Part:BBa_K5366064

AJC7/S125D/T181A/ L140P

AJC7 triple point 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/L140P three-point mutant plasmid. This final plasmid was then transferred to E. coli BL21 (DE3) competent cells for verification via nucleic acid electrophoresis (Figure 2). Verification results are presented in Figure 2.

Fig.1 Mapping of mutant plasmids

Fig.2 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 3).

Result

To enhance substrate transformation capabilities, we introduced a three-point mutation (S125D/T181A/L140P) into AJC7. The results indicated that the product concentrations for this triple mutant were higher compared to the wild-type enzyme. However, its effectiveness was somewhat reduced when compared to the optimal two-point mutant.

Fig.3 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/T181A/L140P, and S125D/T181A/H342L reacted with 100 g/L fructose substrate for 5 h, respectively

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]