Part:BBa_K4927047
F7
We constructed a formaldehyde Dehydrogenase labeled with CL7 (BBa_K4927005). It will form an artificial multi enzyme complex with a protein scaffold designed by us to improve the reaction efficiency of the entire system.
Introduction
Formaldehyde Dehydrogenase is a key enzyme that is widely distributed in living organisms, and its biocatalytic properties are of great significance in the fields of biochemistry and metabolic regulation. The main biological function of the enzyme is to catalyze the reaction of Formaldehyde with oxidant to convert formaldehyde into formic acid, and in addition to the transfer of supported electrons in the REDOX reaction. This critical catalytic process is involved in a variety of metabolic pathways in living organisms, including but not limited to cellular respiration, methyl glucose metabolism, and formic acid metabolism. The catalytic mechanism of formaldehyde dehydrogenase often involves coenzyme synergies, with the most common cofactors including nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors act as electron acceptors in the reaction, thus facilitating the REDOX reaction of formaldehyde and ultimately the formation of formic acid. This reaction plays a key role in energy production and metabolic regulation in living organisms.
Usage and Biology
The formaldehyde dehydrogenase gene (FALDH) we selected was derived from the whole genome of Bacillus subtilis, and the target FALDH fragment was obtained by PCR with corresponding primers. The expression plasmid pET28a-FALDH-CL7 was constructed by fusing FALDH gene with CL7 (BBa_K4927005) at the N-terminal of the vector construction system using T5 exonuclidenase[1].
Figure1: pET28a-FALDH-CL7 plasmid
FALDH will use formaldehyde and NAD+ as substrates to form NADH and formic acid through catalytic oxidation. NADH will produce hydrogen in the final step of the reaction we designed, with the action of hydrogenase.
Figure2:F7 Genetic circuit
Characterization
We transferred the constructed pET28a-FALDH-CL7 (F7) expression plasmid into the receptive state BL21, and after 8h expansion culture, 0.5mM IPTG was used to induce 16h. The bacteria were crushed and purified by nickel column at low temperature and high pressure. Finally, formaldehyde dehydrogenase (F7) was obtained.
Figure3:F7 SDS-PAGE Test result. Lane 1 is marker, lane 2 is the mixed liquid after breaking bacteria, lane 3 is the precipitation after breaking bacteria, lane 4 is the supernatant after breaking bacteria, lane 5 is 10mM imidazole eluent, lane 6 is 30mM imidazole eluent, lane 7 is 50mM imidazole eluent, lane 8 is 80mM imidazole eluent, lane 9 is 100mM imidazole eluent, and lane 10 is 150mM imidazole eluent, 300mM imidazole eluent in lane 11. The red arrow marks the target protein band
We investigated the activity of F7 at different PH values by UV spectrophotometer at room temperature, and recorded the change of light absorption value at 340nm. We found that F7 showed the highest activity at PH=8.05.
Figure 4:Activity test results of F7 under different pH conditions
In order to further explore the optimal conditions for F7, we set different temperatures at pH=8.05 to detect the activity of F7.After detecting the absorbance value at 340nm using a UV spectrophotometer, we found that F7 exhibited the best activity at 40 ℃
Figure 5:Activity test results of F7 under different temperature conditions
References
[1]Yongzhen Xia, Kai Li, Jingjing Li, Tianqi Wang, Lichuan Gu, Luying Xun, T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis, Nucleic Acids Research, Volume 47, Issue 3, 20 February 2019, Page e15.
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]
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