Part:BBa_K5366045

AJC7/S125D/T181A/ I129T/L140P/ H342L-6xHis

AJC7 five-point mutant

Construction

Primers for the S125D point mutation were designed, and PCR was conducted using pET-28a(+)-AJC7 as a template (Fig. 1). Following the PCR reaction, DpnI demethylation was performed. To verify the digestion, 5 µL of the reaction was analyzed using nucleic acid gel electrophoresis. After confirming the correctness of the PCR product, recovery was carried out 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) cells. The cells were incubated in inverted culture at 37°C for 14 hours. From the transformed colonies, single colonies were selected for colony PCR. Following nucleic acid electrophoresis verification, the corresponding colonies with the correct bands were transferred to LB (Kan) liquid medium for preservation. This step completed the S125D single-point mutation process. After the successful creation of the S125D single-point mutant plasmid, this plasmid was further mutated to obtain the S125D/T181A two-point mutant plasmid, following the same single-point mutation protocol. Subsequently, the S125D/T181A two-point mutant plasmid was mutated to generate the S125D/T181A/H342L three-point mutant plasmid. This process continued with the S125D/T181A/I129T three-point mutant plasmid and the S125D/T181A/I129T/L140P four-point mutant plasmid. Finally, the S125D/T181A/I129T/L140P four-point mutant plasmid was mutated to create the S125D/T181A/I129T/L140P/H342L five-point variant mutant plasmid, which was then transfected into E. coli BL21 (DE3) and verified by nucleic acid electrophoresis (Figure 2).

Fig.1 Mapping of mutant plasmids

Fig.2 Nucleic acid gel diagram of colony PCR (4-point mutation on the left and 5-point mutation on the right)

Product Analysis

The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification processes to extract the target proteins, as described in [Experimental]. The volume of the purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration indicated in [Experimental]. The final fructose concentration in the system was set at 100 g/L, along with the addition of 10 µL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, and the final product was analyzed using High-Performance Liquid Chromatography (HPLC) (Figure 3).

Fig.30 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

Result

To further enhance substrate transformation capabilities, we conducted five point mutations on AJC7, specifically substituting S125D, T181A, I129T, L140P, and H342L . The results indicated that the concentration of the product following these mutations was significantly increased compared to that of the wild-type strain. 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]