Continuous computer controlled production of formate dehydrogenase (FDH) and isolation on a pilot scale

Abstract

AbstractA continuous production process has been developed up to pilot scale (300 l) for FDH production with the methylotrophic yeast Candida boidinii. A high cell mass specific FDH activity (50 U/g) is achieved by process computer controlled supply of pure methanol to operate the reactor at an optimum methanol concentration of 10 g/l. The maximum FDH spacetime yield achievable with this process control involves a residence time of 7 h. The FDH space‐time yield (STY) and FDH concentration are a function of the oxygen transfer rate (OTR) of the fermenter (maximum STY = 255 U/(l h) at kLa = 870 l/h). For a reasonable compromise between high FDH space‐time yield and high FDH concentration, an optimum residence time is adjustable by regulating the supply of nutrient salt solution in relation to the OTR of the fermenter. On a pilot scale (200 l continuously stirred tank reactor) roughly 4 million U of FDH were produced within 10 days at a residence time of 14.3 h. Isolation of intracellular FDH enzyme was performed using extraction with an aqueous two‐phase system (PEG/K2HPO4). A technical product quality of 1.2 U/mg FDH was achieved without any chromatographic purification step.