Construction and PCR identification of plasmid containing DPEase
In this study, we constructed the inducible plasmid pET22b(+)-psi(6283 bp, Fig. 2), which contains the DPEase gene, and obtained the recombinant strain BL21/pET22b(+)-psi, screened with 100 µg/mL ampicillin. The plasmid was extracted using a commercial kit, and amplified by PCR with primers psi-F and psi-R. The PCR product’s length was 870 bp, confirming the expected size (Fig. 3).
Schematic representation of pET22b(+)-E. coli.
Plasmin PCR identification; Note: M: Marker; 1, 2, 3: Target electrophoretic bands.
Expression, isolation, and purification of D-allulose 3-epimerase in E. Coli
The recombinant strain BL21/pET22b(+)-psi was induced for protein expression, as shown in the electropherogram in Fig. 4 post-cell lysis. As depicted in Fig. 4, E. coli expressed a protein of approximately 32 kDa, matching the expected size of DPEase, suggesting successful expression; Furthermore, the purified protein’s single band on the gel confirms a molecular weight of approximately 32 kDa, demonstrating effective separation from other proteins and impurities during purification.
Expression of D-allulose-epimerase Note: M: Marker, 1: Unpurified protein, 2: Purified target protein.
D-allulose 3-epimerase activity detection
The recombinant strain was incubated in a medium containing D-fructose as the substrate, and D-allulose production was detected using HPLC. Figure 5a is a liquid-phase chromatogram showing the separate detection of substrates. As shown in Fig. 5b, the presence of D-allulose at 25 min indicates that DPEase is functional in E. coli. It expresses active DPEase, which catalyzes the synthesis of D-allulose.
HPLC detection of recombinant DPEase. (a) The peak of D-allulose; (b) peak diagram of D-fructose catalyzed by whole cell.
Optimization of induction conditions
E. coli is one of the most used expression hosts for producing and expressing recombinant proteins. However, for various reasons, improved efficiency and yield are often required. Therefore, optimizing the E. coli expression system conditions is crucial for efficiently expressing target proteins28.
The effect of culture medium on enzyme activity
Ineffective media use can be prevented by assessing the effectiveness of various media throughout the production process29. As illustrated in Fig. 6a, the evaluation of the relative enzyme activity of DPEase in E. coli revealed that the different compositions of the medium exerted a considerable influence on the enzyme activity. The highest relative enzyme activity of DPEase was observed in TB medium, at 100% composition, followed by SOB medium (59.30%) and LB medium (37.21%). Conversely, the relative enzyme activities in 2×YT and SB media were lower, at 34.40% and 31.01%, respectively.
The effect of induction temperature on enzyme activity
The induction temperature is crucial for enzyme secretion; lower temperatures facilitate proper protein folding into soluble forms but can marginally decrease overall expression levels30. In exploring the effect of induction temperature on the relative enzyme activity of DPEase, it was observed that the enzyme activity showed a specific pattern of change with increasing induction temperature (Fig. 6b). Upon reaching the temperature threshold of 30 °C, the enzymatic activity of DPEase within E. coli cultures achieved a zenith, attaining a relative activity of 100%. In contrast, at the reduced temperatures of 20 °C and 25 °C, the relative enzymatic activities were markedly diminished, recording values of 25.63% and 31.85%, respectively. Upon further elevation of the thermal gradient to 37 °C and 42 °C, a decrement in enzymatic activity was observed, with relative activities plummeting to 54.30% and 39.35%, respectively.
The effect of induction metal ion on enzyme activity
Metal ions are crucial for the conversion of D-fructose to D-allulose, as they stabilize the D-fructose molecule. The DPEase enzymes exhibit significant variation in their dependence on metal ions like Mn2+, Co2+, and Mg2+31,32. In this study, Fig. 6c illustrates that both Fe3+ and Zn2+ significantly enhance DPEase activity, suggesting that this action may play an essential role in regulating DPEase conformation; in contrast, the activities of Ca2+ and Mg2+ are just 15% of the Fe3+ activity (p < 0.0001).
Optimization of plasmid PET22b-E. coli induced conditions (Calculated with a conversion rate of 100% under optimal single factor conditions. (a) The effect of culture medium on enzyme activity. (b) The effect of induction temperature on enzyme activity. (c) The effect of induction Metal ion on enzyme activity. (d) The effect of induction OD600 enzyme activity. (e) The effect of inducer concentration on enzyme activity. (f) The effect of induction time on enzyme activity. ns, no significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are expressed as the mean ± standard deviation (SD) of 3 independent experiments.
