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 (79.5kDa) than that produced from E. coli recombinant (75kDa); 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.
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