In this study, unknown functional proteins derived from Chloroflexota bacterium, Pseudothermotoga hypogea DSM, Candidatus Aerophobetes bacterium, and Thermotogales bacterium were identified to possess tagatose-4-epimerase activity through gene mining. These proteins were designated as MBC, AJC7, TET, and HDM. AJC7 exhibited the highest activity among the wild-type enzymes and was thus selected for further studies involving soluble expression and site-directed mutagenesis. The research process is detailed below.
Using the amino acid sequence UxaE from Thermotoga petrophila RKU-1 as a template, we performed a BLAST search against the non-redundant protein sequence database at the National Center for Biotechnology Information (NCBI). From the 100 sequences retrieved, 80 sequences with a similarity threshold below 70% were selected for further analysis. Multiple sequence alignment was conducted using the MEGA software, and a phylogenetic tree was constructed using the neighbor-joining method (Fig. 1).
Figure 1 The phylogenetic tree was constructed using the amino acid sequence UxaE derived from Thermotoga petrophila RKU-1 as a template for BLAST search
In this study, we used AlphaFold2 to perform structural modeling on the 80 selected sequences. AlphaFold2 is a state-of-the-art deep learning-based tool for predicting protein structures with high accuracy based on their amino acid sequences.
Figure 2 Modeling the structure of Alphafold2 (AJC7 as an example)
After completing protein homology modeling using AlphaFold2, we downloaded the structure file of D-tagatose from the PubChem website. In Discovery Studio 2019, we first opened the three-dimensional structure of the AJC7 protein. Then, in the tool browser, we selected "Receptor-Ligand Interactions" and clicked on "Define and Edit Binding Site" to define AJC7 as the receptor molecule. Next, we clicked "File > Open" to import the D-tagatose structure file downloaded from PubChem, and in the tool browser, we expanded "Small Molecules" under "Prepare or Filter Ligands > Prepare Ligands" to open the corresponding process parameter settings panel. After setting the "Input Ligands" parameters, we clicked "Run" to process the small molecule. Subsequently, we used the C-DOCKER module (a docking tool based on the CHARMM force field) to dock D-tagatose into the active site of AJC7. Candidate conformations were generated through random rigid-body rotation and simulated annealing. The structure of the protein-ligand complex underwent energy minimization using the CHARMM force field. Finally, we retrieved the lowest energy docking conformations using the C-DOCKER module and selected the substrate orientation with the lowest interaction energy with the ligand for further analysis. The conformations were ranked based on CHARMM energy, and the highest-scoring conformations were retained for further study.
We calculated the binding free energy of the receptor-ligand complexes using the CHARMm-based energy function and an implicit solvent model. The binding energy between the receptor and ligand (ΔEBinding) is defined as EComplex = ELigand - EReceptor. To estimate these free energies, we minimized the ligand energy in the presence of the receptor using the steepest descent and conjugate gradient methods. The effective Born radii were computed using the Generalized Born Simple Switching (GBSW) implicit solvent model, replacing the costly molecular surface approximation with a smooth dielectric boundary combined with a van der Waals surface.
Using this approach, we calculated the binding free energy between the selected sequences and fructose, leading to the identification of four proteins with potential D-tagatose-4-epimerase activity
Figure 3 Binding self-energy between selected sequences and fructose
Considering the free energy data and phylogenetic tree analysis, four functional proteins with tagatose-4-epimerase activity were identified: MBC (MBC122911.1), AJC7 (AJC74158.1), TET (TET09249.1), and HDM (HDM70976.1).
Four screened sequences with potential tagatose-4-epimerase activity were synthesized and ligated into the pET-28a(+) vector (Fig.4).
Figure 4 Plasmid maps of plasmids pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, pET-28a(+)-TrxA-AJC7
Plasmids pET-28a(+)-AJC7, pET-28a(+)-HDM, pET-28a(+)-MBC, pET-28a(+)-TET, and pET-28a(+)-UxaE were transformed into E. coli BL21 (DE3) and cultured at 37°C for 14 hours. Individual colonies from the transformation were screened via colony PCR, and correct colonies were transferred to LB (Kan) medium for activation, amplification, and protein purification.
The reaction was conducted with a final fructose concentration of 10 g/L and 1 mM Ni2+ as a catalyst. The volume of the pure enzyme required was determined based on protein concentration. The reaction was carried out at 70℃ for 5 hours, and products were analyzed using high-performance liquid chromatography (HPLC) (Fig.5).
