown in Supplementary Table S2. Among these, mutant N33K showed an activity (20.33 U mL⁻¹) that was significantly higher than the one of the wild type (12.60 U mL⁻¹) and identified as the optimal mutant.

The molecular mass of the recombinant mutase N33K, produced through fermentation, was determined using SDS-PAGE after nickel column purification (Figure 1E). Subsequent characterization of the N33K mutant included assessing its optimal temperature, thermal stability, optimal pH, and pH stability. These investigations revealed that the amino acid changes did not significantly alter these enzymatic performance characteristics (Supplementary Figure S2).

Table 2 presents the kinetic constants using maltose as the substrate for both wild-type BsCGT enzyme and its N33K mutant variant. The wild-type BsCGT showed a catalytic constant (k_cat) of 22.81 s⁻¹ and a Michaelis–Menten constant (K_M) of 16.63 mM, indicating efficient catalytic activity and substrate binding affinity. The N33K mutant exhibited a reduced K_M value (14.47 mM), indicating a stronger binding affinity for maltose, as it requires less substrate concentration to achieve half the maximum reaction velocity. The mutant also showed a higher k_cat (36.17 s⁻¹), indicating an increased turnover number and enhanced ability to rapidly convert substrate to product.

Molecular dynamics simulations were performed to compare the substrate interactions of the CGTase N33K mutant and its wild-type counterpart. The simulations revealed that the N33K mutation, replacing asparagine with lysine at position 33, introduces an additional positive charge in the enzyme’s active site. This charge modification enhances electrostatic interactions with the negatively charged regions of maltose, leading to the formation of more stable hydrogen bonds. Specifically, in the N33K mutant (Figure 1C2), the distance between lysine-33 and maltose was reduced to 2.3 Å, whereas in the wild-type (Figure 1C1), this distance was 2.6 Å. This closer interaction is likely facilitated by lysine’s side chain, which can form hydrogen bonds with the hydroxyl groups of maltose. These interactions are expected to improve substrate binding and stabilize the transition state, potentially lowering the activation energy required for the catalytic reaction.

In industrial processes where high operating temperatures were required, the development of heat-resistant CGTase was of paramount importance. The addition of CaCl₂ was an effective method to enhance the enzyme’s thermal tolerance, which improved CGTase activity and stability. However, the presence of CaCl₂ could complicate downstream processing, posing challenges for industrial applications. CGTase’s stability was strongly influenced by intermolecular interactions, particularly those involving calcium ions. These interactions played a crucial role in maintaining the enzyme’s structural integrity and functional stability under elevated temperatures. However, the specific regions or residues with CGTase that contributed to its thermal stability were still under investigation and required further elucidation. Understanding the molecular mechanisms underlying CGTase’s thermal stability, particularly in the presence of CaCl₂, was essential for developing strategies to engineer enzymes with enhanced thermal tolerance. This knowledge could guide the design of CGTase variants that maintained robust performance under harsh industrial conditions while minimizing the need for additives like CaCl₂ during downstream processing. Calcium ions play a crucial role in stabilizing CGTase through coordination bonds with oxygen-containing amino acid residues such as Asp., Glu, Ser and Thr.

Sequential alignment of CGTase from various sources was conducted to identify critical residues that enhance thermal stability. This approach, depicted in Supplementary Figure S3, emphasized calcium-interaction sites (highlighted in a green box) and specific regions (marked in a red box) previously recognized as crucial for temperature adaptability. The modifications, within these identified areas could significantly prolong the enzyme’s half-life (Leemhuis et al., 2004).

Insights led to the selection of Ser211, Ile212, and His255 for mutagenesis due to their proximity to the calcium-binding site. The hypothesis was that mutating these residues could alter the enzyme’s interaction with calcium ions, potentially affecting its thermal stability. Utilizing directed evolution, five promising mutations were identified: S211G, I212R, H255W, H255F, and H255Y, as shown in Figure 1D1. These changes were anticipated to impact the enzyme’s thermal stability by modulating its calcium-binding affinity.

