The amino acid sequence UxaE, derived from Thermotoga petrophila RKU-1, served as a template for a BLAST search against the non-redundant protein sequence database of the National Center for Biotechnology Information (NCBI). The top 100 sequences exhibiting high sequence similarity were selected. Multiple sequence alignments were conducted using MEGA software, and after removing mismatched sequences, a phylogenetic tree was constructed using the adjacency method (Fig. 1).
Figure 1 The phylogenetic tree was constructed by BLAST search of the amino acid sequence UxaE derived from Thermotoga petrophila RKU-1 as a template
AlphaFold2 was utilized to model the structures of 80 sequences with a similarity threshold of less than 70% (Fig.2).
Figure 2 Alphafold2 structural modeling ( AJC7 as an example)
A molecular dynamics (MD) protocol based on the CHARMM force field was employed to dock the ligand to the receptor binding site. The binding free energy of the acceptor-ligand complex was calculated using a CHARMM-based energy function and the implicit solvent method. The binding free energy between the selected sequence and fructose is displayed in Fig.3.
Figure 3 The binding free energy between the selected sequence and fructose
Considering the binding free energy as well as the branches and sources in the phylogenetic tree, we selected four potential proteins with tagatose-4-epimerase activity, naming them MBC, AJC7, TET, and HDM, respectively. These four enzymes were evaluated for their ability to catalyze the conversion of fructose to tagatose under identical conditions. The resulting tagatose was analyzed using high-performance liquid chromatography (HPLC), and the results are presented in Fig. 4. According to the HPLC data, the activity of wild-type AJC7 significantly surpassed that of wild-type UxaE, with tagatose concentration produced under the same catalytic conditions being approximately four times greater than that of UxaE. Given its promising tagatose-4-epimerase activity, AJC7 was selected for further research.
Figure 4 Concentrations of tagatose produced by wild-type UxaE, AJC7, HDM, MBC, and TET under the same reaction conditions
To improve the wild-type enzyme activity of AJC7, we integrated three fusion-promoting tags with the AJC7 sequence using a GGGGSGGGGS linker. The resulting recombinant plasmids were denoted as pET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, and pET-28a(+)-TrxA-AJC7.
Figure 5 Plasmid maps of recombinant plasmids PET-28a(+)-MBP-AJC7, pET-28a(+)-NusA-AJC7, pET-28a(+)-TrxA-AJC7
The fusion-promoting tag fragment underwent PCR amplification, while the pET-28a(+)-AJC7 vector was linearized via reverse PCR. Gel electrophoresis was employed to recover the correct nucleic acid fragment. After homologously recombining the tag fragment with the vector, heat shock transformation was carried out. Colonies that grew on plates were selected, cultured, and subjected to colony PCR to confirm the presence of recombinant plasmids (Fig. 6). The sizes of the TrxA, NusA, and MBP fragments were 402 bp, 1560 bp, and 1179 bp, respectively. Validated colonies from colony PCR were sequenced to verify the correct construction of plasmids with fusion tags. The accurate sequences were utilized for inducible expression. The strains altered with fusion-promoting tags were named TrxA-AJC7, NusA-AJC7, and MBP-AJC7, respectively. Following expression induction, cell lysates were separated into supernatant and pellet fractions for SDS-polyacrylamide gel electrophoresis analysis.
Figure 6 Nucleic acid gel plot of colony PCR
Figure 7 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 are as follows: AJC7 at 60.4 kDa, TrxA-AJC7 at 70.4 kDa, NusA-AJC7 at 113.4 kDa, and MBP-AJC7 at 99.0 kDa.