Figure 5 Concentrations of tagatose produced by wild-type UxaE, AJC7, HDM, MBC, and TET under the same reaction conditions
HPLC results indicated that the activity of wild-type AJC7 significantly exceeded that of wild-type UxaE, producing approximately four times the tagatose concentration under identical catalytic conditions. AJC7 exhibited considerable potential as a tagatose-4-epimerase, warranting further investigation.
During the experiments, AJC7 exhibited poor soluble expression, predominantly forming inclusion bodies (Fig. 6), complicating subsequent research on its enzymatic properties.
Figure 6 M: Protein marker; Lane 1: AJC7 precipitated Lane 2: AJC7 supernatant
To address this issue, we developed three strategies: fusion tags, molecular chaperones, and fermentation optimization.
Fusion-promoting tags can enhance the soluble expression of proteins by preventing aggregation of misfolded intermediates. We selected three tags to incorporate into AJC7:
1. Thioredoxin A (TrxA): Thermally stable and highly soluble.
2. Transcriptional Anti-Terminating Factor A (NusA):Prevents self-association of target proteins.
3. Maltose-binding protein (MBP): Known for its wide solubilization range and efficiency.
We inserted the gene of interest alongside the fusion-tagged DNA into a plasmid for transcription and translation, yielding a target protein attached to the fusion tag.
AJC7 was combined with the three fusion-promoting tags via the linker GGGGSGGGGS, resulting in recombinant plasmids: pET-28a(+)-MBP-AJC7,pET-28a(+)-NusA-AJC7,and pET-28a(+)-TrxA-AJC7 (Fig.7).
Figure 7 Plasmid profiles of recombinant plasmids pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7 and pET-28a(+)-TrxA-AJC7
1.The fusion tag was amplified by PCR, and the pET-28a(+)-AJC7 vector was linearized using reverse PCR, followed by gel recovery of the correct fragment.
2.The fusion-promoting tag fragment underwent homologous recombination with the vector, and heat shock transformation was performed. Colonies were cultured, and colony PCR confirmed the presence of recombinant plasmids (Fig. 8).
3.Verified colonies were sequenced, and those with correct sequences underwent expression induction. Following induction, the cell lysate was analyzed by SDS-PAGE. The strains modified with the fusion-promoting tag were designated TrxA-AJC7, NusA-AJC7, and MBP-AJC7,respectively.
Figure 8 Nucleic acid gel diagram of colony PCR
Figure 9 SDS-PAGE of recombinant AJC7 with a fusion-promoting tag (M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of TrxA-AJC7; Lane 4: supernatant of TrxA-AJC7; Lane 5: precipitation of NusA-AJC7; Lane 6: Supernatant of NusA-AJC7; Lane 7: precipitation of MBP-AJC7; Lane 8: Supernatant of MBP-AJC7)
The molecular weights of AJC7, TrxA-AJC7, NusA-AJC7, and MBP-AJC7 are 60.4 kDa, 70.4 kDa, 113.4 kDa, and 99.0 kDa, respectively. As illustrated in Fig. 8, there was minimal difference between the supernatant and precipitation of the original AJC7 (lanes 1 and 2) in the cell lysate, suggesting that the soluble expression of the target protein was limited. The concentration of the 70.4 kDa protein band (lane 4) in the supernatant was significantly increased, while the proportion of the precipitated protein band (lane 3) was reduced. Similarly, the concentration of the 113.4 kDa protein band (lane 6) in the supernatant also showed a significant increase, accompanied by a decrease in the precipitated protein band. The 99.0 kDa protein band (lane 8) exhibited a notable increase in the supernatant, with a corresponding reduction in the density of the precipitated protein band. Induced at 20℃ for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:
Figure 10 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid before and after fusion-promoting tag attachment
Figure 11 Percentage comparison of densitometry data for bacterial cell breakage, supernatant, and precipitated protein bands before and after fusion-promoting tag attachment
Figures 10 and 11 demonstrate that under specific induction conditions, the solubility of the protein was significantly enhanced by NusA, TrxA, and MBP, increasing by 6.86, 6.79, and 6.92 times, respectively, compared to AJC7. Additionally, the density of the protein bands in the precipitate was greatly reduced. This provides further validation for the inference that incorporating the fusion tag effectively enhances the solubility of the target protein.
The solubility of proteins in E. coli improved significantly with fusion tags at the N-terminus of AJC7. However, additional proteases are required to cleave the fusion protein for pure enzyme recovery. To enhance expression and solubility while maintaining practicality, we explored molecular chaperones.
Molecular chaperones facilitate proper protein folding and reduce inclusion body formation without compromising protein activity. A Chaperone Plasmid Set (including pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16) was co-introduced into E. coli BL21 (DE3) with the AJC7 plasmid, aiming to improve soluble protein recovery through synergistic folding assistance.
