Part:BBa_K2949013
Carbonic Anhydrase 2(L203K)-C-LCTASR
This part is the coding sequence (CDS) of the C-terminal modified Carbonic anhydrase 2(L203K)(BBa_K2547004).Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups.The terminal aldehyde group of CA2, is fixed to surface active carriers containing amino groups by covalent binding. This site-specific fixation avoids the chemical degeneration that may occur in common covalent fixation and has higher stability than physical adsorption.
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]
In order to the further improve the industrial application of CA2 for CO2 capture, basing on the existing part (BBa_K2547004) we designed last year, we have constructed a new biobrick[CA2(L203K)-C-LCTPSR](BBa_K2949013) by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)](BBa_K2547004) coding sequences with the six-residue sulfatase submotif(LCTPSR) in a way similar to that of CA2(L203K)-N-LCTPSR, to achieve enzyme immobilization and maintain high thermal stability and CA2 reuse by modifying its gene sequence.
The coding sequence of CA2(L203K)-C-LCTPSR(BBa_K2949013) was synthesized, and then cloned into pET-30a(+) expression vector. The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.10) and sequencing(Fig.11). Subsequently, the expression of CA2(L203K)-C-LCTPSR in E. coli were detected by SDS-PAGE. The results showed that CA2(L203K)-C-LCTPSR could be successfully expressed in E.coli.(Fig. 12)
To use for the following CA2(L203K)-C-LCTPSR immobilization, the improve part of CA2(L203K)-C-LCTPSR protein was purified through nickel column and detected by SDS-PAGE, as shown in Fig.13. FGE can selectively identify and oxidize cysteine residues in the sulfatase subunit(LCTPSR) at the end of the protein to form aldehyde-containing formylglycinase,which can be used for enzyme immobilization. Then we immobilized CA2(L203K)-C-LCTPSR protein, and our formula for calculating the enzyme immobilized efficiency is as follows：
η: The efficiency of immobilized CA2(L203K)-N-LCTPSR protein; W1: The concentration of total CA2(L203K)-N-LCTPSR protein; W2:The concentration of free CA2(L203K)-N-LCTPSR protein.
According to the formula, we got the efficiency of immobilized CA2(L203K)-C-LCTPSR protein is 39.09%.
To further demonstrate the activity of our improved part, we compared the enzymatic activity of CA2(L203K)-C-LCTPSR (BBa_K2949013) with CA2(L203K) protein (BBa_K2547004) by esterase activity assay. As shown in Fig.14 and Fig.15, immobilized CA2(L203K)-C-LCTPSR protein retain its activity at 37℃and 50 °C, and free CA2(L203K)-C-LCTPSR protein has a higher activity than CA2(L203K) protein at 50℃, indicating that CA2 (L203K)-C-LCTPSR protein was stable at high temperature and retained its activity. Then, the reuse ability of the immobilized enzyme was tested by our designed simulation model (Fig.16), and the result showed that the immobilized CA2(L203K)-C-LCTPSR could absorb CO2 under the simulation model and showed potential reuse ability as indicated in Fig.17. In conclusion, our results demonstrated that the function of improved part has been improved with higher activity than original part, especially achieved enzyme immobilization, and the immobilized CA2(L203K)-C-LCTPSR protein showed reuse ability, which might be suitable for industrial production.
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