As shown in Fig. 7, there is little difference between the supernatant and pellet of the original AJC7 (lanes 1 and 2) in the cell lysate, indicating limited soluble expression of the target protein. The concentration of the 70.4 kDa protein band (lane 4) in the supernatant significantly increased, while the proportion of the precipitated protein band (lane 3) decreased. Similarly, the concentration of the 113.4 kDa protein band (lane 6) in the supernatant rose significantly, with a corresponding reduction in the precipitated band. The 99.0 kDa protein band (lane 8) also showed a significant increase in the supernatant, and the precipitated band appeared notably lighter. Induced at 20℃for 16 hours, further densitometry analysis of the protein gel was conducted to quantify the increase in protein expression, with the results detailed below:
Figure 8 Histogram of densitometry data for bacterial cell breakage before and after ablation tag attachment, SDS-PAGE gel supernatant, and precipitated protein bands
Figure 9 Percentage comparison of densitometry data of bacterial cell breakage, supernatant, and precipitated protein bands before and after thawing tag attachment
Fig. 9 and 10 demonstrate that, under specific induction conditions, the solubility of the protein was significantly enhanced by NusA, TrxA, and MBP, with increases of 6.86, 6.79, and 6.92 times, respectively, compared to AJC7. Additionally, the density values of the protein bands in the precipitate were greatly reduced. This provides further validation for the inference that the incorporation of the fusion tag effectively improves the solubility of the target protein.
Molecular chaperones were utilized in this study. The chaperone plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16 were co-expressed with the pET-28a(+)-AJC7 plasmid to facilitate correct folding of exogenous proteins in prokaryotic cells, thereby enhancing the solubility of these proteins. This approach aimed to yield a substantial quantity of soluble proteins for exploring their biological functions. Modified strain pairs were denoted as follows: pG-KJE8/AJC7, pGro7/AJC7, pKJE7/AJC7, pG-Tf2/AJC7, and pTf16/AJC7. The chaperone plasmids were introduced into E. coli BL21 (DE3) host cells alongside the pET-28a(+)-AJC7 plasmid via thermomechanical transformation and initially screened on Kanamycin and Chloramphenicol-resistant plates. Single colonies that tested positive through colony PCR were subsequently induced for protein expression and subjected to purification processes.Following induction, the cellular supernatant and precipitate were separately analyzed via SDS-polyacrylamide gel electrophoresis.
Figure 10 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.
Further analysis of the protein gel was conducted using densitometry to quantify the increase in protein expression.
Figure 11 Histogram of densitometry analysis data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid co-expressing the target gene and chaperone protein
Figure 12 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands co-expressed with chaperone proteins
As shown in Fig.11 and 12, the protein solubility of pG-Tf2/AJC7 increased by 2.2 times compared to AJC7, and the density of the protein bands in the precipitate decreased. These results indicate that the addition of chaperones significantly enhances the solubility of the target protein.
Recombinant E. coli BL21 (DE3) was cultivated at 37℃ with agitation at 200 rpm. Upon reaching an optical density of 0.6 at 600 nm, 0.5 mM isopropylthiogalactoside (IPTG) was added, followed by incubation at 16℃, 20℃, 30℃, and 37℃ for 16 hours with agitation at 200 rpm to induce enzyme expression.
1. Calculate the amount of DNA used for recombination reaction:
Figure 13 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℃.
Induced at varying temperatures for 16 hours, further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:
Figure 14 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 15 Comparison of optical densitometry data percentages of bacterial cell breakage, supernatant and precipitated protein bands after different temperature induction
The outcomes are depicted in Fig.14 and 15. These figures illustrate a 1.24-fold elevation in the soluble expression of AJC7 at 20°C compared to induction at 16°C. Moreover, the intensity of protein bands in the precipitate significantly decreased, reinforcing the notion that selecting the appropriate induction temperature effectively enhances the target protein's solubility.
Recombinant E. coli BL21 (DE3) was cultured at 37℃ with agitation at 200 rpm. Upon the culture's optical density reaching 0.6 at 600 nm, final concentrations of 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM isopropylthiogalactoside (IPTG) were added successively. Subsequently, the culture was incubated at 20℃ for 16 hours with agitation at 200 rpm to induce enzyme expression.
Figure 16 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.
At 20℃, AJC7 was induced with different concentrations of IPTG for 16 hours. Further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:
Figure 17 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 18 Percentage comparison of densitometry data of bacterial cell breakage, supernatant and precipitated protein bands after IPTG induction at different concentrations
The results, shown in Fig.17 and 18, revealed a 1.39-fold, 1.43-fold, and 1.48-fold enhancement in protein solubility after induction with 0.2 mM, 0.5 mM, and 1 mM IPTG, respectively. Additionally, a substantial decrease in the density of protein bands in the precipitate was observed.
