Transcript
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APPLICATION OF CHOLESTEROL ESTERASE FOR
DETERMINETION OF
TOTAL CHOLESTEROL IN THE BLOOD AND BIOSENSOR
DESIGN
Supervised by:Prof. Lyubov YotovaEng. Vera Semerdzhieva
Ahmed Boujelben
University of Chemical Technology and Metallurgy -UCTM Sofia, Bulgaria
2016
Prepared by :
PLANIntroduction
Purposes of the thesis
Results and Discussion
Conclusion and Perspectives
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1 INTRODUCTION
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Cholesterol
Cholesterol is an extremely important biological molecule, It plays a vital role in human body,such as in : the membrane structure synthesis of steroid hormones, vitamin D
30%comes from diet and the rest is synthesized in the body
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Cholesterol ester
Cholesterol esters is the product of esterification of cholesterol by cholesterol acyltransferase
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Cholesterol Oxidase • Cholesterol oxidase is an enzyme that catalyzes the oxidation of the
C3-OH group of cholesterol to give cholestone
• Cholesterol oxidase has received great attention due to its wider use in clinical determination of serum cholesterol
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Cholesterol Esterase
• Cholesterol esterase is an enzyme which catalyzes the hydrolysis of cholesterol esters to cholesterol and fatty acid
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Cholesterol Esterase
• Cholesterol esterase is present mainly in pancreas
• liver, epithelial cells, blood
• It is essential in lipoprotein metabolism (hydrolysis of cholesterol esters to free cholesterol and fatty acids)
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Applications of Cholesterol Esterase
Widely used as Medical diagnostic biosensor
• Determination of Total cholesterol in serum and plasma
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Free cholesterol Esterified cholesterol
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Applications of Cholesterol Esterase
• Total cholesterol can be measured enzymatically using cholesterol oxidase cholesterol esterase and proxidase
POLLUTANT REDUCING
FOOD INDUSTRY
CHEMISTRY LAUNDRY DETERGENTS
MEDECAL DIAGNOSTICS
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Applications of Cholesterol Esterase
2 Purposes of the thesis
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Characterization of cholesterol
oxidase in order to optimize the
pH and the temperature
Isolation and partial
purification of Cholesterol
esterase from porcine pancreas
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Characterization of the isolated enzyme using different methods for determination of the activity
3 RESULTS AND DISCUSSION
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STANDARD LINES PREPARATION
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Determination of the total protein by Lowry method
For the determination of total Protein ,we prepared two standard lines with different concentrations
of BSA (Bovine Serum Albumin) ,between [0.01-0.1] mg/ml and [0.1-1] mg/ml ,
Absorbance at 750nm;
Lowry Standard curve [0.1-1] mg/ml BSALowry Standard curve [0.01-0.1] mg/ml BSA
The R2 value is 0.99, which means that we could use the equation of line with high confidence
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Determination of the esterase activity with p-Nitrophenylacetate
0 2 4 6 8 10 120
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
f(x) = 0.0123272727272727 x + 0.0524R² = 0.998664635135448
C [μM]
A 41
0
Standard line [1-10] µM PNP
0 20 40 60 80 100 1200
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
f(x) = 0.00807636363636364 x + 0.000799999999999967R² = 0.997945443351166
C [μM]
Standard line [10-100] µM PNP
The R2 value is 0.99, which means that we could use the equation of line with high confidence
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Cholesterol oxidase Characterization and detection assay
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Optimum spectrum determination
The curve shows the absorbance of COD in different wave length
The results indicate that the optimum wave length for detection of Cholesterol oxidase is around 450 nm
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Optimum pH determination
In order of determine the opt pH , we prepared different solutions of the COD and with different pH, the range of pH is between 5 and 9
o From the graph ,it can be observed that the optimum pH is around 7o Extremely high or low pH values result in complete loss of activity of the
Cholesterol oxidase.
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Optimum Temperature determination
In order to determine the temperature optimum of the enzyme, measurements were conducted in different temperatures.The activity of the enzyme is determined at 450 nm in 300 seconds.
