Understanding the Activity of Glucose Oxidase after Exposure to Organic Solvents

Long-term stability of enzymes in organic solvents is one of the most challenging problems in modern biotechnology and chemical industries. However, the resistance of enzymes to organic solvents is not very well understood so far. Herein, the effects of apolar, chlorinated, and polar organic solvents on the activity and structure of glucose oxidase from Aspergillus niger were systemically investigated using spectrophotometric activity assay of this enzyme and absorption and chiroptical spectroscopy. Molecular dynamics simulations and correlation of the activity with properties of the organic solvents were employed to understand the effects of organic solvents on the enzyme. The experimental and theoretical results showed that apolar solvents reduce the enzyme activity because they facilitate its aggregation through inter-enzymatic salt bridges. Moreover, polar solvents strongly coordinate with amino acid residues in the glucose binding pocket and prevent binding of the substrates. We found that this enzyme is stable in pure apolar and chlorinated solvents and these solvents can be used for the functionalization of its residues. This work provides an in depth understanding at the molecular level of the impact of various pure organic solvents on the structure and dynamics of glucose oxidase and the regulation of its catalytic activity.


S3
Materials. The organic solvents were purchased from Eurochemicals and distilled prior the experiments. The used lyophilized powder enzymes of type VII glucose oxidase (GOx) from Aspergillus niger of ≥100 U mg -1 (without added oxygen) and type I peroxidase from horseradish (HRP) of approximately 150 U mg -1 were purchased from Sigma Aldrich. All inorganic salts for the buffer solutions were purchased from Carl Roth GmbH + Co. KG.
Instrumentation. Ultraviolet-visible (UV-Vis) spectra were measured to evaluate the kinetic constants using an Evolution 300 Security UV-Vis Spectrophotometer (Thermo Fisher Scientific). During the measurements, the samples were thermostated at 25 o C using an integrated thermostat. A JASCO J-815 Circular Dichroism spectrometer (Tokyo, Japan) was used to measure the circular dichroism (CD) spectra of GOx prior and after exposure to organic solvents. These CD spectra were carried out under a stream of Ar gas (5 L min -1 ) and in a wavelength range of 190-500 nm with a scan rate of 50 nm min -1 , the concentration of GOx solutions were 1.0 mg mL -1 . The CD spectra were recorded two times and the results were averaged and the baseline was subtracted. The path length of cell was 0.10 cm.
Hydrodynamic diameter of nanoparticles was measured using Zetasizer µV (Malvern) and its compatible software. DMF, DMSO, THF, AN, CHF and buffer solutions of GOx (concentration of 1 mg mL -1 ) were observed. Prior to the experiment, the solutions were filtered using membrane syringes (CA 0.45 μm) to get reliable particle size data and remove large dust particles. Because GOx nanoparticles were measured in pure DMF and DMSO, the solvent parameters in the software were changed from water to DMF and DMSO, respectively. This required to define the solvents by their temperature, viscosity and refractive index. The measurements were performed 3 times and averaged by the program.

S4
Sample preparation. The native GOx enzyme was dissolved in potassium phosphate buffer (NaH 2 PO 4 -Na 2 HPO 4 ) of pH 7.0 (50 mM) and used as a standard of activity comparison. The where V is an initial velocity (μM s -1 ); is a mathematical differential of UV-Vis Correlation between the solvent properties and the GOx catalytic efficiencies. To estimate this correlation between the catalytic efficiencies and the solvents physical properties, the least squares of the experimental and calculated catalytic efficiencies method by Eq. 1 was used.
Solver as the Microsoft Excel add-in program was employed.  Notably, in the beginning of the run GROMACS, the overwhelming majority of designated histidines are uncharged. However, uncharged histidines often formed hydrogen bonds with negatively charged Asp and Glu during the simulation. It was still counted as salt bridges for counting purposes. In the real system, such neutral histidine in a pair with Glu or Asp will likely be protonated, strengthening the binding between the two residues. However, MD is not able to model change of protonation states of the species in the middle of calculation. Still, the simulations seem to reasonably approximate salt bridge formation even with this approximation.
The quantum chemical calculations were carried out with the Spartan'18 program package (Spartan'18 for windows version 1.4.0 Wavefunction, Irvine, CA, USA). These structures of the complexes were optimized using a MP2 method of the Møller-Plesset's perturbation theory and a 6-311G(d,p) basis set. To calculate the energy of the hydrogen-bonding interaction between the H-bonded fragments, the standard energy difference method was applied. The basis set superimposition error (BSSE) in calculating the energy difference was corrected by applying the S12 counterpoise procedure to compute the H-bond energies, in which the virtual orbitals of the other fragment were included in the basis functions. 9 MP2/6-311++G(d,p) as the best method employed in the literature was used for the single points calculations. 10