Enzymatic proteolysis of peptide bonds by a metallo-endoproteinase under precise temperature control with 5.8-GHz microwave radiation

• The 5.8-GHz microwave device which can perform precise thermal management was proposed.
• Microwave non-thermal effects were examined in Asp-N enzymatic reaction.
• The promoted enzymatic reaction was shown by a microwave magnetic field.

The present article examined the advantages of 5.8-GHz microwaves versus the more commonly used 2.45-GHz microwaves using a modular non-commercial apparatus on the proteolysis of the Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly peptide at the amino side of the aspartic acid (Asp) using the Asp-N metallo-endoproteinase enzyme. The microscale sample was subjected to microwaves’ electric field (E-field) and magnetic field (H-field) radiation; good temperature control for samples at the μL scale was achieved using an apparatus that emitted microwaves at a precise frequency (5.800000 GHz). Temperature-dependent experiments with the Asp-N metallo-enzyme confirmed the activity of this enzyme to be greatest at 37 °C reached within 15 s on irradiation with the 5.8-GHz microwaves’ H-field component with an input power of 1.7 W under air cooling conditions. Enzymatic activity decreased significantly above and below this temperature by a slight temperature change of 1 °C. Proteolysis yields of the peptide by the Asp-N enzyme at 30–42 °C under microwave E-field and H-field heating and under conventional heating revealed that enhancement of the proteolysis of the peptide at 37 °C by E-field irradiation (42.6%) was 1.5 times greater than by conventional heating (27.5%), whereas under microwave H-field irradiation (63.5%) it was 2.3 times greater than conventional heating. The relative dielectric loss factors (ɛr″) of the sample solution (peptide + enzyme) were also determined: 22.19 (5.8 GHz) and 12.78 (2.45 GHz) at 22 °C, which decreased with increasing temperature (faster for the 5.8-GHz microwaves) so that the initial heating efficiency of 5.8-GHz microwaves was nearly twofold greater than for 2.45-GHz microwaves. Results indicated that in addition to a thermal factor, microwave non-thermal factors also had a significant influence as the microwaves considerably enhanced the proteolytic process relative to traditional heating.

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