Part:BBa_K5528004

DsDAE

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Part: BBa_K5528004 (DsDAE)

Profile

Name: DsDAE

Base Pairs: 870 bp

Origin: Dorea sp. CAG 317

Properties: DsDAE is an acidic ketose 3-epimerase, a recombinant of D-allulose 3-epimerase found in Dorea sp. CAG 317.

Usage and Biology

DsDAE is a D-allulose 3-epimerase (DAEase) that converts D-fructose to D-allulose. It is a metal-dependent enzyme requiring Co²⁺ ions for its activity. The ratio of D-fructose to D-allulose conversion is approximately 30:70 (Zhang et al., 2015).This enzyme has been studied for its activity characteristics in E. coli. The recombinant DsDAE shows activity at acidic pH and is dependent on Co²⁺ ions for its catalytic function.

Experimental Data

Figure 1. The gel electrophoresis validation of DsDAE nucleic acids. DsDAE: 870 bp.

To construct the plasmid pPICZαA-DsDAE, the target gene DsDAE was amplified by PCR. As shown in Figure 1, the sample is mainly in the range between 750 bp and 1000 bp, matching the theoretical length of 870 bp, indicating a successful PCR amplification.

Purification and SDS-PAGE

When the culture's optical density (OD600) reached about 0.6-0.8, methanol was added to the BMMY medium for recombinant protein induction. After 24 hours of induction, the bacterial cells were lysed by sonication in phosphate buffer. The recombinant His₆-fused DsDAE was purified using Ni²⁺ affinity chromatography. The protein was detected using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

As shown in Figure 2, the protein band is between 34 kDa and 43 kDa, consistent with the expected size of DsDAE at 37.8 kDa, indicating successful protein expression.

Figure 2. The SDS-PAGE detection of DsDAE protein expression after 24 hours. DsDAE: 37.8 kDa.

References

Zhang, Wenli, et al. "Characterization of a D-Psicose 3-Epimerase from Dorea sp. CAG317 with an Acidic pH Optimum and a High Specific Activity." Journal of Molecular Catalysis B: Enzymatic, vol. 120, Oct. 2015, pp. 68–74, https://doi.org/10.1016/j.molcatb.2015.05.018. Accessed 12 Aug. 2024.