Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers

“…First the so called "negative" separation of the enzyme by depletion of all host cell proteins through binding them unspecifically to an ion exchange material, thus collecting the PGA in the flow-through [25] and second the specific binding of PGA itself to an ion exchange material, keeping all host cell proteins (HCP) in the flow-through and subsequent elution of the bound enzyme [9]. Both strategies were tested in the described microliter scale highthroughput screening (see chapter 2.2) with cell lysate to identify best binding material and evaluate basic process conditions.…”

Secretion of recombinant proteins from E. coli

“…Most early publications on the subject usually present qualitative findings on whether periplasmic proteins were found outside the cell or not [183], more recent publications present more detailed information about the extracellular protein concentrations achieved and/or secretion efficiency (see Table 2). Secretion efficiencies of ß95-96% were stated for penicillin G acylase [186] and for streptavidin [187]. Strains with lpp mutation or deletion have recently been described and are gainined attention for protein production.…”

“…Strains with lpp mutation or deletion have recently been described and are gainined attention for protein production. Secretion efficiencies of ß95-96% were stated for penicillin G acylase [186] and for streptavidin [187]. Moreover, an extracellular protein yield of up to 3.5 g/L was achieved, but as indicated in EP 1903105 B1, the secretion efficiency depends on the protein of interest [188].…”

Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC)

“…1. The proposed purification scheme investigated herein using tangential flow filtration anion-exchange membrane chromatography (TFF-AEMC) to simultaneously clarify and purify extracellular PAC (study 2) compared to an equivalent purification scheme using a traditional AEC column or DEF-AEMC (study 1) [17]. These developed bioprocesses are contrasted with a conventional industrial process for the large-scale production of PAC.…”

“…To eliminate these preparatory steps and to ensure the functional operation of anion-exchange membrane chromatography, the spent medium should have a sufficiently low conductivity and a permissible pH to allow its direct processing. Since a buffer with a low salt concentration is normally inadequate for E. coli cultivation, medium composition and cultivation conditions were previously developed to drive effective extracellular production of recombinant PAC with minimum growth impairment as well as to facilitate protein binding onto the anion-exchange membrane during the subsequent chromatographic operation [17]. Using a bench-top bioreactor, JE5505 harboring pTrcKnPAC2902 was cultivated in LCM3 medium for extracellular production of PAC.…”