Part:BBa_K5528004
DsDAE
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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.
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.
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