Part:BBa_K5528003

NtDAE

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]

NtDAE (BBa_K5528003) Documentation

Part: BBa_K5528003 (NtDAE)

Profile

Name: NtDAE

Base Pairs: 873 bp

Origin: Novibacillus thermophilus

Properties: D-psicose 3-epimerase can convert D-fructose to D-psicose. This gene comes from a thermophilic bacterium, the only known source to produce DAEase.

Usage and Biology

Novibacillus thermophilus DAEase (NtDAE) has been studied for its ability to convert D-fructose to D-allulose in the presence of Co²⁺ ions. The conversion yield of 500 g/L D-fructose to D-allulose at 60°C achieved 29.7% (Dong-Xu J et al., 2015). NtDAE showed optimum activity at 70°C and pH 7.0. Co²⁺ enhanced its activity and stability, with t_1/2 values of 846.3, 539.2, and 47.8 min at 40°C, 50°C, and 60°C, respectively.The study aimed to synthesize D-allulose using NtDAE as a key enzyme.

Experimental Data

Gel electrophoresis of NtDAE nucleic acids — Figure 1. The gel electrophoresis validation of NtDAE nucleic acids. NtDAE: 855 bp.

To construct the plasmid pPICZαA-NtDAE, the target gene NtDAE was amplified by PCR. As shown in Figure 1, our sample is in the range between 750 bp and 1000 bp, matching the theoretical length of 855 bp, indicating successful PCR amplification.

Purification and SDS-PAGE

When the culture's optical density (OD600) reached about 0.6-0.8, methanol was added to BMMY medium for recombinant protein induction. After 24 hours, the bacterial cells were lysed by sonication in phosphate buffer. The recombinant His₆-fused NtDAE 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 NtDAE at 37.9 kDa, indicating successful protein expression.

Figure 2. The SDS-PAGE detection of NtDAE protein expression after 24 hours. NtDAE: 37.9 kDa.

References

Dong-Xu J., Chen-Yi S., Yi-Ting J., et al. "Properties of D-allulose 3-epimerase mined from Novibacillus thermophilus and its application to synthesis of D-allulose." Enzyme and Microbial Technology, 2021, 148109816-109816.

Jia, Dong-Xu, et al. "Properties of D-Allulose 3-Epimerase Mined from Novibacillus thermophilus and Its Application to Synthesis of D-Allulose." Sciencedirect, Aug. 2021, www.sciencedirect.com/science/article/abs/pii/S0141022921000740.