Team:AHUT China/Result
Result
New Part
We found that formylglycine-generating enzymes (FGE) can selectively identify and oxidize cysteine residues in the sulfatase subunit (LCTPSR) at the end of the protein to form aldehyde-containing formylglycine, which can be used for enzyme immobilization.
So, in this year, we constructed a new biobrick [CA2(L203K)-N-LCTPSR] (BBa_K2949012) by connecting the N-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) coding sequences with the six-residue sulfatase submotif (LCTPSR), to immobilize CA2(L203K) for efficient CO2 capture.
1. Engineered E.coli TB1
The coding sequence of CA2(L203K)-N-LCTPSR was synthesized and cloned into the expression vector pET-30a(+), as presented in Fig.1:
The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2) and sequencing (Fig.3).
Lane M: DL15000 marker; Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).
We induced pilot expression of CA2(L203K)-N-LCTPSR in E.coli TB1 by using isopropyl-1-thio-β-Dgalactopyrasonide(IPTG).
Briefly, recombinant plasmid of the CA2(L203K)-N-LCTPSR was transformed into E.coli TB1, and positive clone was screened by kanamycin resistance. Then, the recombinant E.coli TB1 were propagated and CA2(L203K)-N-LCTPSR expression were induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and the fraction was subjected to SDS-PAGE.
The results showed that CA2(L203K)-N-LCTPSR could be successfully expressed in TB1 strain (Fig.4).
Lane 1: CA2(L203K)-N-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-N-LCTPSR protein expression with IPTG induction.
After confirming that CA2(L203K)-N-LCTPSR could be expressed in our chassis E.coli TB1, we successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-N-LCTPSR expression vector into E.coli TB1. Then the protein of CA2(L203K)-N-LCTPSR was further purified with nickel column for the following enzyme immobilization.
The results showed that CA2(L203K)-N-LCTPSR was purified with high purity as indicated by a significant single protein band by SDS-PAGE (Fig.5).
2. Identification of the function for CO2 capture
In order to improve the efficiency of the repeated utilization of the CA2(L203K)-N-LCTPSR protein, we achieved enzyme immobilization.
Briefly, we cultured CA2(L203K)-N-LCTPSR in shaking table with the immobilization support (Unisil 30-100 NH2), then added 10% sodium cyanoborohydrate and continued to immobilize it in shaking table culture.
Our formula for calculating the enzymatic immobilized efficiency is as follows：
$$\eta=\frac{W 1-W 2}{W 1}\times100\%$$
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)-N-LCTPSR protein is 32.35%.
The enzyme activity of immobilized CA2(L203K)-N-LCTPSR protein was tested experimentally by esterase activity assay at 37℃ and 50℃.
As indicated in Fig.6, the immobilized CA2(L203K)-N-LCTPSR protein were stable and retained their activity at high temperature.
Improved Part
In order to further improve the industrial application of CA2 for CO2 capture, basing on the existing part 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.
1. Engineered E.coli TB1
The coding sequence of CA2(L203K)-C-LCTPSR was synthesized, and then cloned into pET-30a(+) expression vector(Fig.7).
The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.8) and sequencing(Fig.9).
Lane M: DL15000 marker; Lane 1: CA2(L203K)-C-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-C-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).
The expression of CA2(L203K)-C-LCTPSR in E.coli TB1 were detected by SDS-PAGE. The results showed that CA2(L203K)-C-LCTPSR could be successfully expressed in our chassis E.coli TB1.(Fig.10)
Lane 1: CA2(L203K)-C-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-C-LCTPSR protein expression with IPTG induction.
We successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-C-LCTPSR expression vector into E.coli TB1 for the following CA2(L203K)-C-LCTPSR immobilization. Then the improve part of CA2(L203K)-C-LCTPSR protein was further purified through nickel column and detected by SDS-PAGE, as shown in Fig.11.
2. Identification of the function for CO2 capture
FGE can selectively identify and oxidize cysteine residues in the sulfatase subunit (LCTPSR) at the end of the protein to form aldehyde-containing formylglycine, 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：
$$\eta=\frac{W 1-W 2}{W 1}\times100\%$$
W1: The concentration of total CA2(L203K)-C-LCTPSR protein;
W2:The concentration of free CA2(L203K)-C-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, the enzyme activity of CA2(L203K)-C-LCTPSR and CA2(L203K) protein of CO2 capture were tested experimentally by esterase activity assay at 37℃ and 50℃.
As shown in Fig.12 and Fig.13, immobilized CA2(L203K)-C-LCTPSR protein was stable at high temperature and retained its activity, and free CA2(L203K)-C-LCTPSR protein has a higher activity than CA2(L203K) protein.
3. Application model for detecting CO2 capture
Because the immobilized CA2(L203K)-C-LCTPSR protein have higher activity than immobilized CA2(L203K)-N-LCTPSR protein, so the reuse ability of the immobilized CA2(L203K)-C-LCTPSR was tested by our designed simulation model (Fig.14). Compared with the original enzyme, the immobilized enzyme still retained 54 percent activity after five times of repeated absorption experiments of CO2, as indicated in Fig.15. The result showed that the immobilized CA2(L203K)-C-LCTPSR could absorb CO2 under the simulation model and showed potential reuse ability.
In conclusion, our results demonstrated that the function of CA2(L203K)-C-LCTPSR 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.
Characterization
Characterization of an existing BioBrick Part BBa_K2762004 (CcaA)
For characterization, we have demonstrated the output of this part BBa_K2762004 in E.coli BL21(DE3) and tested its activity by esterase method which was different form the original method.
The sequence of BBa_K2762004 was synthesized and cloned it into the expression plasmid pET-30a(+) to obtain the recombinant expression vector. Then, the plasmid containing CcaA was introduced into E.coli BL21(DE3) for culturing in the medium containing kanamycin, and IPTG was added to induce CcaA expression for 4h. The CcaA protein was extracted from the bacterial lysates followed by identification via SDS-PAGE gel electrophoresis.(Fig.16)
After protein purification, the activity of the enzyme was determined by esterase method different from the original method. As carbonic anhydrase can catalyze the hydrolysis of p-nitrophenyl acetate. Therefore, we intended to use the esterase activity of carbonic anhydrase to catalyze the p-nitrophenyl acetate, and obtain the enzyme activity data by the change of absorbance before and after a certain reaction time. The result showed that CcaA also showed high enzyme activity. (Fig. 17)
References
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