Part:BBa_K5528001

TcDAE

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]

40

Part: BBa_K5528001 (TcDAE)

Profile

Name: TcDAE

Base Pairs: 870 bp

Origin: Thermoclostridium caenicola

Properties: D-psicose 3-epimerase can convert D-fructose to D-psicose. A putative DAEase from Thermoclostridium caenicola was identified and characterized. It is metal-dependent and exhibits maximum activity at pH 7.5 and 65°C in the presence of 1 mM Co²⁺. The enzyme's thermostability is greatly enhanced by Co²⁺.

Usage and Biology

In the current literature, the DAEase of Thermoclostridium caenicola has been expressed in Escherichia coli, and the enzymatic properties of DAEase were further studied. DAEase is used to convert D-fructose to D-psicose, and it is strictly dependent on Co²⁺ for its function. In the presence of Co²⁺, it displayed enhanced thermostability with a 5.4-fold increase in half-life at 55°C.The novel DAEase displayed maximum activity at pH 7.5 and 65°C in the presence of 1 mM Co²⁺, as studied by Chen et al. (2021).

Experimental Data

Figure 1. The gel electrophoresis of TcDAE nucleic acids. The length of TcDAE is 870bp.

To construct the plasmid pPICZαA-TcDAE, the target gene TcDAE was amplified by PCR. In Figure 1, our sample is mainly between 750 bp and 1000 bp, accurately matching the theoretical length of 870 bp, indicating a successful polymerase chain reaction.

Purification and SDS-PAGE

When the cultures reached an optical density (OD₆₀₀) of about 0.6-0.8, methanol was added to the BMMY medium to induce the recombinant protein. After 24 hours of induction, the bacterial cells were lysed by sonication in phosphate buffer. The recombinant His₆-fused TcDAE was purified using Ni²⁺ affinity chromatography.

The protein was then detected using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In Figure 2, a protein band was observed between 34kDa and 43kDa, consistent with the expected size of TcDAE at 37.8kDa, indicating successful protein expression after 24 hours of induction.

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

Reference

Chen, Jiajun, et al. “Characterization of a Recombinant D-Allulose 3-Epimerase from Thermoclostridium Caenicola with Potential Application in D-Allulose Production.” Molecular Biotechnology, vol. 63, no. 6, 29 Mar. 2021, pp. 534–543, https://doi.org/10.1007/s12033-021-00320-z. Accessed 22 Apr. 2022.