Five chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 were co-expressed with pET-28a(+)-AJC7 plasmid, respectively, to promote the correct folding of exogenous proteins in prokaryotic cells and increase the solubility of exogenous proteins, in order to obtain a large number of soluble proteins and explore the biological functions of the proteins. The modified strain pairs were named pG-KJE8/AJC7, pGro7/AJC7, pKJE7/AJC7, pG-Tf2/AJC7, pTf16/AJC7.
In this study, five chaperone plasmids were co-transformed into E. coli BL21 (DE3) along with the pET-28a(+)-AJC7 plasmid using thermomechanical transformation methods. The transformed cells were initially screened on plates containing kanamycin (Kan) and chloramphenicol (Cm) to ensure successful plasmid incorporation. Single colonies that tested positive via colony PCR were induced for protein expression. Post-induction, the cells were disrupted, and both the supernatant and precipitate were analyzed using SDS-PAGE.
In this study, five chaperone plasmids were co-transformed into E. coli BL21 (DE3) along with the pET-28a(+)-AJC7 plasmid using thermomechanical transformation methods. The transformed cells were initially screened on plates containing kanamycin (Kan) and chloramphenicol (Cm) to ensure successful plasmid incorporation. Single colonies that tested positive via colony PCR were induced for protein expression. Post-induction, the cells were disrupted, and both the supernatant and precipitate were analyzed using SDS-PAGE.
Figure 12 SDS-PAGE co-expressed by chaperone and pET-28a(+)-AJC7 (M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of pG-KJE8/AJC7; Lane 4: supernatant of pG-KJE8/AJC7; Lane 5: precipitation of pGro7/AJC7; Lane 6: supernatant of pGro7/AJC7; Lane 7: precipitation of pKJE7/AJC7; Lane 8: supernatant of pKJE7/AJC7; Lane 9: precipitation of pG-Tf2/AJC7; Lane 10: supernatant of pG-Tf2/AJC7; Lane 11: precipitation of pTf16/AJC7; Lane 12: supernatant of pTf16/AJC7)
Subsequent analysis of the protein bands on the gel was conducted using densitometry (see Figures 13-14).
Figure 13 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid co-expressed with chaperone proteins
Figure 14 Comparison of the percentage of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands co-expressing the target gene and chaperone protein
The results indicated that the solubility of the target protein pG-Tf2/AJC7 increased by 2.2 times compared to AJC7, as evidenced by the higher density of protein bands in the supernatant and a reduction in the precipitate. This suggests that the inclusion of chaperone proteins significantly enhanced the solubility of the target protein.
Different combinations of chaperones appear to influence specific classes of proteins, indicating that their effectiveness in enhancing soluble expression can vary based on the target protein and the expression conditions. This study aims to explore whether modifications in culture conditions can further improve protein solubility. The focus will be on optimizing inducers, induction temperatures, and media composition.
Induction temperature and IPTG concentration are critical factors that can influence the expression level and folding quality of target proteins, as well as the overall growth of E. coli. The use of self-inducing media can mitigate cytotoxic effects on the expressed protein, as well as reduce contamination risks by eliminating the need to add inducers midway through the process. This study aims to optimize fermentation parameters including temperature, IPTG concentration, and medium composition.
E. coli BL21 (DE3) was cultured at 37℃ with shaking at 200 rpm. Once the optical density (OD) reached 0.6 at 600 nm, IPTG was added to a final concentration of 0.5 mM. The cultures were then incubated at varying temperatures (16℃, 20℃, 30℃, and 37℃) for 16 hours, shaking at 200 rpm, to induce protein expression.
The same initial conditions were maintained at 37 ℃ with shaking. Upon reaching OD 0.6, cultures received IPTG at varying final concentrations (0.1 mM, 0.2 mM, 0.5 mM, and 1 mM). The cultures were then incubated at 20 ℃ for 16 hours with shaking at 200 rpm to induce protein expression.
To assess the effects of different carbon sources on protein expression, 16 variations of self-inducing medium were prepared, incorporating different combinations of carbon sources along with 10 g/L of NaCl. The recombinant E. coli BL21 (DE3) was inoculated and cultured at 37℃ for 3 hours, then shifted to 20℃ for 20 hours, shaking at 200 rpm to induce enzyme expression. As a control, cultures were induced with a final IPTG concentration of 0.1 mM once OD 0.6 was achieved.