From the graph , it can be observed that the optimum temperature of
COD is around 45-50 oC
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Fractional analysis of the protein mixture from the porcine pancreas extract
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This part of our work aimed to investigate the proteins contain in the pancreas extract in both the precipitate and the supernatant
SDS-PAGE saturated 30% Ammonium Sulfate
(A)Standard protein (B) Precipitate(C) Supernatant
As we can see ,there are protein bands with molecular weight around 75-85kDa in the precipitate , which is the molecular weight of the cholesterol esterase, isolated from porcine pancreas according to the literature.
It is clear that the Cholesterol esterase is mainly present in the precipitate
Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to separate and characterize proteins
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Focusing on the precipitate(B), the results of SDS-PAGE saturated 60% with ammonium sulfate confirm that there are protein bands with molecular weight between 70-80kD,which is the molecular weight of the cholesterol esterase
(B) Precipitate
SDS-PAGE saturated 60% with ammonium sulfate
(A)Standard protein
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Determination of Cholesterol esterase activity
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Cholesterol esterase activity against p-NPA
p-Nitrophenylacetate(p-NPA) is a common substrate for measuring esterase activity.The activity was assayed by measuring the rate of p-Nitrophenol formation (µM) at room temperature.We have measured cholesterol esterase activity against p-NPA with three different conditions :
Activity [U/mg] Protein
22.7 60% Saturation with Ammonium Sulfate
32.17 30% Saturation with Ammonium Sulfate
39.01 Purified with Dialyze Membrane
The activity Cholesterol esterase activity with 30% ammonium sulfate saturation is equal to (32.17 U/mg) and it’s higher than with 60% saturation ammonium sulfate (22.7 U/mg)
The best result for the activity of CE was obtained after removing the ammonium sulfate with dialyze membrane (39.01 U/mg).
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cholesterol esterase activity determination with o-Dianisidine
The graph shows a Plot of the Cholesterol esterase activity in the presence of 5mM Cholesteryl Stearate as a substrate
One unit of enzyme will hydrolyze 1.0μM of cholesteryl stearate per min at pH 7 at 37 oC
The activity of Cholesterol esterase obtained was equal to (6.45 U/mg)
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Determination of the cholesterol esterase activity with Phenol and 4-AAP
Concentration of the substrate [mM] Activity [U/mg]
5 6.2948
10 36.904
7 4.196
6 22.464
The Activity of Cholesterol esterase was measured with different concentrations of the substrate
The maximal enzyme activity was obtained in the presence of 10mM substrate
4 CONCLUSION AND PERSPECTIVES
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The determination of esterase activity, showing the presence of the enzyme in the protein sample. The activity with a specific substrate was determined with two similar methods and the optimal concentration of the substrate for the reaction was obtained with 10mM substrate
The obtained pancreas extracts were fractionally characterized with SDS-PAGE. Molecular weight in the range of 70-85kDa, indicate the possible presence of pancreatic cholesterol esterase. For further studies a native electrophoresis with a specific activity staining for cholesterol esterase can be done.
Biosensors have recently gained much attention in the field of health care, and development of total cholesterol biosensor is possible by coupling cholesterol oxidase and cholesterol esterase together
For the purposes of future construction of Cholesterol biosensor a standard enzyme Cholesterol oxidase was investigated, however , there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized.
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ACKNOWLEDGMETS
This study was conducted in the Department of biotechnology at the
University of Chemical Technology and Metallurgy -UCTM Sofia, and supported
by Erasmus Mundus – GreenIT Project.
I would like to express my gratitude to Prof. Lyubov Yotova , Head of biotech
depart for the guidance, assistance and support during the whole period of my
study at the University.
Special thanks to Prof. Rossica Betcheva, the Erasmus Coordinator at UCTM,
for her assistance and encouragement during the period of stay in Bulgaria
I am grateful to Eng. Vera Semerdzhieva, PhD Student at biotech depart for
her encouragement, support and for all the time she spent in the laboratory
helping me to achieve this work.
Ahmed Boujelben June, 2016
Thank you for your
attention
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Ahmed Boujelben June, 2016
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