Enzymatic synthesis of L-cysteine

O-acetylserine sulfhydrase in the form of a crude extract from Salmonella typhimurium LT2 was used for the production of L-cysteine from L-O-acetylserine and sodium hydrosulfide at pH 7.0 and 25 degrees C. The two substrates have quite different pH stability relationships. O-Acetylserine readily rearranges to N-acetylserine and the rate of this O --> N acyl transfer reaction increases at higher pH, temperature, and concentration of O-acetylserine. On the other hand, sodium hydrosulfide is more soluble at a higher pH. A stirred-tank bioreactor with a continuous substrate feed was employed to overcome this problem. The O-acetylserine feed was stored at its saturation level (2.05M) at pH 5.0, and the sodium hydrosulfide feed was dissolved at 2.05-2.3M without pH adjustment (pH >or= 11.5). Both substrates were simultaneously introduced into the bioreactor. The performance of the bioreactor was optimized by employing an automatic feedback control system to regulate the concentration of O-acetylserine in the bioreactor. This feedback control system was based on the fact that as the bioconversion proceeds, protons are produced along with cysteine. A pH controller thus detected the decrease in pH and activated the substrate pumps. After mixing in the bioreactor, these two substrate solutions behaved as a base due to the high alkalinity of sodium hydrosulfide. Thus, substrate infusion started when the pH was lower than the set point, i.e., the reaction pH, and stopped when the pH was raised higher than the set point. The amount of substrate introduced was determined by the alkalinity of the mixture of the two substrates, which in turn was controlled by the concentration of sodium hydrosulfide. After optimizing the sodium hydrosulfide concentration and the substrate feed rate, the bioconversion gave a productivity of 3.6 g L-cysteine/h/g dry cell weight S. typhimurium, an L-cysteine titer of 83 g/L and a molar yield based on O-acetylserine of 94%.