Volume 16, Issue 62 (5-2026)
NCMBJ 2026, 16(62): 86-98 |
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Araghi F, Hosseinkhani S, Jafary H, Hosseini M. Kinetic study of the creatininase enzyme and evaluation of the peroxidase-mimicking activity of gold nanoclusters. NCMBJ 2026; 16 (62) :86-98
URL: http://ncmbjpiau.ir/article-1-1824-en.html
URL: http://ncmbjpiau.ir/article-1-1824-en.html
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Abstract: (9 Views)
Background and Aim: Peroxidases are vital enzymes in biochemical processes, but high purification costs and instability limit their applications. This study investigated the synthesis of gold nanoclusters and optimized reaction conditions, including the concentration and volume of the substrate and the effect of the inhibitor on the peroxidase reaction.
Materials and Methods: Gold nanoclusters based on the creatininase (Crn-AuNCs) were synthesized using a modified protocol. The kinetics of creatininase were analyzed to measure Km and Vmax values, and the optimization of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) concentrations was performed using a UV-Vis spectrophotometer.
Results: In this study, (Crn-AuNCs) were synthesized, yielding Km and Vmax values of 0.0882 M and 24.5 u.ml-1, respectively. The gold nanoclusters exhibited peroxidase-mimicking activity, effectively catalyzing peroxidation reactions with TMB and H2O2. The optimal concentrations and volumes of TMB and H₂O₂ were determined to be 10 mM (20 µL) and 0.1 M (2 µL), respectively.
Conclusion: This study focused on synthesizing (Crn-AuNCs) that exhibited green fluorescence. It was found that the enzymatic activity of creatininase decreased significantly due to high temperature and alkaline pH. The investigation into the pseudo-enzymatic (peroxidase) activity of the nanoclusters revealed that the presence of Tris during synthesis significantly affected this activity. Additionally, variations in the concentrations of creatine, creatinine, and urea did not influence the fluorescence intensity of the nanoclusters. Overall, the findings suggest the potential application of Crn-AuNCs in biotechnology, particularly in biosensor development.
Materials and Methods: Gold nanoclusters based on the creatininase (Crn-AuNCs) were synthesized using a modified protocol. The kinetics of creatininase were analyzed to measure Km and Vmax values, and the optimization of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) concentrations was performed using a UV-Vis spectrophotometer.
Results: In this study, (Crn-AuNCs) were synthesized, yielding Km and Vmax values of 0.0882 M and 24.5 u.ml-1, respectively. The gold nanoclusters exhibited peroxidase-mimicking activity, effectively catalyzing peroxidation reactions with TMB and H2O2. The optimal concentrations and volumes of TMB and H₂O₂ were determined to be 10 mM (20 µL) and 0.1 M (2 µL), respectively.
Conclusion: This study focused on synthesizing (Crn-AuNCs) that exhibited green fluorescence. It was found that the enzymatic activity of creatininase decreased significantly due to high temperature and alkaline pH. The investigation into the pseudo-enzymatic (peroxidase) activity of the nanoclusters revealed that the presence of Tris during synthesis significantly affected this activity. Additionally, variations in the concentrations of creatine, creatinine, and urea did not influence the fluorescence intensity of the nanoclusters. Overall, the findings suggest the potential application of Crn-AuNCs in biotechnology, particularly in biosensor development.
Keywords: Creatininase enzyme, gold nanoclusters, peroxidase mimicking activity, colorimetric detection
Type of Study: Research Article |
Subject:
Biochemistry
Received: 2026/06/21 | Accepted: 2026/05/31 | Published: 2026/05/31
Received: 2026/06/21 | Accepted: 2026/05/31 | Published: 2026/05/31
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