Exploring the Potential of Glutathione Reductase Overexpression to Improve Tellurium Nanoparticle Production in Escherichia coli

Abstract

Tellurium nanoparticles (TeNPs) are emerging as valuable materials in various technological and biomedical applications due to their unique physicochemical properties. In general, TeNPs are prepared using chemical methods based on a redox reaction in which strong reducing agents are employed which are often toxic and harmful to the environment. Biological biosynthesis provides a green strategy for substituting the commonly used reducing chemical agents with microorganisms or enzymes. Among the enzymes noted as key players in microbial tellurite reduction, glutathione reductase (GR) has been identified; however, its specific role in enhancing TeNP biosynthesis has yet to be fully elucidated. In this study, we aimed to evaluate the impact of GR overexpression on TeNP production in Escherichia coli (E. coli). For this purpose, four GR enzymes from different microorganisms identified as tellurite resistant were heterogeneously expressed and purified from E. coli. The kinetic parameters for NADPH and oxidized glutathione (GSSG), the native substrates of GR, were determined to evaluate their TR activity under saturated NADPH concentrations. The GR from Pseudomonas lini strain BNF22 presented the highest catalytic efficiency for NADPH and exhibited greater TR activity. This enzyme was overexpressed in E. coli MG1655 (DE3) and cells overexpressing GR increased the yield of TeNPs in those cells, presenting an increased elemental cell tellurium composition. Our results provide valuable insights for the development of engineered E. coli as a platform for TeNP biosynthesis. Using microorganisms as a green strategy for TeNP production, the results of this study highlight the enzymatic mechanisms underlying the role of GR in the biosynthesis of TeNPs.

Keywords: biosynthesis TeNPs; enzyme overexpression; glutathione reductase; tellurite reductase activity; tellurium nanoparticles (TeNPs).

Figure 1

Alignment of the amino acid sequence of BNF08 GR with other GR enzymes from various organisms. The yellow circles indicate the conserved motif for FAD binding, the blue squares mark the NADPH binding motif, and the green triangles highlight the catalytic redox-active cysteine residues.

Figure 2

Spectral and molecular weight evaluation for purified GR enzymes: E. coli GR, BNF22 GR, BNF08 GR, and MF01 GR. (A) SDS-PAGE gel. (B) CD spectra. (C) Results of the size exclusion chromatography. The molecular weight of GR enzymes was determined to be ≈110 kDa. (D) Absorption spectra under reduced conditions. (E) Fluorescence emission spectra.

Figure 3

Effect of (A) pH and (B) temperature on the TR activity of the purified enzymes.

Figure 4

Characterization of the in vivo reduction of tellurite in E. coli MG1655 (DE3) overexpressing BNF22 GR (right) or with an empty vector (left). (A) Growth (black), residual tellurite (green), and flow cytometry FCM cell count at 18 h (brown). (B) Energy-Dispersive X-Ray (EDX) spectra for cells harboring the empty vector (left) and BNF22 GR (right) showing cell tellurium content (red box). (C) Scanning Electron Microscopy microphotographs for cells harboring the empty vector and BNF22 GR and corresponding images of the EDX elemental mapping results showing the tellurium localization (green color). (D) Transmission electron microphotographs of ultrathin cell sections of E. coli overexpressing the empty vector or BNF22 GR. The red boxes in the TEM images provide higher magnification views highlighting the TeNPs aggregates formed inside the cells.

Figure 5

Characterization of TeNPs purified from E. coli cultures overexpressing GR from BNF22. (A) UV–visible spectrum of TeNPs. (B) Transmission electron microphotograph of purified TeNPs. Insert picture shows average size distribution for TeNPs from TEM microphotograph. (C) XPS spectra of TeNPs produced. Insert picture shows Te 3d core-level spectrum of TeNPs. (D) XRD pattern for biosynthesized TeNPs in orange color and reported XRD pattern of hexagonal tellurium phase (h-Te) in red color (RRUFF ID: R070376). (E) FTIR spectra of TeNPs generated from E. coli cultures.