FIG. 3.

Heterologous overexpression of the can gene from R. eutropha H16 in E. coli (A) and mass spectrometric assays of CA activities in a crude cell extract of transformant E. coli JW1(pCAN8210) (B and C). (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14% [wt/vol] acrylamide) of cell lysates of transformants harboring pUC19 (lane a) or pCAN8210 (lane b). The arrow indicates the Can protein overproduced in E. coli JW1(pCAN8210). Molecular masses of reference proteins are indicated. (B) Representation of a CA activity assay in a cell extract of E. coli JW1(pCAN8210). The initial slope represents the uncatalyzed exchange reaction between ¹³C¹⁸O₂ and unlabeled CO₂ after addition of NaH¹³C¹⁸O₃. Arrows mark the time points at which cell extract or EZA (0.5 mM) was added to the assay mixture. (C) Dependence of the CA activity on the presence of MgSO₄.