The effect of induction OD600 on enzyme activity
The amount of inoculum significantly affects the enzyme activity: too little leads to insufficient activity, too much to metabolic competition17. Figure 6d shows the relative enzyme activities of E. coli when induced by the addition of inducers at different cell densities. The activity was 47.75% at OD600 of 0.1, which increased to 61.55% at 0.3 and peaked at 100% at 0.5. Subsequently, the activity decreased to 75.23% at 0.7 and 0.9 and further decreased to 67.31% at 0.9. This trend indicates that the enzyme activity is not proportional to the cell density, with a maximum observed at an OD600 of 0.5, followed by a slight decrease at higher densities.
The effect of inducer concentration on enzyme activity
This study utilized pET22b as an inducible promoter, requiring the addition of an inducer to the recombinant strain for protein expression. IPTG, a commonly used inducer, effectively induces protein expression but is relatively expensive and has specific toxicity to humans33,34. Therefore, we selected lactose as an alternative to IPTG. Figure 6e demonstrates that lactose achieves optimal induction effects at a concentration of 2.0 g/L. However, subsequent experimental results showed that higher concentrations of lactose did not improve the enzyme activity yield when the lactose concentration exceeded 2.0 g/L. The relative enzyme activity was 78.20% at a lactose concentration of 2.5 g/L, and plummeted to 51.70% when the concentration increased to 3.0 g/L.
The effect of induction time on enzyme activity
Induction duration is directly proportional to protein yield, up to an optimal point. Prolonged induction enhances DPEase expression but may elicit negative feedback, reducing secretion once protein levels reach their peak. This study investigates the impact of induction time on conversion rates35. As shown in Fig. 6f, at an induction temperature of 30 °C, the relative conversion rate increased with the increase of induction time within 24 h. The optimal equilibrium of protein yield and activity was reached at the 24th hour, and its enzyme activity also reached its peak. Subsequently, the relative enzyme activity gradually decreased, and the relative conversion rate dropped to 65.23% at 30 h of induction time.
Optimization of incubation conditions
After inducing the expression of the recombinant strain with the inducible plasmid pET22b(+)-psi under the previously mentioned optimal conditions, we added a substrate to provide a suitable environment for DPEase activity. The DPEase family is characterized by highly conserved sequences, consistent structural homology, and substrate specificity. These characteristics include the optimal reaction pH range of 7.0–9.0 and temperature range of 40–70 °C, critical for enzyme activity and stability36. A thorough understanding of DPEase’s intrinsic characteristics is essential for strategically designing reaction conditions to fully harness the enzyme’s potential. Therefore, we have designed the fermentation conditions for DPEase, focusing on reaction pH, temperature, and substrate concentration. This targeted approach maximizes the conversion rate while ensuring the quality and consistency of the final product, crucial for subsequent large-scale industrial applications.
Effect of permeating agent on enzyme activity
The E. coli system, a leading expression vector, encounters issues with rare codons and complex proteins even under optimal expression conditions. Permeating agent enhances protein solubility, facilitates bacterial material exchange, and promotes proper folding, thus enabling the extraction of heterologous proteins from inclusion bodies37. Figure 7a shows that the relative enzyme activities of E. coli in the presence of osmotic agents were 83.7% for N, N-Dimethylformamide (DMF), 100% for Tween-80, 76.50% for glycine (Gly), and 65.06% for dimethyl sulfoxide (DMSO), with the most pronounced promotion by Tween-80, compared to no osmotic agent, Tween 80 and Gly increased the relative conversion rate compared to no addition, while DMSO inhibited the enzyme activity.
Effect of incubation temperature on enzyme activity
The thermostability of recombinant enzymes is attributed to numerous hydrophobic interactions and a rigid amino acid sequence configuration12. The reported optimal temperature range for DPEase in catalysis is 40 °C to 70 °C. The optimal temperature for the enzymatic reaction was found to be 65 °C. The catalytic efficiency declines rapidly if temperatures deviate from 65 °C. At 60 °C, the conversion efficiency drops to 55.4% of the optimum, and at 70 °C, it is 66.92%, showing that this DPEase is highly temperature-sensitive. Strictly control the temperature during the reaction (as shown in Fig. 7b).
