Immobilization of Escherichia coli JM103[pUC8] in κ‐Carrageenan Coupled with Recombinant Protein Release by in Situ Cell Membrane Permeabilization

Abstract

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.

Strategies for Enhancing Product Yield: Design of Experiments (DOE) for Escherichia coli Cultivation

“…The recombinant plasmid is relatively unstable when the cloned gene products are inhibitory to the host cells. Phenotypic instability of plasmid is due to the disappearance of the entire plasmid or the deletion of a specific region [108]. Both plasmid copy number and plasmid loss rate are features affected by factors such as media composition growth rate and culture strategy [109] and other factors such as temperature, agitation rate, and pH [110].…”

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides

“…This permeabilisation method can be applied because TDFE is thermostable, thus allowing selective denaturation of mesophilic proteins. A further advantage of this method is that it avoids the use of organic solvents, which are frequently exploited for breaking down the selective barrier of the cell membrane [4,14,17]. The high recovery of TDFE activity permitted the immobilisation of whole cells instead of purified free enzyme, thereby avoiding cumbersome and expensive downstream procedures (e.g.…”