ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

Kao Pang Hsu, Shih I. Tan, Chen Yaw Chiu, Yu Kaung Chang, I-Son Ng

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 languageEnglish
Article numbere2834
JournalBiotechnology Progress
Volume35
Issue number5
DOIs
Publication statusPublished - 2019 Sep 1

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Carbonic Anhydrases
Escherichia coli
Industry
Carbon Sequestration
Ions
Phenolsulfonphthalein
Enzymes
Bicarbonates
Carbon Dioxide
Heating
Zinc
Catalytic Domain
Stem Cells
Metals
Water
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

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title = "ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli",
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.",
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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 journalArticle

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