Team:AHUT China/Design
Design
Our project is to improve the heat resistance and stability of human Carbonic Anhydrase 2(CA2) so as to control the carbon dioxide emission in the factory. Last year, we improved thermal stability of wild-type human CA2, but the lack of data on enzyme fixation limited its further industrial applications. So in this year, based on the previous year's project, we hope to achieve enzyme immobilization and maintain high thermal stability by modifying its gene sequence.
Overview
Formex glycine enzymes
Prokaryotic formylglycine - producing enzyme (FGE) was first discovered in 2003 and is used to label proteins. FGE recognizes LCTPSR and selectively oxidizes cysteine residues in the peptide to form aldehyde-containing formyl glycine (FGly) residues. CA2, which is terminal aldehyde group, 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.
So this year we intend to construct a new biobrick(CA2(L203K)-N-LCTPSR) for silver medal by linking the N-terminal of the mutant human CA2(CA2(L203K)) (BBa_K2547004) with the LCTPSR, and construct a new biobrick (CA2(L203K)-C-LCTPSR) for gold medal by linking the C-terminal of CA2(L203K) with the LCTPSR. Then, the inserted LCTPSR tag was recognized by FGE to form aldehyde-containing formyl glycine (FGly) residues, which was used for the followed CA2 immobilization.
The overall design for our project is as follows:
Construction of Vectors
We constructed CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR recombinant plasmids by using genetic engineering technology. The coding sequences of CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR were synthesized, then cloned into the expression vector pET-30a(+), respectively. The correctness of the obtained recombinant vectors were identified by restriction enzyme digestion and sequencing.
Expression and Purification of Proteins
We induced CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR expression in E.coli TB1 by using IPTG. The crude extracts were centrifuged to separate the supernatant and precipitate. The precipitate was used for SDS-PAGE.
After confirming that CA2(L203K)-N-LCTPSR, CA2(L203K)-C-LCTPSR and CA2(L203K) could be expressed in E.coli, these proteins will be further purified with nickel column.
Enzyme Immobilization
FGE can oxidize the terminal cysteine residues of the target protein to form aldehyde labels at the end. So we expected to construct a dual plasmid system containing the aldehyde-tagged CA2(L203K)-N-LCTASR and CA2(L203K)-C-LCTASR can be immobilized via forming covalent bond with amino functionalized carriers (Unisil 30-100 NH2) through the Schiff base reaction.
Identification of the Function
Esterase activity analysis
CA2 can catalyze the hydrolysis of p-nitrophenyl acetate. Therefore, we intend to use the esterase activity of CA2 to catalyze the p-nitrophenyl acetate, and obtain the enzyme activity data by the change of absorbance before and after a certain reaction time.
Application Model
This year, we designed an experimental device to measure the application of CO2 capture and the ability of the reuse of fixed enzyme. The overall designed model is as follows:
