P23: Production of carbonic anhydrase in Pichia pastoris for utilizing the enzyme as a medium component in succinic acid fermentation process

Succinic acid, a four-carbon dicarboxylic acid, is produced as a major fermentation product during anaerobic metabolism by bacterial Actinobacillus succinogenes. Succinic acid is used in a wide range of applications such as surfactants, detergents, green solvents, food and pharmaceutical industries. In the previous paper, we reported that succinic acid productivity was significantly enhanced when CO₂ gas was optimally supplied into the 5L stirred-tank bioreactor system, and also when the commercial enzyme of carbonic anhydrase (of human origin) was supplemented as a medium component into the production medium (about 40% higher productivity than the parallel fermentations with no addition of the enzyme). These impressive results had been assumed to be due to the enhanced CO₂ gas mass transfer rate(k_La), and also due to the increased reaction rate catalyzed by the supplemented enzyme (i.e. rapid conversion of aqueous CO₂ to HCO₃^-, which can be far more easily transported into the producing cells than the aqueous CO₂). In this study, because of too high price of the commercial carbonic anhydrase of human origin, it was undertaken to economically overproduce a cyanobacterial carbonic anhydrase by the use of a recombinant Pichia pastoris. For this purpose, we constructed a expression vector system with the carbonic anhydrase gene PCR-cloned from Cyanobacterium synechocystis sp., and introduced it into the host cells of P. pastoris for fermentation studies. Various fermentation results for the production of the cyanobacterial carbonic anhydrase will be presented in this paper as well.