Kinetic study of cholesterol oxidation by cholesterol oxidase enzyme as application for cholesterol biosensor

Kinetic study of cholesterol oxidation by cholesterol oxidase enzyme as application for cholesterol biosensor

Cite as: AIP Conference Proceedings 2092, 030027 (2019); https://doi.org/10.1063/1.5096731 Published Online: 09 April 2019

Meka Saima Perdani, Muhamad Sahlan, Siti Farida, Dwini Normayulisa Putri, Sri Angky Soekanto, and Heri Hermansyah

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Kinetic Study of Cholesterol Oxidation by Cholesterol Oxidase Enzyme as Application for Cholesterol Biosensor

Meka Saima Perdani

¹

, Muhamad Sahlan

^1,2

, Siti Farida

³

, Dwini Normayulisa Putri

¹_,

Sri Angky Soekanto

⁴

, Heri Hermansyah

^1,a)

1Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, West Java, 16424, Indonesia

2Research Center of Biomedical Engineering, Universitas Indonesia, Depok, West Java, 16424, Indonesia

3Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Central Jakarta, DKI Jakarta, 10430 Indonesia

4Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Depok, West Java, 16424, Indonesia

a)Corresponding author: heri@che.ui.ac.id

Abstract. Cholesterol oxidase enzyme well known as oxidoreductase enzyme which able to catalyze the transfer of electron from one molecule (the oxidant) to another molecule (the reductant). The kinetic model for cholesterol oxidation by cholesterol oxidase has been demonstrated using first order irreversible reaction with Euler’s method. The aim of this study is to determine the kinetic parameter of cholesterol oxidation using enzyme cholesterol oxidase. For this kinetic study, experimental data was obtained from several literatures. The affecting factors of kinetic rate constant in this study were solvent addition, stabilizer concentration, granule concentration, temperature and immobilized enzyme. In the oxidation reaction, there were some variables condition based on the experimental data, CA-1, CA-2, CA-3, respectively. The modelling result showed that the first order reaction was able to modelling the cholesterol oxidation with high accuracy.

The smallest relative error of kinetic value was showed in the immobilized enzyme with the temperature variable at 37°C with k value 0.0290 h^-1. This kinetic study is useful for further developing of application for biosensor based enzyme as catalyzer in the oxidation reaction. Moreover, kinetic study is also useful for choosing the operating factor for cholesterol oxidation.

Keywords: biosensor, cholesterol, cholesterol oxidase, kinetic, modelling, oxidation

INTRODUCTION

Cholesterol is an unsaturated alcohol of the steroid family of compounds; it is essential for the normal function of all animal cells and is a fundamental element of their cell membranes. It is also a precursor of various critical substances such as adrenal and gonadal steroid hormones and bile acids. In human’s body, cholesterol as nonpolar lipid substance is packaged in blood circulation as the complex of lipid and protein, lipoprotein. Cholesterol are classified by density, electrophoretic mobility, size and their relative content of cholesterol and protein into LDL (low density lipoprotein) and HDL (high density lipoprotein). High cholesterol levels in blood caused the accumulation and blockage of the artery (atherosclerosis), which is one of the major cause of coronary heart disease [1].

Being one of the most important health parameter, cholesterol level is appealed to be monitored annually by quantification of blood cholesterol level. Determination of cholesterol by enzymatic method are preferred, because of its specificity and sensitivity. Determination of cholesterol by enzymatic method generally involves 2 enzymes, cholesterol oxidase and cholesterol esterase [2].

Cholesterol oxidase (CO) is an enzyme which catalyzes the oxidation of cholesterol and converts 5-cholesten-3β- ol into 4-cholesten-3-ones [3]. Cholesterol oxidase has been isolated from a variety organism sources such as fungi

3rd Biomedical Engineering’s Recent Progress in Biomaterials, Drugs Development, and Medical Devices AIP Conf. Proc. 2092, 030027-1–030027-10; https://doi.org/10.1063/1.5096731

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and bacteria, mainly fungi. This enzyme can be produced by a bacterium in three forms: intracellular, extracellular and membrane bound. Based on the wide applications of CO, screening, isolation and kinetic oxidation study are of great importance. CO has received considerable attention around the world due to its use in enzymatic cholesterol analysis, especially biosensor as a new advance method for analysis.