Table 1 Recipe table of self-inducing medium
| Horizontal factors | Tryptone（g/L） | Yeast powder（g/L） | Glycerol（g/L） | Glucose（g/L） | Lactose（g/L） |
| 1 | 5 | 5 | 3 | 0.3 | 1 |
| 2 | 10 | 10 | 5 | 0.5 | 2 |
| 4 | 20 | 20 | 9 | 0.9 | 4 |
Table 2 Table of factor levels of self-inducing medium
| Experimental group factors | Tryptone | Yeast powder | Glycerol | Glucose | Lactose |
| 1 | 1 | 1 | 1 | 1 | 1 |
| 2 | 1 | 2 | 2 | 2 | 2 |
| 3 | 1 | 20 | 3 | 3 | 3 |
| 4 | 1 | 4 | 4 | 4 | 4 |
| 5 | 2 | 1 | 2 | 3 | 4 |
| 6 | 2 | 2 | 1 | 4 | 3 |
| 7 | 2 | 3 | 4 | 1 | 2 |
| 8 | 2 | 4 | 3 | 2 | 1 |
| 9 | 3 | 1 | 3 | 4 | 2 |
| 10 | 3 | 2 | 4 | 3 | 1 |
| 11 | 3 | 3 | 1 | 2 | 4 |
| 11 | 3 | 3 | 1 | 2 | 4 |
| 12 | 3 | 4 | 2 | 1 | 3 |
| 13 | 4 | 1 | 4 | 2 | 3 |
| 14 | 4 | 2 | 3 | 1 | 4 |
| 15 | 4 | 3 | 2 | 4 | 1 |
| 16 | 4 | 4 | 1 | 3 | 2 |
Figure 15 SDS-PAGE of recombinant AJC7 induced at different temperatures（M：marker； Lane 1: precipitation of enzyme AJC7 after induction at 16℃; Lane 2: precipitation of enzyme AJC7 after induction at 20℃; Lane 3: precipitation of enzyme AJC7 after induction at 30℃; Lane 4: precipitation of enzyme AJC7 after induction at 37℃; Lane 5: supernatant of enzyme AJC7 after induction at 16 ℃; Lane 6: supernatant of enzyme AJC7 after induction at 20℃; Lane 7: supernatant of enzyme AJC7 after induction at 30℃; Lane 8: supernatant of enzyme AJC7 after induction at 37℃)
The molecular weight of AJC7 is 60.375 kDa, as shown in Figure 14. After induction at 16 ℃, the concentration of protein bands in the supernatant (lane 5) was higher than that in the precipitation (lane 1). Similarly, after induction at 20 ℃, the concentration of protein bands in the supernatant (lane 6) exceeded that of the precipitation (lane 2). However, after induction at 30 ℃, the concentration of protein bands in the supernatant (lane 7) was lower than in the precipitation (lane 3). After induction at 37 ℃, the concentration of protein bands in the supernatant (lane 8) was also lower than that in the precipitation (lane 4).
Induced at various temperatures for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:
Figure 16 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid after induction at different temperatures
Figure 17 Comparison of optical densitometry data percentages of bacterial cell breakage, supernatant and precipitated protein bands after different temperature induction
Figures 16 and 17 indicate that the soluble expression of AJC7 protein increases at both 16 ℃ and 20 ℃, with a 1.24-fold higher expression at 16 ℃ compared to that at 20 ℃. Additionally, the density of the protein bands in the precipitate is significantly reduced. This provides further validation for the inference that selecting an appropriate induction temperature can effectively enhance the solubility of the target protein, with 16 ℃ identified as the optimal temperature for induction.
Figure 18 SDS-PAGE of recombinant AJC7 induced by different concentrations of IPTG (M: protein marker; Lane 1: precipitation of enzyme AJC7 after 0.1 mM induction, lane 2: precipitation of enzyme AJC7 after 0.2 mM induction, lane 3: precipitation of enzyme AJC7 after 0.5 mM induction, lane 4: precipitation of enzyme AJC7 after 1 mM induction, lane 5: supernatant of enzyme AJC7 after 0.1 mM induction, lane 6: supernatant of enzyme AJC7 after 0.2 mM induction, lane 7: supernatant of enzyme AJC7 after 0.5 mM induction, lane 8: supernatant of enzyme AJC7 after 1 mM induction)
AJC7 has a molecular weight of 60.375 kDa. As shown in Figure 18, the concentration of protein bands in the supernatant (lane 5) of the cell disruption solution after 0.1 mM IPTG induction is higher than that in the precipitation (lane 1). Similarly, after 0.2 mM IPTG induction, the concentration of protein bands in the supernatant (lane 6) exceeds that of the precipitation (lane 2). This trend continues with 0.3 mM IPTG induction, where the concentration in the supernatant (lane 7) is also higher than in the precipitation (lane 3). After 1 mM IPTG induction, the concentration of protein bands in the supernatant (lane 8) remains greater than that in the precipitation (lane 4).
