Part:BBa_K5366037

AJC7/S125D/ T181A

AJC7 two-point mutant

Molecular Docking

Fig.2 Docking model of D-fructose in the S125D/T181A mutant.

Construction

The mutation site of T181A is located far from S125D, allowing for the direct construction of the S125D/ T181A double mutant using pET-28a(+)-AJC7-S125D as a template along with the primers designed for single-point mutation as outlined in [Design 1]. Following the mutation, the PCR products were verified using nucleic acid gel electrophoresis. The plasmids that exhibited the correct bands were 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 two-point 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 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 concentration of fructose in the system was set at 100 g/L, with the addition of 10 μL of Ni²⁺ 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).

Result

The results demonstrated that the concentration of products obtained from the AJC7 enzyme after the two-point mutation of S125D/T181A, under the conditions of a 70℃ reaction for 5 hours, was significantly higher than the enzyme activity enhancement observed in the wild-type AJC7-S125D. Furthermore, the catalytic efficiency of the enzyme was enhanced by nearly two-fold compared to that of the wild-type AJC7. This indicates that the S125D/T181A mutation is an exceptionally effective double mutation for enhancing the performance of AJC7.

Fig.3 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/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]