Numerous researches proofed that the catalyzer in the first step of the cholesterol degradation was Cholesterol oxidase. Moreover, Cholesterol oxidase is useful for biotechnological implementation as well as industrial implementation, for example determination of cholesterol serum in blood, insecticides, larvacides, and antibiotic for pathogen bacteria Rhodococcus equi [4]. Cholesterol oxidase has been used as a biocatalyst that provides valuable steroidal compounds and non-steroidal chiral compounds. Cholesterol oxidase from Chromobacterium sp. DS-1 can be used to reduce oxysterol cytotoxicity [5]. Therefore, the aims of this research is to determine the kinetic parameter of cholesterol oxidation by enzymatic route by several factors so that this study could become a reference for maximizing the use of cholesterol oxidase in industry.

Cholesterol oxidase is a Flavin-enzyme (with a FAD prosphetic group) that produces hydrogen peroxide.

Enzymatic oxidation of cholesterol proceeds with two electron reduction of the cholesterol oxidase Flavin adenine dinucleotide (FAD) prosthetic group to FADH2 [6]. The common reaction of cholesterol oxidation as shown below:

Cholesterol +O2 + Enzyme  4-Cholesten-3-one + H2O2 (1) The cholesterol oxidation can be determined by following the appearance of the conjugated ketones, formation of hydrogen peroxide by measuring the oxygen consumption. This method able to identified using analytical devices based on enzymatic biosensor. However, there were several factors that affecting to the oxidation value, such as microorganism source of enzyme, solvent, concentration of enzyme, temperature and pH [7].

Cholesterol oxidation by enzymatic catalyzed are consist of several mechanisms including oxidation and isomerization of cholesterol molecules. The oxidation of cholesterol is indicated by the depletion of substrate concentration. The experimental data that we used are from six different parameter kinetic data. The kinetic study of enzymatic reaction of cholesterol oxidation are based on enzymatic first order irreversible reaction using Euler’s numerical method for differential equations. The effect of reaction’s parameters such as solvent variants, temperature and substrate concentration are studied based on the kinetics parameter. A kinetic model considering the differences between the enzyme and substrate concentration was proposed to obtain the accurate parameter for further implementation [8].

METHODS Kinetic Study

The general method of this study are divided into three prominent step, collecting experimental data by literature, kinetic modelling and analysis, respectively. The aims of this study was to determine an exact parameter based on oxidation enzymatic reaction. The first step has done by literature review and preparing the cholesterol oxidation experimental data. The experimental data that used in this study based on the research by N.Doukyu (1998), Lv (2002), M.T.Lee (1999), Salva (2000), Huang (2015), and G.K.Kouassi (2005) [5,13,14,16,11,9]. The second step was derivation of kinetic equation from the exact reaction model, the kinetic model that used in this study was first-order irreversible reaction. In this step was conducted mathematical process from kinetic equation then it was obtained analytical equation which is able to use for modelling the cholesterol oxidation reaction. Then for the third step was fitting curve with an adjustment value between numerical data and analytical data. The aim of curve fitting process was to estimate the value of the unknown parameters in the kinetics equations. The fifth step was simulating the modelling result to validating the value of estimation results. The last step was analyzing the data. Figure 1 was described the overall process of this study as shown below:

FIGURE 1. Flow diagram process to estimating the parameter value

Kinetic Model

The cholesterol oxidation was evaluated upon consumption of cholesterol dissolved in various parameters. This simulation is used first-order irreversible reaction and Euler method. The reaction model and kinetic equation are described below:

Z

A  

^k (2)

A

kC

A

dt dC 



(3)

Separating the variables and integrating between limit as equivalent forms of the integrated first-order rate equation:



 ^{ }

 

^{C t}

C A

A A

A

k dt

C kC dC dt

dC

^A

A 0

0

(4)

C kt C

A

A

 

ln

0 (5)

kt A

A

C e

C 

^{0 } (6)

Estimation of Reaction Rate Constant

The curve changes of substrate concentration against time has been known from the data obtained through several literatures, then it can be a fitting with the curve and the kinetic equations. There was an unknown constant reaction rate or parameters in the mechanism or known as “k”. The estimation method is done by fitting from a derived analytical equation to the oxidation data that already exists. The first step was determining initial concentration of

Abbreviation:

A = Cholesterol Z = 4-Cholesten-3-one CA = Concentration A k = Rate constant t = Time

substrate by the literature itself. Then, calculate every concentration of oxidation reaction start from initial concentration (t=0) until the reaction has ended up. The analytical concentration calculated by using equation (6).

