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Updated: Oct 10, 2026
Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
Enzymatic Characterization and Application Study of Fructosyl Peptide Oxidase with Improved Thermal Stability and
Kehong Lou1,2, Feibo Dong1, Guosheng Gao1
1Ningbo No. 2 Hospital, Department of Clinical Laboratory, No. 41, Northwest Street, Haishu District, Ningbo 315010, Zhejiang Province, China.
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.
