Part:BBa_K3771000
JJU
Description
JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide[1].
Biology
The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].
<img src="圖片網址" style="width:35%;">
Figure.1 Taurine pathways in E. coli [1,2].
JJU is an enzyme that is part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC[1].
Usage
<img src="圖片網址" style="width:35%;">
Figure 2. Biobrick of trc-JJU
JJU enzyme was used in in vitro testing of taurine production. The sequence for JJU enzyme and trc promoter (BBa_K864400) were ligated and transformed into E. coli. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentrations were determined using high-performance liquid chromatography (HPLC).
Characterization
The jju sequence synthesized by IDT was amplified by PCR and ligated to trc promoter (BBa_K864400) in the pET28a cloning vector.
<img src="圖片網址" style="width:35%;">
Taurine Production of JJU in E. coli BL21(DE3) strain
In our in vitro testing of taurine production by JJU, JJU was transformed into E. coli BL21(DE3) strain. Supernatant (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE.
<img src="圖片網址" style="width:35%;">
Figure 4. Confirmation of JJU expression by SDS-PAGE
Whole cell and supernatant JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentrations were lower, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 2, 2:1 ratio of JJU to CoaBC supernatant volume had the highest taurine concentration of around 95 mg/L.
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]
None |