High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

Abstract

BackgroundPenicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits.ResultsThis work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively.ConclusionsTo the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

T4 lysozyme fused with cellulose-binding module for antimicrobial cellulosic wound dressing materials

“…), and complex medium (containing per litre: 10 g glucose, 40 g glycerol, 10 g tryptone, 5 g yeast extract, 0·5 g MgSO 4 ·7H 2 O, 3·4 g KH 2 PO 4 , 9 g Na 2 HPO 4 ·12 H 2 O, 2·7 g NH 4 Cl and 0·7 g Na 2 SO 4 ) (Vèlez et al . ). The respective frozen glycerol stocks of E. coli T4Lyz‐CBM and E. coli pLysS‐T4Lyz‐CBM (100 μ l each) were used to inoculate 10 ml of LB medium supplemented with appropriate antibiotics, and incubated overnight at 37°C and 200 rev min −1 to prepare the inocula.…”

Metabolic fluxes-oriented control of bioreactors: a novel approach to tune micro-aeration and substrate feeding in fermentations

“…In all the experiments, data acquisition and monitoring/control of the instruments were achieved using SuperSys_Ferm, an in-house computational tool for flux-based control of micro-aerated fermentation processes. SuperSys_Ferm was implemented in LabView, via a Compact FieldPoint (Model cFP-2020, National Instruments), and was based on SuperSys_HCDC, which was developed to assist fed-batch bioreactor operation during high cell density cultivations [52, 53].…”

On-Line Monitoring of Biomass Concentration Based on a Capacitance Sensor: Assessing the Methodology for Different Bacteria and Yeast High Cell Density Fed-Batch Cultures

“…Modified complex auto-induction media formulations (Studier, 2005, Santos, 2012, containing yeast extract and Tryptone or Phytone as nitrogen sources and glycerol, glucose and lactose as carbon sources, were used in cultivations of E. coli "a" and "e"; a modified defined HDF medium (Seeger et al, 1995;Sargo, 2011) was used in cultivations of E. coli "b" and "c" and both media were used for cultivations of E. coli "d" (Vélez et al, 2014). Commercial S. cerevisiae was cultivated in complex medium containing glucose (10 g/L) as main carbon source and KH 2 PO 4 (5.0 g/L), (NH 4 ) 2 SO 4 (4.5 g/L), MgSO 4 (0.5 g/L) and yeast extract (30 g/L).…”