Biocalorimetric and respirometric studies on production of Penicillin G acylase from Bacillus badius pac in E. coli DH5α
An isothermal heat flux biological reaction calorimeter is employed to monitor the production of Penicillin G acylase (PAC) from E. coli DH5α harboring recombinant construct pPROPAC (pET-30b carrying pac gene from Bacillus badius with its own promoter) cultivated in fermentation medium earlier with different flow rates of oxygen. Three oxygen flow rates were studied. At 0.66 vvm (volume of gas per volume of liquid per minute [l l−1 min−1]) a maximum enzyme yield of 43 U/ml was achieved with fructose as carbon source. Metabolic heat flow rate profile and heat yields at different levels helped to conclude that the recombinant construct was an obligate aerobic. Oxygen uptake rate correlated well with enzyme activity, biomass growth and heat production rate at exponential and stationary growth phases in optimized conditions. It was observed that metabolic heat, enzyme activity, substrate utilization rate and oxygen uptake rate matched well. The heat yields due to biomass growth, 15.26 kJ/g, substrate consumption, 16.52 kJ/g and oxygen uptake, 413.2 kJ/mol led to understanding the process behavior and physiological activity of the organism under study. The results can contribute to scale up studies, possibly.
► The growth of E. coli DH5α containing Bacillus badius pac gene can be calorimetrically monitored. ► Reliable information on growth dynamics and production of Penicillin G acylase obtained. ► A maximum PAC activity of 43 U/ml was achieved for the first time at 0.66 vvm oxygen supply with fructose as carbon source. ► Change in metabolic heat flow rate profiles and heat yields indicated that the recombinant construct is an obligate aerobic. ► The heat yields due to biomass, 15.26 kJ/g, substrate, 16.52 kJ/g and OUR, 413.2 kJ/mol reflects physiological activity of the organism.
Journal: Biochemical Engineering Journal - Volume 55, Issue 3, 15 August 2011, Pages 223–229