Induced at 20 ℃with varying IPTG concentrations for 16 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:
Figure 19 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid induced by different concentrations of IPTG
Figure 20 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands after IPTG induction at different concentrations
Figures 19 and 20 illustrate that the solubility of the protein increased by 1.39-fold, 1.43-fold, and 1.48-fold following induction with IPTG at concentrations of 0.2 mM, 0.5 mM, and 1 mM, respectively. Additionally, the density of the protein bands in the precipitate was significantly reduced. Therefore, at an IPTG concentration of 0.5 mM, protein expression was found to be the most abundant, resulting in optimal solubility.
Figures 21 SDS-PAGE of recombinant AJC7 after induction in autoinduction medium with different combinations of carbon sources(M: protein marker; Lane C: supernatant of enzyme AJC7 after induction in normal LB control group; Lanes 1-16: supernatant of enzyme AJC7 after induction in experimental groups 1-16; Lane C': precipitation of enzyme AJC7 after induction in the normal LB control group, and lanes 1'-16': precipitation of enzyme AJC7 after induction in experimental groups 1-16)
The molecular weight of AJC7 is 60.375 kDa, as shown in Figure 21. The supernatants of the cell disruption solutions after induction in experimental groups 1, 2, 14, 15, and 16 exhibited higher concentrations of protein bands compared to those induced with standard LB medium, with the highest concentration observed in the supernatant of experimental group 16. Additionally, the concentration of protein bands in the cell disruption solutions for experimental groups 2, 14, 15, and 16 was lower than that observed after induction with ordinary LB.
Induced at 20°C for 20 hours, further densitometric analysis of the protein gel was conducted to quantify the increase in protein expression, and the results are summarized as follows:
Figures 22 Histogram of densitometry data of bacterial cell breakage fluid SDS-PAGE gel supernatant and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources(0: common LB control group; 1-16: Test group 1-16)
Figures 23 Comparison of optical density analysis data of bacterial cell breakage, supernatant, and precipitated protein bands after induction in autoinduction medium with different combinations of carbon sources(0: common LB control group; 1-16: Test group 1-16)
Figures 22 and 23 demonstrate that the solubility of the target protein increased by 1.82 times, 2.33 times, 2.15 times, 2.36 times, and 2.15 times, respectively, in experimental groups 1, 2, 14, 15, and 16 compared to ordinary LB medium. Additionally, the density of the protein bands in the precipitate was significantly reduced.
In the field of synthetic biology, selecting the optimal system requires careful consideration of several factors. The optimization of fermentation conditions has established a foundation for the efficient soluble expression of AJC7 in E. coli. Through extensive literature research, we found that mutations are a common method to enhance the activity of the enzyme AJC7. Consequently, in the subsequent experimental process, we decided to employ a mutagenesis strategy to increase the activity of AJC7.
In this study, the UxaE amino acid sequence from Thermus neapolitana served as a template for gene mining. We identified four enzyme sequences with potential tagatose-4-epimerase activity, named MBC, HDM, TET, and AJC7, based on phylogenetic analysis and binding free energy considerations. Given their soluble and wild-type enzyme activities, AJC7 was selected for further investigations involving single-point and iterative mutations. The catalytic mechanism of tagatose-4-epimerase, which converts fructose to tagatose, has been well-characterized (Fig. 24). Focusing on the structure and function of the AJC7 protein, we constructed a series of mutants: S125D, T181A, H342L, I129T, L140P, and various combinations thereof.
Figures 24 Catalytic mechanism of tagatose-4-epimerase catalyzing fructose production of tagatose[1]
Figures 25. （A）Docking model of D-fructose in the H342I mutant,（B）Docking model of D-fructose in the I129T mutant,（C）Docking model of D-fructose in the L140T mutant,（D）Docking model of D-fructose in the S125D mutant,（E）Docking model of D-fructose in the T181A mutant.
