Part:BBa_K5366062

AJC7/S125D/ L140P

AJC7 two-point mutant

Construction

The mutation site of L140P is located far from S125D, allowing for the direct construction of the S125D/L140P 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 showed that the concentration of products obtained from the S125D/L140P two-point mutation of AJC7 was decreased, and the catalytic efficiency was lower compared to that of the wild-type AJC7-S125D under the reaction conditions of 70℃ for 5 hours. These findings indicate that the S125D/L140P two-point mutation is not effective, confirming that this particular mutant does not enhance enzyme performance.

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]