Permeabilizing Escherichia coli for whole cell biocatalyst with enhanced biotransformation ability from l-glutamate to GABA

Abstract

Abstract γ-Aminobutyric acid (GABA) is widely used as a pharmaceutical, nutraceutical, and as a precursor for synthesizing materials for industrial use. Bacterial cells can be exploited for use as biocatalysts with the potential to synthesize compounds such as GABA with greater efficiency, safety, and economy. However, efforts to use biocatalysts must overcome the permeability barrier of the cell envelope. Therefore, to produce a whole-cell biocatalyst with enhanced cell-associated glutamate decarboxylase (GAD) activity, we overexpressed the Glutamate (Glu)-GABA antiporter (GadC) in Escherichia coli (BL21(DE3)-pET28a- gadB ). The cell-associated GAD activity of the transformants was higher by a factor of 2.6 in comparison; however, expression of GadC inhibited growth. Therefore, we permeabilized the cells using either organic solvents, surfactants, or heat. Permeabilization with organic solvents increased cell-associated GAD activity as a function of their hydrophobicity, and hexane was the most effective, increasing cell-associated GAD activity by a factor of 9.65 (6.72 U/mg). The surfactants Triton X-100, CHAPS, NP-40, OGP, and Brij-35 enhanced cell-associated GAD activity, and Triton X-100 was the most effective, increasing cell-associated GAD activity by a factor of 10.8 (7.53 U/mg). Heating BL21(DE3)-pET28a- gadB at 70 °C for 30 min increased cell-associated GAD activity by a factor of 13.1. GAD did not leak from the permeabilized cells under optimum conditions. When the heat-permeabilized cells were immobilized on Ca-alginate gel beads, the biotransformation ability of beads maintained over 60% of their initial ability after 20 consecutive batches, and the beads retained 90% of their initial activity after 30 days of storage. These approaches for improving cell permeability to enhance cell-associated GAD activity show great promise for decreasing the cost of industrial production of GABA.