An orthogonal experimental design was employed to create sixteen groups of self-inducing media with varying carbon source combinations. Recombinant E. coli BL21 (DE3) was initially inoculated at 37℃ for 3 hours with agitation at 200 rpm, followed by incubation at 20℃, and 200 rpm for 20 hours to induce enzyme expression. Upon reaching an optical density of 0.6 at 600 nm in standard LB medium, a final concentration of 0.1 mM IPTG was introduced for induction control purposes.
Subsequent to induction at 20℃ for 20 hours, densitometry analysis of the protein gel was conducted to quantify the upsurge in protein expression, with the results scrutinized as outlined below:
Figure 19 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 the 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; Lanes 1'-16': precipitation of enzyme AJC7 after induction in experimental groups 1-16.
Induced at 20℃ for 20 hours, further densitometry analysis of the protein gel was performed to quantify the increase in protein expression, with the results detailed below:
Figure 20 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
Figure 21 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
Fig.20 and 21 indicate 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 in experimental groups 1, 2, 14, 15, and 16, respectively, compared to the standard LB medium. Additionally, the density values of the protein bands in the precipitate were significantly reduced.
1. Point mutations were introduced using designed primers for S125D, T181A, H342L, I129T, and L140P on the pET-28a(+)-AJC7 template to obtain single-point mutants (Fig.22). The PCR reaction system was followed by DpnI-mediated demethylation, nucleic acid gel electrophoresis verification, and subsequent recovery of PCR products. The concentration of each single-point mutant plasmid was measured before transfer to E. coli BL21 (DE3) and incubation at 37℃ for 14 hours invertedly. Individual colonies were selected post-transformation, subjected to colony PCR, and validated using nucleic acid gel electrophoresis as presented in Figure 23. After validation, colonies with correct bands were transferred to LB (Kan) liquid medium, and sequencing confirmed successful construction of all five single-point mutant plasmids.
2. The three distantly located mutation points, T181A, H342L, and L140P, were used with the template pET-28a(+)-AJC7-S25D to construct S125D/T181A, S125D/H342L, and S125D/L140P two-point mutants. Given the proximity of I129T to S125D, primers from the I129T spike served as the template for constructing the S125D/I129T mutant.
3. Three-point mutants, S125D/T181A/I129T, S125D/T181A/L140P, and S125D/T181A/H342L, were successfully developed using the method detailed in [engineering].
4.The four-point mutant, S125D/T181A/I129T/L140P, and the five-point mutant, S125D/T181A/I129T/L140P/H342L, of AJC7 were successfully created with the method outlined in [engineering].
Figure 22 Nucleic acid gel diagram of PCR of single-point mutant colonies
Figure 23 Nucleic acid gel diagram of PCR with two-point mutant colonies
Figure 24 Nucleic acid gel diagram of PCR with two-point mutant colonies
Figure 25 Nucleic acid gel diagram of PCR of three-point mutant colonies
Figure 26 Nucleic acid gel map of colony PCR (four-point mutation on the left and five-point mutation on the right)
Mutant and wild-type strains were activated, amplified, and cultured, followed by a series of protein purification operations to extract the protein of interest using the method outlined in [Experiment]. The volume of purified enzyme solution required for the 500 uL reaction system was determined based on the protein concentration, as shown in [Experiment]. The final concentration of fructose in the system was set at 100 g/L, with 10 uL of Ni2+ used as a catalyst. The reaction was conducted at 70℃ for 5 hours, and the product was analyzed using high-performance liquid chromatography (HPLC) (Fig.27).
Figure 27 The concentrations of tagose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h
The results indicated that the concentrations of AJC7 after five single-point mutations—S125D, T181A, H342L, I129T, and L140P—were higher than those of wild-type AJC7. The enhancements observed with the H342L, I129T, and L140P mutants were not significant. Notably, the catalytic efficiency of the S125D mutant was twice that of the wild type under identical reaction conditions and substrate concentrations.Two point mutations.