Table 3 Reasons for mutation site selection
| Mutation point | Reasons for choosing |
| S125D | The conversion of fructose to D-tagatose begins with the formation of a glyceraldehyde intermediate. When serine is mutated to aspartic acid, the carboxyl group of aspartic acid can interact with the terminal aldehyde group of the glyceraldehyde intermediate, facilitating the protonation of the aldehyde and thereby aiding in the catalysis and production of D-tagatose. Furthermore, the negatively charged aspartic acid may enhance interactions with the positively charged substrate and alter the charge distribution in the binding pocket, thereby increasing the catalytic activity of the enzyme. |
| T181A | By mutating from a large amino acid to a smaller one and from a polar to a non-polar amino acid, the mutation increases the hydrophobicity within the active pocket, thereby contributing to the stability of the protein. |
| H342L | Replacing the positively charged histidine with the uncharged and more hydrophobic leucine alters the charge properties of the amino acid residue, reconfiguring the charge distribution within the substrate pocket. This enhances interactions with the substrate. Additionally, the increased hydrophobicity within the active pocket improves the enzyme's stability. |
| I129T | After replacing the nonpolar amino acid isoleucine with the polar amino acid threonine, the hydroxyl group in the threonine side chain can form a new hydrogen bond interaction with serine at position 125. This enhanced interaction may increase the enzyme's stability and alter its conformation, thereby creating a more favorable environment in the active pocket for substrate binding through a transmission effect, ultimately improving catalytic efficiency. |
| L140P | Proline has a cyclic structure, and its unique secondary amine limits the rotational freedom of the residue. This restriction leads to changes in the enzyme's three-dimensional structure, which, through a transmission effect, alters the interactions between the active pocket and the substrate, thereby enhancing catalytic activity. |
We designed primers for point mutations S125D, T181A, H342L, I129T, and L140P, using pET-28a(+)-AJC7 as a template to amplify single-point mutants (Fig. 26). This design was pivotal for assessing the impact of each mutation on enzyme activity.
Figures 26 Point mutation localization and primer design
Figures 27 Nucleic acid gel diagram of colony PCR
The PCR mutation protocol is outlined in the experimental section. Following PCR amplification, the products underwent demethylation using DpnI. Verification of the PCR products involved nucleic acid gel electrophoresis. The successful constructs were then transformed into E. coli BL21 (DE3) and incubated at 37°C for 14 hours. Individual colonies were picked for colony PCR, as depicted in Fig. 27. After confirmation through electrophoresis, the positive colonies were transferred to LB (Kan) liquid medium. Subsequent sequencing confirmed the successful construction of all five single-point mutants.
We activated and cultured both the mutant strains and the wild-type strain, followed by a series of purification steps to extract the target protein using methods outlined in the experimental section. The volume of purified enzyme solution for the 500µL reaction system was determined based on protein concentration. A final fructose concentration of 100 g/L was established, with 10µL of Ni2+ as a catalyst. Reactions were conducted at 70°C for 5 hours, and the products were analyzed via high-performance liquid chromatography (HPLC) (Fig. 28).
The results demonstrated that the concentrations of fructose from the mutants—S125D, T181A, H342L, I129T, and L140P—were higher than those from the wild-type AJC7. Notably, the H342L, I129T, and L140P mutations showed less pronounced improvements. The S125D mutant exhibited a catalytic efficiency that was twice that of the wild type under identical reaction conditions and substrate concentrations.
Figures 28 The concentrations of tagatose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h
The results showed that the concentrations of AJC7 after five single-point mutations, namely S125D, T181A, H342L, I129T and L140P, were higher than those of wild-type AJC7. The improvement effect of H342L, I129T, and L140P is not obvious. The catalytic efficiency of the S125D mutant was twice as high as that of the wild type under the same reaction conditions and substrate concentration.
Because the catalytic efficiency of S125D was significantly improved compared with WT type, we decided to use S125D as a template to construct two-point mutants of S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P to further improve the enzyme activity.
The three mutation points, T181A, H342L, and L140P, are located at a considerable distance from S125D. Therefore, pET-28a(+)-AJC7-S125D can be utilized as a template for the construction of the double mutants S125D/T181A, S125D/H342L, and S125D/L140P using the primers previously employed for single-point mutations in Design 1. However, due to the close proximity of I129T to S125D, the primers for the I129T mutation were redesigned using pET-28a(+)-AJC7-S125D as a template to create the S125D/I129T mutant.
The same methodology as outlined in Construction 1 was applied to generate the double mutants S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P.
Figures 29 Nucleic acid gel diagram of colony PCR
Same method as [Test 1].
Figures 30 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively
The results indicated that the concentrations of AJC7 following the mutations S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P were significantly higher than those of both wild-type AJC7 and S125D. Notably, the S125D/T181A double mutant exhibited the most substantial increase in AJC7 enzyme activity, with its catalytic efficiency nearly doubling that of wild-type AJC7.
