RESUMO
Penicillin G acylase from E. coli TA1 was immobilized by Cross-Linked Enzyme Aggregates [CLEA], a new method for immobilization. This biocatalyst and commercial immobilized penicillin G acylase [PGA-450] were used to study the effect of pH, temperature and substrate concentration on the synthesis of ampicillin from phenyl glycine methyl ester [PGME] and 6-aminopenicillanic acid [6-APA]. Compared with PGA-450, this immobilized enzyme showed a high synthesis activity. The optimum conditions for synthetic activity was at pH 6, 25°C and 2:6 [6-APA:PGME] substrate ratio
Assuntos
Penicilina Amidase/biossíntese , Imobilização , Escherichia coli , Cromatografia Líquida de Alta Pressão , TemperaturaRESUMO
Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics. The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized. The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature. A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media. The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined
Assuntos
Bacillus megaterium/enzimologia , Penicilina Amidase/biossíntese , Bacillus megaterium/crescimento & desenvolvimento , Bacillus megaterium/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Penicilina Amidase/análise , Temperatura , Fatores de TempoRESUMO
Fermentation parameters for the production of penicillin G acylase by Escherichia coli NCIM 2400 have been evaluated. The bacterium produced the enzyme intracellularly when grown in nutrient broth containing PAA. PAA stimulated the enzyme synthesis by 8-10 fold and reduced the lag period. The optimum concentration of PAA for induction was 20 mM and addition of PAA prior to inoculation gave maximum production of PGA. Glucose, lactose, sorbitol, acetate and lactate even at 0.1% concentration catabolically repressed the enzyme formation. Peptone was the best utilised 'N' source for the enzyme production. Phosphate and yeast extract were found to be essential for both the growth and for enzyme biosynthesis. Temperature between 22-24 degrees C was optimum and under ideal condition E. coli NCIM 2400 produced 0.45-0.55 U/ml of penicillin G acylase.