Abstract
Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high-throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo-stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino-pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3–5. The kinetic analysis by ART system showed that the kcat/Km of free enzyme was 4.4-folds that that of whole cell. On the other hand, the screening platforms as Wilbur–Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high-throughput screening platform is support in direct evolution of CA and further used in the industry.
| Original language | English |
|---|---|
| Article number | e2834 |
| Journal | Biotechnology Progress |
| Volume | 35 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2019 Sep 1 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
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ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli. / Hsu, Kao Pang; Tan, Shih I.; Chiu, Chen Yaw; Chang, Yu Kaung; Ng, I-Son.
In: Biotechnology Progress, Vol. 35, No. 5, e2834, 01.09.2019.Research output: Contribution to journal › Article
TY - JOUR
T1 - ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli
AU - Hsu, Kao Pang
AU - Tan, Shih I.
AU - Chiu, Chen Yaw
AU - Chang, Yu Kaung
AU - Ng, I-Son
PY - 2019/9/1
Y1 - 2019/9/1
N2 - Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high-throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo-stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino-pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3–5. The kinetic analysis by ART system showed that the kcat/Km of free enzyme was 4.4-folds that that of whole cell. On the other hand, the screening platforms as Wilbur–Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high-throughput screening platform is support in direct evolution of CA and further used in the industry.
AB - Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high-throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo-stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino-pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3–5. The kinetic analysis by ART system showed that the kcat/Km of free enzyme was 4.4-folds that that of whole cell. On the other hand, the screening platforms as Wilbur–Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high-throughput screening platform is support in direct evolution of CA and further used in the industry.
UR - http://www.scopus.com/inward/record.url?scp=85066470407&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85066470407&partnerID=8YFLogxK
U2 - 10.1002/btpr.2834
DO - 10.1002/btpr.2834
M3 - Article
C2 - 31074194
AN - SCOPUS:85066470407
VL - 35
JO - Biotechnology Progress
JF - Biotechnology Progress
SN - 8756-7938
IS - 5
M1 - e2834
ER -