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

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 k _cat /K _m 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.