Part:BBa_K5366038
AJC7/S125D/ H342L
AJC7 two-point mutant
Construction
The mutation site of H342L is located far from S125D, allowing for the direct construction of the S125D/H342L 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 indicated that the concentration of products obtained from the AJC7 enzyme after the two-point mutation of S125D/H342L showed an increase compared to the enzyme activity of the wild-type AJC7-S125D under the reaction conditions of 70℃ for 5 hours; however, the enhancement observed was not significant.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 501
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1003
- 1000COMPATIBLE WITH RFC[1000]
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