Carbonic anhydrase (CA) is an enzyme which can be commonly seen in human erythrocytes, it helps the respiratory system to remove CO2 by increasing CO2 solubility in the bloodstream. The CA catalyzes the gas form of CO2 to the ionic form of bicarbonate as a dissolved inorganic compound.

Gene cloning and protein expression

Human carbonic anhydrase II (BBa_K2932001) is well-studied and is one of the most efficient enzymes catalyzing the rapid conversion of CO2 to bicarbonate (HCO3-), and we used it to improve the dissolution rate of CO2 in the algal culture medium. We got the gene sequence from NCBI and synthesized the DNA fragment by Twist Bioscience. Then, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3. CA was tagged with 6xHis at C terminus for further protein purification if needed.

Construct of PliaI-RBS-CA-Tr/pSB1C3

To express genes of CA in Bacillus subtilis, we transfer the DNA fragments of PliaI-RBS-CA-Tr to pBS0E vector and transformed Bacillus subtilis 168 with the resulting plasmids.

Protein induction procedure

↓ culture Bacillus Subtilis 168 carrying the plasmid of PliaI + RBS + CA + terminator/pBS0E in LB + antibiotics O/N at 37°C, shaking at 170 rpm, supplied with 60μM of ZnSO4
↓ transfer 3 ml to 50 ml LB+Antibiotics in 250 ml flask with 60μM of ZnSO4
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 400μM of ZnSO4 and 50μl of 30μg/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr
After protein induction by bacitracin, total lysates of Bacillus expressing were subjected to SDS-PAGE and stained with Coomassie blue. CA protein was observed after induction around 30kDa as the same size of predicted molecular weight.

CA protein purification

↓ Equilibrate Ni-NTA resin in the column with 20mM Tris-HCH, 200mM NaCl, pH7.5
↓ Load protein lysates onto the column. The flow-through was collected.
↓ Wash the column with 20mM Tris-HC, 200mM NaCl, 5mM imidazole, pH7.5 The wash-through was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 20mM imidazole, pH7.5. The Elution #1 was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 200mM imidazole, pH7.5. The Elution #2 was collected.
↓ Elute the column with 20mM Tris-HC, 200mM NaCl, 500mM imidazole, pH7.5 The Elution #3 was collected.
The total lysates, flow-through, wash-through and Elution #1, #2, #3 were subjected to SDS-PAGE and Coomassie Blue staining as well as Western Blotting with the anti-His antibody. The data presented below gave a clear evidence of the CA protein induction, expression and purification. (The work of purification, SDS-PAGE and WB for CA protein was collaborated with team CSMU-Taiwan)

CA functional assay

To test CA function, we added the purified CA into ddH2O and pump the ambient CO2 (~450 ppm) into the water. The pH level and CO2 dissolution rate were measured by pH meter and CO2 sensor, respectively. Based on the catalyzation reaction of CA, if CA works, CO2 level will drop and pH value of water should decrease.

The calibration curve presented in the figure below is made in the scatter plot. The concentration of phenol (mg/L) (Y) can be calculated from the values of OD580 by the equation of Y = 0.00003*e^(16.202*X)

CYP2E1 functional assay

Benzene is one of the substrates of CYP2E1. Because of the toxicity of benzene, a collegiate iGEM team, Tunghai-TAPG helped us conduct the analysis in a specialized laboratory in the Department of Chemistry in the university.

Experiment procedure

↓ prepare CYP2E1 and WT lysates
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution with 10X serial dilution to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I (1% of 4-aminoantipyrine in KOH solution, pH=9~10
↓ then add 45μl of solution II (4% of K3[Fe(CN)6])
↓ measure at OD580

The data showed that the values measured at OD580 are higher in the group of benzene plus CYP2E1 compared to controls of benzene without CYP2E1, indicating that the phenol is converted from benzene by CYP2E1. The benzene has a basal effect in Emerson reaction. The values of OD580 of Benzene are regarded as background and are subtracted for study the phenol generation.

Next, according to the calibration curve of phenol analysis in Emerson reaction. We converted the OD values of phenol formation to benzene degradation. The result suggested CYP2E1 in the bacterial lysate can convert 0.09 - 0.47 μg/L of benzene to phenol, implying the possibility of CYP2E1 application in benzene removal in our microalgae purification system.

Microalgae purification system demonstration

Finally, we’d like to know the phenol consumption by algae in our system. We incubated algae with a serial 10x dilution of phenol solution as did in the analysis of the calibration curve, followed by culturing algae with light at 37°C for 1hr. Then, the algae culture media was centrifuged to discard the algal cells, and the supernatants were subjected to Emerson reaction assay.

Summary

We successfully demonstrated the CYP2E1 can oxidize benzene to phenol, which can be absorbed by algae. These findings extend the possibility of applying the enzymes to microalgae purification system to remove various pollutants or toxic chemicals, which are naturally unable to be absorbed by algae.

Discussion

Cytochrome P450 2E1 (CYP2E1) used in benzene and chloroform removal is first studied by Sharon L. Doty, et al. in 2007 and published on the distinguished PNAS journal. The authors demonstrated that CYP2E1-transgenic tree, Populus alba, has the ability to remove benzene at 0.28 μg/h. Based on our study in microalgae purification system, it is for the first time to demonstrate algae is able to take up phenol converted from benzene in the process of CYP2E1 catalyzation. The algae can indirectly absorb benzene at 0.95 μg/h, which improve the efficiency up to 4.7 fold. Our result showed that the microalgae system has great potential to remove unwanted pollutants efficiently with extracellular enzymes, which convert the pollutants to substrates absorbed by algae.

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