Final value of analytical concentration using Euler’s method was compare with the experimental or numerical data from experiment. Model constant estimation has done using trial and error method to obtain the minimum sum relative error. The sum relative error (SRE) between analytical and numerical was calculated using the equation (7) as shown below:



^



 





 









^{t n}



t tanalytical tnumerical numerical t analytical t

C C

C SRE C

0

2

, ,

, ,

2 )

(

⁽⁷⁾

Sensitivity Analysis

The parameter sensitivity test of estimation parameter result is done by calculating the sum value of the relative error of the final parameter’s result then calculated the sum relative error parameter which are plus 100% and minus 50%. A comparison between these three parameter results will show the accuracy of estimation parameter value. The deviation value was calculated by delta percentage error divided with percentage initial parameter value. High deviation percentage means good accuracy of parameter value.

RESULTS AND DISCUSSIONS Kinetic Matrices

Based on the literature screening, we found that six experimental data to be examined (Table 1). The experimental data is used to evaluate the parameter value of each condition or operating factor.

TABLE 1. Literature of cholesterol oxidation for kinetic modeling

Author Enzyme Substrate Immobilization Oxidation Factor N.Doukyu (1998) [5] Cholesterol Oxidase

Pseudomonas sp. Cholesterol - Addition of organic solvent variants

Lv (2002) [13] Cholesterol Oxidase Brevibacterium

Yolk Cholesterol

- Granule disrupter solution

Salva (2000) [16] Cholesterol Oxidase

Brevibacterium Cholesterol - Concentration of stabilizer (Glycerol)

M.T.Lee (1999) [14] Cholesterol Oxidase Rhodococcus equi no.23

Cholesterol - Temperature

Huang (2015) [11] Cholesterol Oxidase Stenotrophomonas sp.

Cholesterol Fe3O4-SiO2 Free enzyme and immobilized enzyme, time, concentration of substrate

G.K. Kouassi (2005) [9] Cholesterol Oxidase Nicordia sp.

Cholesterol Fe3O4 Temperature factor of immobilized enzyme

Numerously researchers have been evaluated the oxidation of cholesterol using cholesterol oxidase enzyme as catalyzer. In spite of it, the kinetic data such as rate constant of its reaction was not show in their study. For modelling of kinetic, it is needed to collecting the experimental data of cholesterol oxidation such as concentration of cholesterol and time of reaction. The details of experimental data that had chosen as shown in Table 2:

TABLE 2. Experimental detail of cholesterol oxidation

Data Description

1. N.Doukyu (1998)

[5] Cholesterol (10 mol/l) was suspended in 2.5 ml assay solution with the absence of organic solvent (CA-1), adding of 0.25 ml cyclooctane (CA-2) or diphenylmethane and p-xylene (CA-3) and added to assay solution consisting of Cholesterol oxidase. The reaction was in 7.4 pH

2. Lv (2002) [13] The substrate was yolk cholesterol with initial concentration 0.1 mol/L. The operating factor in this reaction was temperature at 39°C, pH at 7.5, granule disrupter and time variations. The variants of granule disrupter were (NH4)2CO3, NaCl and Pepsin with the same concentration. In the simulation process for every granule variables were abbreviated with CA-1, CA-2 and CA-3, respectively 3. Salva (2000) [16] The oxidation of cholesterol using electrophoresis method and utilizing a membrane as the receptor.

The electrophoresis reaction was dialyzed for 18 hours with initial substrate concentration was 0.012 mol/l. The variants of solution component was done by the adding of 1mM phosphate buffer solution containing 0.7% Triton X-100 added by Glycerol 0% (CA-1) and Glycerol 20% (CA-2)

4. M.T.Lee (1999) [14]

The free enzyme directly subjected to the substrate with 0.8 mol/l for initial concentration. The reaction was analyzed high performance liquid chromatograph with an ultraviolet detector at 210 and 240 nm. The effect of temperature was analyzed at 39°C (CA-1), 34°C (CA-2) and 34.2°C (CA-3), respectively

5. Huang (2015) [11] The effect of immobilization process on cholesterol oxidase enzyme. The cholesterol oxidation of free enzyme (CA-1) and Fe3O4-SiO2 immobilized on cholesterol oxidase (CA-2) was determined with different time at 60°C and pH at 7.0. The overall reaction conducted in 10-4 mol/l of initial concentration of substrate, 15 mg nanoparticles Fe3O4-SiO2 and redispersed in 3.68 mL phosphate buffer solution (PBS0.01 mol/l, pH = 7.3)

