Enzymatic Characterization and Application Study of Fructosyl Peptide Oxidase with Improved Thermal Stability and Activity

Abstract

Abstract Fructosyl peptide oxidase (FPOX) is clinically valuable for glycated hemoglobin (HbA1c) quantification, but current variants suffer from low activity and thermal stability, limiting their applications. A novel consensus sequence (FPOX-Con) was designed by analyzing the structural and sequence data of existing FPOXs. The FPOX-Con mutant was expressed in Escherichia coli BL21 (DE3) and purified via Ni2+-NTA chromatography, yielding high-purity protein. Enzymatic assays revealed an activity of 31.2 U/mg, significantly surpassing the wild-type (15.3 U/mg). FPOX-Con exhibited an optimal pH of 7.5 with stability across pH 5–9, and optimal activity at 45 °C, maintaining over 90% activity after heating at 50 °C for 10 minutes. Spectral analysis confirmed distinct flavin cofactor characteristics. Clinical testing with FPOX-Con showed excellent agreement with conventional HPLC methods (R2 = 0.9879), with relative deviations mostly under 5%. This high-activity FPOX mutant offers promising potential for rapid and precise HbA1c clinical detection.