Journal ArticleDOI
Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105
TLDR
Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene showed that the performance of E.'s coli G271 depends both upon the host strain properties and the plasmid structure.Abstract:
Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by itsmore » transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.« lessread more
Citations
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Journal ArticleDOI
Current state and perspectives of penicillin G acylase-based biocatalyses
Helena Marešová,Martina Plačková,Martina Plačková,Michal Grulich,Michal Grulich,Pavel Kyslík +5 more
TL;DR: The application potential of the enzyme goes beyond the β-lactam biocatalysis due to its enantioselectivity and promiscuity: the PGA can be used for the production of achiral and chiral compounds convenient for the preparation of synthons and active pharmaceutical ingrediences, respectively.
Journal ArticleDOI
Recent biotechnological interventions for developing improved penicillin G acylases
TL;DR: An overview of recent advances in the production, stabilization and application of PAC is provided, highlighting the recent biotechnological approaches for the improved catalysis of PAC.
Journal ArticleDOI
Genetic manipulation to identify limiting steps and develop strategies for high-level expression of Penicillin acylase in Escherichia coli
TL;DR: This is the first report demonstrating that expression of PAC could be limited by various steps, such as transcription, translation, and post-translational steps (i.e. translocation and periplasmic processing), depending on the host/vector systems.
Journal ArticleDOI
Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3(pKA18)
TL;DR: The high-expression system developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chromosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19 led to an increase in the specific activity of PGA.
Journal ArticleDOI
Molecular biology of β-lactam acylases.
TL;DR: Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.
References
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Journal ArticleDOI
Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar
Journal ArticleDOI
Preparation and General Properties of Crystalline Penicillin Acylase from Escherichia coli ATCC 11 105
Journal ArticleDOI
Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli
S. Birnbaum,James E. Bailey +1 more
TL;DR: The results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.
Journal ArticleDOI
Factors influencing productivity of fermentations employing recombinant microorganisms
D.W. Zabriskie,E.J. Arcuri +1 more
TL;DR: Representative efforts in these areas indicate the broad scope of considerations involved in developing fermentation processes for recombinant microorganisms and the opportunities for further research in this field.
Book ChapterDOI
Microbial Penicillin Acylases
Erick Vandamme,J.P. Voets +1 more
TL;DR: The chapter examines methods to isolate and purify penicillin acylases and to stabilize these enzyme preparations, and describes the reaction of cysteine and related compounds with penicillins, resulting in a loss of antibacterial activity.
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