SUMO-G5C23-D208G@ZIF-F: A Novel Immobilized Enzyme with Enhanced Stability and Reusability for Organophosphorus Hydrolysis

Abstract

Organophosphorus hydrolase (OPH) is a highly effective bioscavenger for detoxifying hazardous organophosphorus compounds. However, its practical application is hindered by low yield and poor stability. In this study, we employed Small Ubiquitin-like Modifier (SUMO) fusion expression to enhance the solubility of the OPH mutant G5C23-D208G and, for the first time, immobilized the enzyme on a zeolitic imidazolate framework-F (ZIF-F) carrier to improve its stability. The SUMO-G5C23-D208G fusion protein was successfully expressed in Escherichia coli, resulting in a yield that was 2.4 times higher than that of native OPH and an 11-fold increase in solubility. The purified protein achieved a purity of 95%. The immobilized enzyme, SU-MO-G5C23-D208G@ZIF-F, exhibited a farfalle-shaped structure with a diameter of approximately 3-5 μm. Compared to the free enzyme, the immobilized enzyme maintained high catalytic efficiency (kcat/Km = 8.9 × 10⁴ M^-1·s^-1) and demonstrated enhanced thermal stability, pH stability, and reusability. This study has significantly improved the yield and stability of OPH, thereby supporting its potential for industrial applications.

Keywords: SUMO fusion expression; ZIF-F; enzyme immobilization; enzyme kinetic parameters; organophosphorus hydrolase.

Figure 1

Expression and purification of SUMO-G5C23-D208G. (A) Schematic diagram illustrating the construction of the expression vector pET28a-SUMO-G5C23-D208G. (B) Double enzymatic digestion (BamH I/EcoR I) verification of the recombinant plasmid pET28a-SUMO-G5C23-D208G. M: super DNA marker; 1, 2: double digestion product. (C) Protein solubility analysis. The S lane represents soluble cellular lysate; the IB lane represents the inclusion body. (D) Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of purification by nickel column. M: protein ladder; 1: centrifugated supernatant; 2: sample effluent; 3: 50 mmol·L⁻¹ imidazole eluate; 4: 100 mmol·L⁻¹ imidazole eluate; 5: 300 mmol·L⁻¹ imidazole eluate of the 1st tube; 6: 300 mmol·L⁻¹ imidazole eluate of the 2nd tube; 7: 300 mmol·L⁻¹ imidazole eluate of the 3rd tube; 8: 300 mmol·L⁻¹ imidazole eluate of the 4th tube.

Figure 2

Characterization of immobilized enzymes. Scanning electron microscopic images of ZIF-F at (A) 5000× (2 μm bar scale) and (B) 10,000× (1 μm bar scale) magnifications. Scanning electron microscopic images of SUMO-G5C23-D208G@ZIF-F at (C) 5000× (2 μm bar scale) and (D) 10,000× (1 μm bar scale) magnifications. (E) The Fourier Transform Infrared Spectroscopy (FT-IR) spectra of SUMO-G5C23-D208G, ZIF-F and SUMO-G5C23-D208G@ZIF-F. (F) The X-ray Diffraction (XRD) patterns of the synthesized ZIF-F and SUMO-G5C23-D208G@ZIF-F.

Figure 3

Determination of enzyme activity and kinetic properties. (A) Enzyme activity test results. (B) Enzyme activity curves. (C) Lineweaver–Burk plot. (Mean ± SD, n = 3, * p < 0.05).

Figure 4

pH (A) and temperature (B) on the activities of free and immobilized SUMO-G5C23-D208G. (Mean ± SD, n = 3).

Figure 5

Stability performance analysis of SUMO-G5C23-D208G and SUMO-G5C23-D208G@ZIF-F with various organic solvents or different temperature environments: (A) methanol; (B) acetonitrile; (C) DMSO. Thermal stability results at (D) 40 °C and (E) 50 °C. (F) Storage stability of immobilized enzymes at 25 °C. (Mean ± SD, n = 3).

Figure 6

Reusability results of the immobilized enzyme. (Mean ± SD, n = 3).