Influence of controlled-pH and uncontrolled-pH operations on recombinant benzaldehyde lyase production by Escherichia coli

Abstract

To select the host microorganism having the highest benzaldehyde lyase (BAL) production capacity, pUC18∷ bal gene was transferred into four Escherichia coli strains. As the highest enzyme activity was obtained with E. coli K12 (ATCC 10798) carrying pUC18∷ bal gene, BAL production medium was designed for K12. Using the designed medium containing 8.0 kg m −3 glucose, 5.0 kg m −3 (NH 4) 2HPO 4 and the salt solution, the effects of uncontrolled-pH and controlled-pH operations were investigated at uncontrolled-pH pH UC 7.2 and controlled-pH values pH C 5.0, 6.4, 6.7, 7.0, 7.2 and 7.8 in 3.0 dm 3 bioreactor systems with a V R = 1.65 dm 3 working volume at the air inlet rate of Q o/ V R = 0.5 vvm and agitation rate of N = 500 min −1. The uncontrolled-pH (pH UC 7.2) operation produced the highest cell concentration and BAL activity as C X = 2.3 kg m −3 and A = 860 U cm −3, respectively. Among the controlled-pH operations, the highest cell concentration and enzyme activity were obtained at pH C 7.0 operation, respectively, as C X = 2.1 kg m −3 and A = 775 U cm −3. The accumulation of the metabolic by-product acetic acid and the total organic acid concentrations were the highest at pH C 7.2 and 7.8 operations. On the other hand, there was no significant difference in the acetic acid concentration profiles of pH C 5.0, 6.4, 6.7 and 7.0 operations; however, the highest total organic acid concentration was attained at pH C 5.0 because of the lactic acid excretion, and the lowest total organic acid was obtained at pH C 6.7. K L a values varied between 0.01 and 0.03 s −1. To compare the mass transfer and biochemical reaction rates, the maximum possible oxygen utilization rate, possible mass transfer rate, Damköhler number, and effectiveness factor, were also calculated. Damköhler number increased with the cultivation time indicating that mass transfer resistances were becoming more effective than biochemical reaction resistances. The yield and maintenance coefficients as well as the kinetic constants for BAL production process were also reported.