Cloning, Expression and Characterization of Two Beta Carbonic Anhydrases from a Newly Isolated CO 2 Fixer, Serratia marcescens Wy064

Abstract

Bacterial strains from karst landform soil were enriched via chemostat culture in the presence of sodium bicarbonate. Two chemolithotrophic strains were isolated and identified as Serratia marcescens Wy064 and Bacillus sp. Wy065. Both strains could grow using sodium bicarbonate as the sole carbon source. Furthermore, the supplement of the medium with three electron donors (Na₂S, NaNO₂, and Na₂S₂O₃) improved the growth of both strains. The activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) could be detected in the crude enzyme of strain Wy064, implying that the strain Wy064 might employ Calvin cycle to fix CO₂. S. marcescens genome mining revealed four potential CA genes designated CA1–CA4. The proteins encoded by genes CA1–3 were cloned and expressed in Escherichia coli. The purified recombinant enzymes of CA1 and CA3 exhibited CO₂ hydration activities, whereas enzyme CA2 was expressed in inclusion bodies. A CO₂ hydration assay demonstrated that the specific activity of CA3 was significantly higher than that of CA1. The maximum CO₂ hydration activities for CA1 and CA3 were observed at pH 7.5 and 40 °C. The activities of CA1 and CA3 were significantly enhanced by several metal ions, especially Zn²⁺, which resulted in 21.1-fold and 26.1-fold increases of CO₂ hydration activities, respectively. The apparent K_m and V_max for CO₂ as substrate were 27 mM and 179 WAU/mg for CA1, and 14 mM and 247 WAU/mg for CA3, respectively. Structure modeling combined with sequence analysis indicated that CA1 and CA3 should belong to the Type II β-CA.

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