Thermo-alkali-stable α-carbonic anhydrase of Bacillus halodurans : heterologous expression in Pichia pastoris and applicability in carbon sequestration

Abstract

Recombinant α-carbonic anhydrase of the polyextremophilic bacterium Bacillus halodurans TSLV1 (rBhCA) has been produced extracellularly in active form in Pichia pastoris under methanol inducible (AOX1) as well as constitutive (GAP) promoters. A marked improvement in rBhCA production was achieved by developing a P. pastoris recombinant that produces rBhCA constitutively as compared to that under inducible promoter. The purified rBhCA from P. pastoris is a glycosylated protein that displays a higher molecular mass (79.5 kDa) than that produced from E. coli recombinant (75 kDa); the former has a Tm of 75 °C, which is slightly higher than that of the latter (72 °C). The former rBhCA exhibits higher thermostability than the latter. The former sequestered CO₂ efficiently similar to that of the native BhCA and the latter. This is the first report on the production of recombinant carbonic anhydrase extracellularly in P. pastoris.