Figure 28 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 showed that the concentrations of AJC7 after the S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P mutations were higher than those of both wild-type AJC7 and S125D. Notably, the S125D/T181A two-point mutant exhibited a significant increase in AJC7 enzyme activity, with its catalytic efficiency nearly twice that of wild-type AJC7.Three-point mutation.
Figure 29 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 indicated that the concentrations of AJC7 after the S125D/T181A/I129T and S125D/T181A/H342L three-point mutations were higher than those of wild-type AJC7, S125D, and S125D/T181A. However, the S125D/T181A/L140P three-point mutant did not significantly enhance enzyme activity. Among the mutants, the S125D/T181A/I129T three-point variant demonstrated the greatest increase in AJC7 enzyme activity, with its catalytic efficiency nearly three times that of wild-type AJC7.Four-point, five-point mutation.
Figure 30 The concentrations of tagosse in WT, S125D, S125D/T181A, S125D/T181A/I129T, S125D/T181A/I129T/L140P,S125D/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 of AJC7 showed improvements compared to wild-type AJC7. However, the enzyme activity of the S125D/T181A/I129T mutant was not satisfactory when compared to that of the S125D/T181A/I129T mutant.
a.Relative activity was calculated under various pH conditions considering maximum activity at pH 9.0.
Figure 31 The relative activities of AJC7-S125D/T181A/I129T at different pH. The activity at pH 9.0 was set as 100%
Examining Fig.31 reveals that AJC7 shows minimal catalytic activity for fructose to tagatose conversion at pH 4.0-5.0. The enzyme's activity increases with rising pH (6.0–9.0), peaking at pH 9.0 and declining beyond pH 9.0 to 11.0. Thus, AJC7 is most effective for fructose to tagatose conversion under slightly alkaline conditions.
b.The activity at 70℃ was set as 100%, with relative activity calculated for other temperature ranges.
Figure 32 AJC7-S125D/T181A/I129T triple mutant enzyme activity as a function of reaction temperature
Analysis of Fig.32 indicates that, at the optimum pH of 9.0, the enzyme activity of the AJC7-S125D/T181A/I129T three-point mutant rises gradually within the 50–70℃ range, reaching a peak at 70℃. Further increase in temperature leads to a decline in enzyme activity. Therefore, the optimal temperature for catalyzing fructose to tagatose conversion by the AJC7-S125D/T181A/I129T mutant is 70℃.
c. To determine the conversion rate of tagatose-4-epimerase AJC7, the substrate conversion rate of the optimal mutant was evaluated under optimal reaction conditions (70℃, pH 9.0, 1 mmol/L Ni2+).
Figure 33 Conversion rate of the best AJC7 mutant under optimal conditions
With an enzyme concentration of 23 mg/mL, the optimal mutant catalyzed the conversion of 100 g/L fructose to yield 37 g/L tagatose in 120 minutes, achieving a 37% conversion rate and a yield of 18.5 g/(L·h). At 80 minutes, 33 g/L tagatose was produced, translating to a 33% conversion rate and a yield of 24.1 g/(L·h), the highest recorded.
Figure 34 Nonlinear regression equations for substrate concentration and reaction rate
In determining the Michaelis constant of the optimal mutant S125D/T181A/I129T of AJC7, a fructose concentration gradient was set (5g/L, 10g/L, 20g/L, 40g/L, 50g/L, 60g/L, 80g/L, 100g/L) with a final pure enzyme concentration of 0.1 mg/mL in the presence of 1mM Ni2+ catalytic metal ion and Tris-HCl buffer at 50mM pH=8.0. The reaction was conducted at 70℃ in a metal shaker for 20 minutes. Post-reaction, the yield of tagatose and residual fructose was determined by high-performance liquid chromatography to calculate the conversion rate. Using Origin software nonlinear regression, Km was calculated as 99.8 mM and Vmax as 27.30 h/mM, indicating the strong substrate affinity of the AJC7 S125D/T181A/I129T mutant.