Effect of incubation pH on enzyme activity
In the isomerization reaction of D-fructose to D-allulose, the optimal pH range is generally 7.0 to 9.0. A limited number of DPEase sources, such as Dorea sp, are adapted to weakly acidic conditions38. Extremes in pH can lead to the denaturation and inactivation of the enzyme. Consequently, this study discusses the impact of substrate pH on enzyme activity. Figure 7c shows that the relative enzyme activity of DPEase showed fluctuations in the range of pH 5 to 9. The highest enzyme activity of 100% was reached at neutral pH 7. As the pH deviated from neutral, the enzyme activity decreased, especially at pH 5 and 9 to 33.64% and 52.23%, respectively.
Effect of substrate concentration on enzyme activity
Based on substrate differences, isomerases for synthesizing D-allulose are categorized into distinct families, including D-labeled enzyme-3-epimerases (DTEase) and D-allulose-3-epimerases (DPEase). Members of the DPEase family exhibit strong substrate specificity39. We used a gradient concentration of D-fructose as the substrate. Figure 7d illustrates a negative correlation between substrate concentration and enzyme activity found in this concentration range. The enzyme activity peaked at the lowest concentration of 50 g/L, recorded as 100%. When the substrate concentration was increased to 100 g/L, the relative activity decreased to 48.39%. Further increase in concentration to 150 g/L and 200 g/L resulted in 36.68% and 28.05% enzyme activity, respectively. At the peak concentration of 250 g/L of substrate, the enzyme activity decreased abruptly to 26.06%.
Effect of cell concentration on enzyme activity
The essence of whole-cell catalysis lies in intracellular enzyme action, with varying bacterial concentrations indicating different levels of enzyme addition40. The relative enzyme activity of the reaction system determined at different initial inoculum ratios showed a significant difference, as depicted in Fig. 7e. The relative enzyme activity of E. coli cultures showed a significant increase with the increase of the initial inoculum ratio. At the lowest inoculum ratio of 1%, the enzyme activity was 58.47%, reflecting a weak catalytic ability. When the inoculation ratio was increased to 3%, a significant increase in enzyme activity was observed to 94.51%. When the inoculum ratio was further increased to 5% and 7%, the activity reached 95.43% and 100%, respectively, indicating that the enzyme expression reached the optimal range. Interestingly, at the highest inoculum ratio of 9%, a slight decrease in activity was observed at 98.03%.
Effect of incubation time on relative enzyme activity
Incubation time refers to the duration of substrate-enzyme contact, during which substrate conversion rates are expected to increase theoretically. However, for reversible reactions such as DPEase-catalyzed conversion of D-fructose to D-allulose, equilibrium restricts additional product formation and may lead to a decrease41. As depicted in Fig. 7f, the relative enzyme activity of E. coli showed some differences at different incubation times. At 6 h incubation time, the enzyme activity was 58.47%, and as the incubation time was extended to 12 h, a significant increase in the enzyme activity was observed up to 94.51%. After extending the incubation time to 18 h, the activity increased slightly to 95.43%. The peak activity of 100% was reached at 24 h incubation time, which indicates that the enzyme expression was at its optimum. At the most extended incubation of 30 h, the activity slightly decreased to 98.03%.
Optimization of plasmid PET22b-E. coli in conditions (Calculated with a conversion rate of 100% under optimal single-factor conditions). (a) Effect of penetrant on enzyme activity. (b) Effect of Incubation temperature on enzyme activity. (c) Effect of Incubation pH on enzyme activity. (d) Effect of substrate concentration on enzyme activity. (e) Effect of bacterial concentration on enzyme activity. (f) Effect of incubation time on enzyme activity. ns, no significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are expressed as the mean ± standard deviation (SD) of 3 independent experiments.
D-allulose preliminary purification
After gradually optimizing the fermentation conditions, we achieved a conversion rate of 33.91%, with an expected D-allulose concentration of 16.95 g/L. However, due to LAB’s consumption of D-allulose as a carbon source, the final concentration is 15.53 g/L after accounting for losses. After a 16-hour reaction (Fig. 8), the purity reached 64.73%, with a D-allulose content of 13.28 g/L. By the 24th hour, the purity had increased to 75.60%, yet the D-allulose content was down to 11.34 g/L, This indicates that LAB, similar to E. coli, consumes a portion of D-allulose, hindering the accumulation of the final product. Consequently, the purification was halted at 16 h.
D-fructose content in the system and purity of D-allulose.
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- Source: https://www.nature.com/articles/s41598-024-80561-5