6. G.K.Kouassi (2005) [9]

The enzyme was immobilized using Fe3O4 nanoparticles with different temperatures, there was 37°C (CA-1), 50°C (CA-2), 60°C (CA-3) and 70°C (CA-4), respectively. The reaction of CA-3 and CA-4 has ended up in reaction time of 1.2 h and 0.8 h, respectively. Overall reaction was prepared by chemical co-precipitation with 50 mg nanoparticles, 1 mL of phosphate buffer (0.05 M pH 7.4) at different times 0-2 hours)

Curve Fitting Result

The advantage of this curve fitting was to estimating the unknown parameters value in an equation’s model. In The parameter of first order irreversible reaction is k (reaction rate constant). There were plotting result between experimental data (numerical) and modelling method (analytical) as shown below:

FIGURE 2. Fitting model result for Data I (Experimental by N.Doukyu, 1998 [5])

FIGURE 3. Fitting model result for Data II (Experimental by Lv, 2002 [13])

FIGURE 4. Fitting model result for Data III (Experimental by Salva, 2000 [16])

Experimental by N. Doukyu (1998), Lv (2002), and Salva (2000) had similarity on the variation of the affecting reaction solvent. Cholesterol is poorly soluble in water. Thus, enzymatic H2O2 generation reactions were carried out using cholesterol emulsified with stabilizer, organic solvent or solubilizer to obtained good oxidation reaction [11].

Based on Figure 2, the constant rate in every organic solvent showed different constant rate also. By means, the addition of organic solvent should be affected to the kinetic behavior. As same way like Figure 3, the addition of granule disrupter such as (NH4)2CO3 (CA-1), NaCl (CA-2) and Pepsin (CA-3) on cholesterol conversion was affected to the kinetic value. NaCl greatly enhance cholesterol conversion, while (NH4)2CO3 has a markedly adverse effect on conversion rate. The reaction rate constant of NaCl (CA-2) was the highest than other granule disrupter.

FIGURE 5. Fitting model result for Data IV (Experimental by MT Lee, 1999 [14])

FIGURE 6. Fitting model result for Data V (Experimental by Huang, 2015 [11])

Another parameter factors such as temperature and typical of enzymes treatment was described in the experimental by MT. Lee (1999), Huang (2005), and G.K. Kouassi (2005). As described in Figure 5, the temperature variation 39°C (CA-1), 34°C (CA-2), and 34.2°C (CA-3) showed that the highest reaction rate constant was on the highest temperature. It proofed that in the temperature of 39°C, the enzyme still able and actively to catalyze the cholesterol itself. If we increase greatly the temperature, there should be other supporting system to the enzyme, due to the enzyme sensitivity to the temperature.

FIGURE 7. Fitting model result for Data VI (Experimental by G.K.Kouassi, 2005 [9])

The problem of enzyme activity was solved by G.K. Kouassi (2005) which is added metal materials to the enzyme then immobilized it [9]. The temperature effect on the activity of immobilized enzyme was highly increased. As shown in Figure 7, the more temperature increase, the reaction rate constant showed greater than other temperatures. This all supported by Huang (2015) who described the differences of cholesterol oxidation of free enzyme and immobilized enzyme [11]. Based on the simulation that showed in Figure 6, the CA-1 (immobilized enzyme) showed greater reaction rate constant than CA-2 (free enzyme). Furthermore, an immobilization process was able to support the enzyme properties as described by H. Hermansyah (2015) on the kinetic study of immobilized enzyme as biocatalyst [10].

Kinetic Constant Analysis

As shown in Figure 2 to Figure 7 the parameter value was depend on the operating of the reaction such as variant solvent, temperature, immobilization process, solubilizer or stabilizer. In Figure 2, it shown that the parameter value which had minimum error at third condition (CA-3) was a reaction added by p-xylene’s solvent. The conversion of cholesterol in organic solvent is proofed the different parameter value. The percentage error in every reaction as shown in Figure 2 to Figure 7 was in minimize percentage which are able to estimate the parameter value accurately. In Figure 3, the effect of granule solubilizer on cholesterol conversion was revealed the different parameter value also.

CA-1 as solubilizer of (NH4)2CO3 , CA-2 NaCl solubilizer and CA-3 Pepsin group solubilizer, respectively. The parameter percentage was smalll and able to implemented to the application.

