Part:BBa_K5366041

AJC7/S125D/T181A/ I129T

AJC7 triple point mutant

Molecular Docking

Fig. 1 Docking model of D-fructose in the S125D/T181A/I129T mutant

Construction

1. Plasmid Construction
S125D point mutation primers were designed, and PCR was performed using pET-28a(+)-AJC7 as a template for the mutation (Fig. 1). Following the PCR reaction, demethylation was carried out using DpnI. To verify the mutations, 5 μL of the reaction mixture was taken for analysis by nucleic acid gel electrophoresis. After confirming the correctness of the PCR product, the product was recovered to obtain the single point mutant plasmid. The concentration of the single-site mutant plasmid was measured, and it was subsequently transformed into E. coli BL21 (DE3) competent cells. The cells were incubated in an inverted culture at 37°C for 14 hours. Single colonies were selected from the transformed colonies, and colony PCR was performed. After verification through nucleic acid electrophoresis, the corresponding single colonies displaying the correct bands were transferred to LB (Kan) liquid medium for preservation. This completed the S125D single-site mutation step. After successfully obtaining the S125D single-point mutant plasmid, this plasmid was further mutated to construct the S125D/T181A two-point mutant plasmid, following the same steps as for the single-point mutation. Next, the S125D/T181A two-point mutant plasmid underwent additional mutation to create the S125D/T181A/I120T three-point mutant plasmid. This final plasmid was then transferred to E. coli BL21 (DE3) competent cells for verification via nucleic acid electrophoresis (Figure 3). Verification results are presented in Figure 2.

Fig. 2 Mapping of mutant plasmids

Fig.3 Nucleic acid gel diagram of colony PCR

2. Product Analysis
The mutant and wild-type strains were activated, cultured for amplification, and subjected to a series of protein purification operations to extract the target proteins, as outlined in the [Experimental] section. The volume of the purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration specified in [Experimental]. The final fructose concentration in the reaction system was set at 100 g/L, and 10 µL of Ni²⁺ was included as a catalyst. The reaction was conducted at 70°C for 5 hours, after which the products were analyzed using High-Performance Liquid Chromatography (HPLC) (Figure 4).

Result

The results indicated that the concentration of products following the three-point mutation (S125D/T181A/I120T) of AJC7 was significantly higher compared to that of the wild-type AJC7, as well as the S125D and S125D/T181A variants. Notably, the S125D/T181A/I120T three-point mutant exhibited a substantial enhancement in enzyme activity, with the catalytic efficiency of AJC7 increasing by nearly threefold compared to that of the wild-type enzyme.

Fig.4 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

Determination of Enzymatic Properties and Enzyme Kinetic Parameters

Enzymatic Properties Studies
a.Calculate the relative activity under other pH conditions separately with a maximum activity of 100% at pH 9.0.

Fig.5 The relative activities of AJC7-S125D/T181A/I129T at different pH. The activity at pH 9.0 was set as 100%.

Figure 5 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 (see Fig. 6).

Fig.6 AJC7-S125D/T181A/I129T triple mutant enzyme activity as a function of reaction temperature

As illustrated in Fig. 5, 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⁺).

Figure 7 Conversion rate of the best AJC7 mutant under optimal conditions

As shown in Figure 6, 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). Enzyme Kinetic Parameter Determination

Fig. 8 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. <h>Conclusion</h1> Upon reviewing the iGEM part library, we discovered that past teams, such as XJTU-China 2018, had also provided sequences for enzymes capable of catalyzing the generation of tagatose from fructose. However, these teams did not present any experimental data. In contrast, our team proposes an enzyme that catalytically generates tagatose from fructose, supported by wet experimental data for the first time in iGEM history. Furthermore, we modified this enzyme through protein engineering, and the three-point mutant of this enzyme resulted in a maximum conversion of 37%, achieving peak yield rates of 24 g/(L·h). While comparable data from previous iGEM entries is lacking, literature(Fig.9) review indicates that our proposed enzyme remains competitive among others with similar functionalities. This advancement presents promising prospects for the industrial-scale production of Tagatose.

Fig. 8 Enzymes related to catalysing the production of tagatose from fructose and their conversion rates in the literature in recent years

Reference

[1]Wang Lifei, Tan Zi nuclei, Xie Xixian, etc. New enzyme mining and enzymatic properties of Tagsugar-4-isomerase [J]. Journal of Microbiology, 2023,63(11):4197-4207.DOI:10.13343/j.cnki.wsxb. 20230182. [2]Shin K C, Lee T E, Seo M J, et al. Development of tagaturonate 3-epimerase into tagatose 4-epimerase with a biocatalytic route from fructose to tagatose[J]. Acs Catalysis, 2020, 10(20): 12212-12222. [3]Xia, Wenhao, et al. "Reshaping the binding pocket of D-tagaturonate epimerase UxaE to improve the epimerization activity of C4-OH for enabling green synthesis of d-tagatose." Molecular Catalysis 566 (2024): 114439.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 501
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1003
  
• 1000
  COMPATIBLE WITH RFC[1000]