Putrescine Oxidase (PutOx) is a flavoprotein enzyme that catalyzes the oxidation of polyamines, such as putrescine, which serves as a biomarker for various diseases including cancer, Alzheimer’s, and gestational diabetes. To enhance its diagnostic potential, we have engineered PutOx by tagging the enzyme with a gold-binding peptide (AuBP2) and developed a fusion enzyme (PutOx-AuBP) to self-assemble on gold surfaces. In this study, we explore the self-assembly ability of the fusion enzyme and investigated the enzyme stability at varying temperature and pH values. The activity of PutOx-AuBP was investigated using an HRP-coupled assay with ABTS as a colorimetric reagent and compared its activity to the control wild-type PutOx (WT-PutOx). Thermal stability was assessed by quantifying enzyme activity from 25 to 90°C, compared to the optimal activity of the enzyme at 37°C. PutOx-AuBP maintained comparable activity to WT-PutOx at temperatures up to 37°C and demonstrated increased activity at temperatures 40-70°C. Enzyme activity was also assessed over pH values 4-9, where it was observed that PutOx-AuBP maintained its pH stability compared to WT-PutOx. The engineered enzyme demonstrated increased activity compared to WT-PutOx at pHs 4-6.5, and the highest retained activity observed was at pH 7.4 (112.16%). Our results suggest that PutOx-AuBP is more adaptable to temperature and pH variations, making it a promising candidate as diagnostic enzyme. Future work will explore the use of this fusion protein in gold nanoparticle (AuNP) systems. Our biomimetic approach facilitates novel approaches for enzyme immobilization and enables their use in developing advanced biosensors for early, non-invasive disease detection. The funding was supported by NSF-CHEM 2108448 and UG Research Fellow Program at the School of Engineering, KU.
