High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations.
BACKGROUND
Penicillin 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.


RESULTS
This 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.


CONCLUSIONS
To 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.
Ana Maria
AM
Vélez
Adilson José
AJ
da Silva
Antonio Carlos
AC
Luperni Horta
Cintia Regina
CR
Sargo
Gilson
G
Campani
Gabriel
G
Gonçalves Silva
Raquel
R
de Lima Camargo Giordano
Chemical Engineering Department, Federal University of São Carlos (UFSCar), Rodovia Washington Luís, km 235, C,P, 676, CEP 13565-905 São Carlos, SP, Brazil. teresacz@ufscar.br.
Teresa Cristina
TC
Zangirolami
D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D010399:Penicillin Amidase / Q000096:biosynthesis*; D013696:Temperature
D003470:Culture Media; D010399:Penicillin Amidase