Part:BBa_K5366054

T7 promoter-RBS-AJC7/S125D-6xHis-T7 termonator

AJC 7 single-point mutation expression plasmid

Molecular Docking

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

S125D plays a consistent role in all mutations due to the first step in the conversion of D-fructose to D-tagatose is the production of a glyceraldehyde intermediate.When serine is mutated to aspartic acid, the carboxy group of aspartic acid can interact with the terminal aldehyde group of the glyceraldehyde intermediate, promoting the protonation of the aldehyde and thus facilitating the catalysis and production of D-tagatose. Furthermore, the negatively charged aspartic acid may enhance interactions with the positively charged substrate and alter the charge distribution in the binding pocket, thereby improving the catalytic activity of the enzyme.

Construction

Primers were designed for the S125D point mutation, and the plasmid containing the pET-28a(+) vector was amplified using PCR. Following the mutation, the PCR products were verified by nucleic acid gel electrophoresis to confirm the presence of the desired bands. The plasmid containing the correct bands was subsequently transformed into E. coli BL21 (DE3) competent cells.

Fig.1 Point mutation localisation and primer design

Fig. 2 Nucleic acid gel plot of colony PCR

Indicator

The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification procedures to extract the target proteins, as outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The final concentration of fructose in the reaction mixture was 100 g/L, and it included 10 μL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, and the resulting products were subsequently analyzed using High-Performance Liquid Chromatography (HPLC).

Result

The catalytic efficiency of the S125D mutant was nearly doubled compared to the wild type when both were subjected to a 5-hour reaction at 70°C under identical reaction conditions and substrate concentrations. This finding suggests that the S125D mutation represents a very effective modification for AJC7.

Fig.3 Fig.3 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

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1534
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 540
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1042
  
• 1000
  COMPATIBLE WITH RFC[1000]