The integrity of purified CGTase was confirmed via SDS-PAGE (Fiure 1E). The enzymatic activities of the thermostable double mutants containing mutation N33K (Table 3) show that disproportionation activity was reduced in mutants N33K/H255W, N33K/H255F and N33K/H255Y, where His255 was substituted. This suggests that His255 plays a critical role in either substrate binding or in catalysis. In contrast, the activities of the N33K/S211G and N33K/I212R mutants remained largely unchanged.

The optimal pH and pH stability for N33K, N33K/S211G, and N33K/I212R mutants was similar to that of the wild type enzyme (Supplementary Figure S2).

Enhancing the optimal temperature and temperature stability of enzymes is crucial for their application, noting that enzymatic activity peaks at this optimum temperature. Setting the activity at the optimal temperature for mutants and wild-type BsCGT to 100%, the optimal temperature for N33K/S211G was 60°C, whereas for N33K, N33K/I212R, and wild-type BsCGT, it remained at 55°C (Figure 2). The introduction of the N33K/S211G mutation effectively raised the enzyme’s optimum temperature to 60°C. The same figure compares the thermal stability of these mutants which were subjected to various temperatures for 1 h. Whereas mutant N33K/I212R and N33K showed similar thermal stability profiles, N33K/S211G showed higher stability. Specifically, the half-life of N33K/S211G at temperatures of 50, 55, 60, and 65°C was 2.4 h, 2.0 h, 55.4 min, and 38.4 min, respectively. For N33K/I212R, the half-lives at these temperatures were 1.9 h, 1.4 h, 25.2 min, and 21.0 min (Supplementary Figure S4), respectively. Notably, at the optimal temperature of 60°C, the half-life of N33K/S211G was 2.3-fold longer than that of N33K (24.0 min). This highlights the significant contribution of the Ser211 mutation (Figure 1D) in N33K/S211G to structural stabilization and improved thermal stability of CGTase. Mutations at this site critically influence the enzyme’s overall activity and temperature adaptability to high temperatures, offering insights into enhancing its performance for industrial applications.

The thermal stability of the mutants was also assessed using Differential Scanning Calorimetry (DSC), which measures heat change associated with conformational transitions in a temperature ramp. The T_m value, derived from the DSC curve, represents the temperature at which the protein transitions from a folded state to an unfolded state, which is a critical indicator of thermal denaturation. For the wild-type BsCGT, T_m was 65.2°C, whereas it was higher for mutant N33K/S211G (67.8°C).

Trehalose synthesis was achieved using laboratory-prepared MTSase and MTHase enzymes with the incorporation of either wild-type BsCGT or mutant enzyme N33K/S211G toward the end of the reaction schematically shown in Figure 3. Addition of CGTase increased trehalose production by 20% (Figure 4), and trehalose yield was higher using the N33K/S211G mutant compared to BsCGT. For example, at an enzyme concentration of 1.5 U mL⁻¹, the mutant achieved a conversion of 69%, higher than 66% achieved by wild-type BsCGT. Additionally, the analysis of the reaction product of trehalose preparation by BsCGT indicated that the maltose content in the N33K/S211G mutant (5.01 g L⁻¹) was about 33.6% lower than that in the wild-type BsCGT (7.54 g L⁻¹). This may be attributed to the mutant’s enhanced affinity for maltose, allowing it to convert maltose to maltodextrin more efficiently, thereby improving substrate utilization. Therefore, this is also consistent with the difference in trehalose yield in the reaction product composition analysis, indicating that the mutant can increase trehalose yield compared with the wild enzyme (Table 4).

Overall, integration of homology modeling, docking analysis and virtual mutagenesis is a robust approach to understand and improve the structural and functional characteristics of BsCGT, and to advance enzyme engineering in industrial biotechnological applications.