Compared to wild-type AJC7, the enzyme activity of the S125D/T181A double mutant was significantly enhanced. Consequently, we decided to use S125D/T181A as a template to construct additional superimposed mutants, specifically S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L, in order to further improve enzyme activity.
The single-point mutagenesis primers used in Design 1 were directly applied in this phase.
Construction 1 was utilized to successfully generate the three-point mutants S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L.
The procedures were consistent with those outlined in Test 1.
Figures 31 Nucleic acid gel diagram of colony PCR
The procedures were consistent with those outlined in Test 1.
Figures 32 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/T181A/I129T, and S125D/T181A/H342L reacted with 100 g/L fructose substrate for 5 h, respectively
The results demonstrated that the concentrations of AJC7 following the S125D/T181A/I129T and S125D/T181A/H342L three-point mutations were higher than those observed for wild-type AJC7, S125D, and S125D/T181A. However, the S125D/T181A/L140P three-point mutant did not yield a significant improvement in enzyme activity. Among the mutants, the S125D/T181A/I129T exhibited the highest increase in AJC7 enzyme activity, with a catalytic efficiency nearly three times greater than that of wild-type AJC7.
The S125D/T181A/I129T three-point mutant demonstrated further improvements in enzyme activity. Consequently, we utilized S125D/T181A/I129T as a template for saturation mutagenesis, constructing the S125D/T181A/I129T/L140P four-point mutant and the S125D/T181A/I129T/L140P/H342L five-point mutant to enhance enzyme activity further.
We directly employed the single-point mutagenesis primers outlined in [Design 1].
The same method as in [Construction 1] was used to construct S125D/T181A/I129T/L140P four-point mutants and S125D/T181A/I129T/L140P/H342L five-point mutants.
Figures 33 Nucleic acid gel diagram of colony PCR (4-point mutation on the left and 5-point mutation on the right)
Same method as [Test 1].
Figures 34 The concentrations of tagatose in WT, S125D, S125D/T181A, S125D/T181A/I129T, S125D/T181A/I129T/L140PS125D/T181A/I129T/L140P/H342L after reacting with 100g/L fructose substrate for 5 h
The results indicated that the S125D/T181A/I129T/L140P four-point mutant and the S125D/T181A/I129T/L140P/H342L five-point mutant exhibited improved enzyme activity compared to wild-type AJC7. However, the enzyme activity of the S125D/T181A/I129T was found to be unsatisfactory relative to the S125D/T181A mutant.
The enhancements in enzyme activity for the S125D/T181A/I129T/L140P four-point mutant and S125D/T181A/I129T/L140P/H342L five-point mutant did not meet our expectations. In future experiments, we will explore additional mutation sites to facilitate further superimposed mutations of the S125D/T181A/I129T three-point mutants to improve enzyme activity, or we may pursue directed evolution of AJC7 to enhance its enzymatic performance. To gain deeper insights into the S125D/T181A/I129T three-point mutants, we conducted further assays on their enzymatic properties and kinetic parameters.
In this study, we focused on the iterative mutation of the AJC7 enzyme, specifically identifying the three-point mutant S125D/T181A/I129T, which exhibited a nearly threefold increase in the conversion rate of fructose to tagatose. To further understand the enzymatic properties and industrial applicability of this mutant, we investigated the optimal pH and temperature for its activity. Additionally, we examined the relationship between the enzyme's conversion rate over time and determined its kinetic parameters, particularly the Michaelis constant (Km), to assess the enzyme's activity and affinity for the substrate.
a.In order to study the optimal pH of the reaction, the enzyme solution was reacted in 50mM buffer at pH 3~11 (in which the concentration of fructose in the substrate was 100g/L and the final concentration of the pure enzyme solution was 0.1mg/ml), 70 °C for 3h, containing 1 mM Ni2+. The buffer system is as follows: pH3-5: citric acid-sodium citrate; pH6-7：Na2HPO4-NaH2PO4;pH8-9：Tris-HCl; pH10-11：NaOH-NaH2PO4.
b. The optimal temperature for the enzyme reaction was assessed by incubating the reaction mixtures containing 100 g/L fructose, 0.1 mg/mL enzyme, and 1 mM Ni2+ in Tris-HCl buffer (pH 9.0) for 2 hours at varying temperatures: 50°C, 60°C, 70°C, 80°C, and 90°C. After incubation, the reaction products were analyzed via HPLC, and an optimal temperature curve was generated to determine the temperature at which the enzyme activity was maximized.
c. To evaluate the maximum conversion rate of the S125D/T181A/I129T mutant, we set up a reaction system with a final concentration of 23 mg/mL of the pure enzyme in a 500µL volume, along with a fructose concentration of 100 g/L and 1 mM Ni2+. The buffer was adjusted to 50 mM Tris-HCl at pH 9.0. The reaction was conducted at 70℃ in a metal shaker, and samples were taken every 20 minutes over a total period of 180 minutes. After the reaction, the concentrations of tagatose produced and remaining fructose were quantified using HPLC, enabling us to calculate the conversion rate.