In the first-order reaction, a plot of ln CA vs t is linear which is the first-order rate constant can be obtained from the slope. Unfortunately, this way showed numerously high % error. So that, the Euler method was applicated to obtain the rate constant to reduce the correction. Based on the kinetic simulation, the cholesterol consumption rate was higher in the presence of various organic solvent (cyclooctane, diphenylmethane or p-xylene) than that found in the absence of organic solvent, as shown in Figure 2. In general conditions, such a behavior could be due to any kind of rate-limiting process preceding the formation of oxidized Flavin. Assuming the absence of organic solvent or Flavin, such a step would have to precede the reaction with dioxygen.

The changes of conformation involving complex between reduced enzyme intermediate/product prior to reaction with dioxygen was explained by kinetic mechanism. The decreasing of substrate concentration was possibility from the rate of oxygen or enzyme escape from the micelles and its availability to be limiting [12]. In the same condition of initial concentration of substrate such as reaction condition CA-1, CA-2 and CA-3 could arise from the micelles solution and affected to the rate constant.

Moreover, the temperature variation in the oxidation reaction also affected to the parameter, as shown in Figure 4 and Figure 7. In Figure 4, the reaction was without any immobilized enzyme or well known as free enzyme. The free enzyme in the different temperature which indicated as 39°C (CA-1), 34°C (CA-2), and 34.2°C (CA-3) showed the decreasing k value was because of the decreasing of temperature as well. In addition, the consumption rate of cholesterol increased as the temperature increased up to as 39°C.

Based on 4^th experimental by Salva (2000), an addition of another glycerol solution to the oxidation reaction was able to stabilize the enzyme activity [16]. The variation amount of concentration was 0% Glycerol (CA-1) and 20%

Glycerol (CA-2). The constant rate of reaction was decreased as well as increasing adding solution to the reaction.

High concentration of glycerol was inhibit the oxidation process and made it slower than CA-1.

In spite of temperature and solvent, the method of enzyme process was also affected to the reaction. The free enzyme and immobilized enzyme was showed different behavior in kinetic study. In Fig.6, the kinetic model of free enzyme (CA-1) and immobilized enzyme (CA-2) was observed. A kinetic modelling was showed that the reaction rate constant of immobilized enzyme was higher than free enzyme. Otherwise, the percentage error of CA-1 was higher than CA-2. Assuming, the immobilized enzyme should be greater activity to oxidate the cholesterol rather than free enzyme due to the material properties which support the enzyme and binding to the enzyme covalently [9].

Sensitivity Analysis

Sensitivity parameter test has done by calculated the sum relative error from obtained parameter then calculated the objective of sum relative error parameters. The parameter was estimated as 0. 5k, k and 2k. These three parameters are shown the accuracy of estimation modelling. As shown in Table 3, the overall sensitivity analysis result was good accuracy, signed by high deviation percentage. There was an exception value in the 4^th data by Salva (2000) which is showed smallest deviation (+814%). Generally, the positive deviation value means that high accuracy of estimation parameter. Moreover, overall simulation data was good accuracy due to the minimum percentage error and high deviation.

TABLE 3. Sensitivity parameter of estimation value

CONCLUSION

The cholesterol oxidation was determined by monitoring the consumption of hydrogen peroxide and other oxidation products. In the enzymatic reaction, the adding of another solvent to the reaction was able to increase the oxidation result with rate constant value was 0.095 h^-1. The rate constant value of free and immobilized enzyme was 0.11665 h^-1 and 0.05160 h^-1, respectively. Kinetic modelling of cholesterol oxidation revealed that the high rate constant affected by organic solvent in the reaction and it was probably due to the stability in the presence of various organic solvent. The rate constant value was described the correlation between the concentration of the reacting substances and the rate of reaction. An immobilized enzyme was greater consumption rate constant reaction rather than free enzyme. Moreover, the kinetic modelling of cholesterol oxidation is useful for determining operating factor or determining the value of oxygen consumption by using analytical sensor devices. By the rate constant value, it is shown to monitoring the maximum concentration of reaction substrate.

ACKNOWLEDGMENTS

The authors would like to thank for the support provided by the United States Agency for International Development (USAID) through the Sustainable Higher Education Research Alliance (SHERA) Program for Universitas Indonesia’s Scientific, Modelling, Application Research and Training for City-centered Innovation and Technology (SMART CITY) Project, Grant #AID-497-A-1600004, Sub Grant #IIE-00000078-UI-1.

No.0139/UN2.R3.SC/HKP.05.01/2018

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