Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

Abstract

The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cell-bound glutamate decarboxylase (GAD) activity of recombinant Escherichia coli (BL21(DE3)-pET28a-gadB) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5μg·ml−1. Using this concentration, the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21 (DE3)-pET28a-gadB cells with 5μg·ml−1 ampicillin resulted in structural changes to the cell envelope, but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol (PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid (GABA) at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30days of storage at 4°C.