final present uctm

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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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• 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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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

final present uctm

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