Volume 12, Issue 48 (10-2022)
NCMBJ 2022, 12(48): 47-58 |
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Jalili S, Rahmati F, Abdollahzadeh Parsa M. Extracting the creatininease enzyme and investigating the effect of different concentrations of gold nanoparticles on the enzyme activity. NCMBJ 2022; 12 (48) : 4
URL: http://ncmbjpiau.ir/article-1-1561-en.html
URL: http://ncmbjpiau.ir/article-1-1561-en.html
Department of Biochemistry, Faculty of Biological Sciences, North-Tehran Branch, Islamic Azad University, Tehran, Iran. , f.rahmati@iau-tnb.ac.ir
Abstract: (1555 Views)
Aim and Background: The use of creatininase is an enzymatic method for measuring amount of creatine and creatinine in body fluids to assess kidney health. The aim of this study was to investigate the effect of gold nanoparticles on the activity and stability of creatininase enzyme for use in creatine and creatinine assay kits.
Materials & Methods: In this study, E. coli BL21 containing pET28a amplification vector containing creatininase gene was first cultured in liquid LB medium and then induced at 28°C. After sonication, purification of enzyme was performed by affinity chromatography. After confirmation of expression by SDS-PAGE, The expressed enzyme concentration was measured by Bradford method. Then the activity of the enzyme was examined and compared by colorimetric method in the presence and absence of gold nanoparticles. Finally, the effect of gold nanoparticles on the structure of creatininase enzyme was investigated by fluorescence spectroscopy.
Results: Creatininase activity in the absence of gold nanoparticles 14.6 U/ml and in the presence of gold nanoparticles with concentrations of 1.97, 3.94, 7.88, 11.82, 15.76, 19.7 and 39.4 ngr/l 14/1, 12.4, 11.2, 10.1, 8.4, 7.5, 4.7 U/ml were calculated which indicates a decrease in creatininase activity in the presence of gold nanoparticles. On the other hand, fluorescence spectroscopy of creatininase in the presence and absence of gold nanoparticles at the excitation wavelength of 295 nm showed a decrease in fluorescence intensity in the presence of gold nanoparticles.
Conclusion: Gold nanoparticles reduced the activity of the creatininase enzyme. Gold nanoparticles also reduced the fluorescence intensity of creatininase enzyme, which could indicate a change in the microenvironment of the amino acid tryptophan in the structure of the creatininase enzyme, so using gold nanoparticles is not a good option for stable structure and activity of creatininase for use in commercial kits.
Materials & Methods: In this study, E. coli BL21 containing pET28a amplification vector containing creatininase gene was first cultured in liquid LB medium and then induced at 28°C. After sonication, purification of enzyme was performed by affinity chromatography. After confirmation of expression by SDS-PAGE, The expressed enzyme concentration was measured by Bradford method. Then the activity of the enzyme was examined and compared by colorimetric method in the presence and absence of gold nanoparticles. Finally, the effect of gold nanoparticles on the structure of creatininase enzyme was investigated by fluorescence spectroscopy.
Results: Creatininase activity in the absence of gold nanoparticles 14.6 U/ml and in the presence of gold nanoparticles with concentrations of 1.97, 3.94, 7.88, 11.82, 15.76, 19.7 and 39.4 ngr/l 14/1, 12.4, 11.2, 10.1, 8.4, 7.5, 4.7 U/ml were calculated which indicates a decrease in creatininase activity in the presence of gold nanoparticles. On the other hand, fluorescence spectroscopy of creatininase in the presence and absence of gold nanoparticles at the excitation wavelength of 295 nm showed a decrease in fluorescence intensity in the presence of gold nanoparticles.
Conclusion: Gold nanoparticles reduced the activity of the creatininase enzyme. Gold nanoparticles also reduced the fluorescence intensity of creatininase enzyme, which could indicate a change in the microenvironment of the amino acid tryptophan in the structure of the creatininase enzyme, so using gold nanoparticles is not a good option for stable structure and activity of creatininase for use in commercial kits.
Article number: 4
Keywords: Creatininase, Expression, Gold nanoparticles, Colorimetry, Fluorescence spectroscopy, Iau Science.
Type of Study: Review Article |
Subject:
Microbiology
Received: 2022/03/16 | Accepted: 2022/10/2 | Published: 2022/10/2
Received: 2022/03/16 | Accepted: 2022/10/2 | Published: 2022/10/2
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