To determine the Michaelis constant (Km) of the optimal mutant S125D/T181A/I129T of AJC7, a concentration gradient of fructose was established, with concentrations of 5 g/L, 10 g/L, 20 g/L, 40 g/L, 50 g/L, 60 g/L, 80 g/L, and 100 g/L. The final enzyme concentration was set at 0.1 mg/mL, and 1 mM Ni2+ was added as a catalytic metal ion. The remaining volume was adjusted to 50 mM Tris-HCl buffer at pH 8.0. The reaction was conducted at 70°C in a metal shaker for 20 minutes. After the reaction, the yield of tagatose and the residual fructose concentration were analyzed using high-performance liquid chromatography (HPLC), and the conversion rate was subsequently calculated.
a.Calculate the relative activity under other pH conditions separately with a maximum activity of 100% at pH 9.0.
Figures 35 The relative activities of AJC7-S125D/T181A/I129T at different pH. The activity at pH 9.0 was set as 100%
Figure 35 illustrates that AJC7 exhibits minimal activity in catalyzing the conversion of fructose to tagatose at pH levels between 4.0 and 5.0. However, enzyme activity increases with rising pH values within the range of 6.0 to 9.0, reaching a maximum at pH 9.0. Beyond this point, specifically from pH 9.0 to 11.0, enzyme activity begins to decline. These findings suggest that AJC7 is more effective in facilitating the conversion of fructose to tagatose in a weakly alkaline environment.
b. The maximum activity observed at 70°C was designated as 100%, and relative activity at other temperature conditions was calculated accordingly (Fig. 36).
Figures 36 AJC7-S125D/T181A/I129T triple mutant enzyme activity as a function of reaction temperature
As illustrated in Fig. 36, at the optimal pH of 9.0, the enzyme activity of the AJC7-S125D/T181A/I129T three-point mutant increased gradually with rising temperatures within the range of 50–70°C, peaking at 70°C. Beyond this temperature, enzyme activity began to decline. Therefore, the optimal reaction temperature for the AJC7-S125D/T181A/I129T three-point mutant in catalyzing the conversion of fructose to tagatose is determined to be 70°C
c. To determine the conversion rate of tagatose-4-epimerase AJC7, the substrate conversion of the optimal mutant was assessed under optimal reaction conditions (70°C, pH 9.0, and 1 mmol/L Ni2+).
Figures 37 Conversion rate of the best AJC7 mutant under optimal conditions
As shown in Figure 36, when the final enzyme concentration was 23 mg/mL, the optimal mutant of AJC7 catalyzed the conversion of 100 g/L fructose to produce 37 g/L tagatose within 120 minutes, resulting in a conversion rate of 37% and a yield of 18.5 g/(L·h). Notably, at 80 minutes, 33 g/L of tagatose was generated, corresponding to a conversion rate of 33% and a peak yield of 24.1 g/(L·h).
Figures 38 Nonlinear regression equations for substrate concentration and reaction rate
The values of Km (mM) and Vmax (h/mM) were determined by Origin soft nonlinear regression with Vm of 27.3086 and Km of 99.805. It can be seen that the best mutant of AJC7, S125D/T181A/I129T, has a good affinity with the substrate.
The conversion rate of the most effective mutant of AJC7, S125D/T181A/I129T, has improved; however, it still does not meet the requirements for industrial production. To address this limitation, we aim to obtain mutant strains with even higher conversion rates through directed evolution. Before proceeding, it is essential to develop an efficient and rapid high-throughput screening method to replace traditional high-performance liquid chromatography. Consequently, we focused on designing a biosensor that exhibits specific green fluorescence in response to tagatose.
Currently, the developed biosensor demonstrates a specific response to tagatose. However, due to time constraints during the competition and certain challenges encountered in the experimental process, the sensor's sensitivity to changes in tagatose concentration is limited, hindering the screening of high tagatose-4-epimerase activity based on fluorescence values. In future work, we aim to construct new plasmids and mutate promoters to enhance sensitivity. We also plan to explore and improve novel methodologies, ultimately striving to provide a practical tool for high-throughput screening. This work will also offer valuable insights and components for the iGEM team to employ high-throughput screening methods for directed evolution in future projects