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Development of enzymatic membrane for the detection of cholesterol in serum.

Introduction

The formation of enzyme membranes with entrapped of enzymes in non-aqueous solution is well accepted procedure and is a versatile way for fundamental studies of enzyme function and structure. These enzyme- membranes are helpful to stabilize enzymes, enhance their biocatalytic availability, facilitate their recovery and achieve continuous operation without product inhibition. The behavior of enzyme membranes varies markedly from the behavior of the free enzymes. The enzymes which are highly unstable in non-aqueous solvent could be stabilized when it immobilized into solid supports (1 & 2). These artificial enzyme-membranes can be used in the study of heterogeneous enzyme kinetics and for modeling biological membranes (3).

A number of methods for enzyme membrane preparation is available in the literature (4, 5 & 6) but no single method is perfect for the all the enzymes. However, application and preparation of enzyme membranes have been limited due to (i) short life of enzymes (ii) possibility of severe leaching in long term operation. That's why there is always demand of new suitable materials for enzyme membrane development. The immobilization of enzymes on polymer could be a better answer to retain their maximum activities and stability because when an enzyme is immobilized to a polymer , either in solution or hydrogel, a drastic changes occur during the polymer formation. Thus, enzyme membranes show more interesting properties than the free enzymes such as thermal stabilization (7 & 8).

The main objective of this work was to prepare an enzymatic membrane where, cholesterol oxidase and cholesterol esterase were immobilized for the determination of cholesterol in serum. Considering that cholesterol is very important parameter for life because its higher level in plasma is responsible for cardiac arrest, (9) atherosclerosis (10) and gal stone (11) formation. Numerous methods are available to estimate cholesterol in the serum such as Thin layer chromatography (12) Gas chromatography / mass spectrometry (13) HPLC --mass spectrometry (14) and Enzymatic --colorimetric methods (15) where COE and COD were used. The enzymatic-colorimetric method is simple sensitive, specific and rapid over the other methods and therefore, appropriate for the clinical analysis. However high cost of enzymes restricts the use of enzymatic method for routine work. To over come this situation COE and COD were co-immobilized on different substrate because once the enzymes were immobilized they can be reused for many times in the reaction mixture. (16, 17 & 18) In the present work an enzymatic membrane were made where COD and COE were immobilized and has potential to estimate total cholesterol in serum.

Material and Methods

Chemicals

Cholesterol oxidase, cholesterol esterase, peroxidase and 25% glutaraldehyde were purchased from M/s Fluka Chemicals Germany. Polyvinyl resin (Commercial name Formvar), TritonX-100, cholesterol oleate and taurocholic acid were purchased from M/s Sigma Chemicals ( St. Louis Mo, USA). All other reagents were of analytical grade and were purchased locally. Enzo-kit for cholesterol determination was from M/S Miles India Ltd., Baroda India. Water for preparing reagents and buffers, was purified through Millipore-Q-Reagent System ( Millipore. Inc. Bedford, MA).

Preparation of Enzyme Membrane

Enzyme membrane was prepared from 2ml of 4% (w/v) polyvinyl formal resin (formvar) using chloroform and ethylene dichloride (1:1 ratio, v/v). Cholesterol esterase and cholesterol oxidase (5 units/ml of each) were added to this mixture and stirred for two minutes at room temperature (25[degrees]C) to achieve a homogenous distribution of enzymes within the polymer. The membrane was cast on the plane glass surface which was cleaned with ethanol and then rinsed thoroughly with distilled water. Polymerization was carried out at room temperature resulting a formation of thin membrane with entrapped enzymes. The enzyme membrane was allowed to air dry for four to six hours. Any unbound enzymes were removed by washing the membrane with 0.1M phosphate buffer followed by a distilled water. The enzyme membrane was stored at room temperature for further studies or at 4[degrees]C for longer studies. The enzyme membrane was then incubated with glutaraldehyde a cross-linking agent at different concentrations ranging from 0.25 % to 2.5% for 4-5 hours at room temperature.

Measurement of Enzyme Activities

Cholesterol oxidase

Cholesterol oxidase activity was measured by the method based on the conversion of cholesterol to 4 cholesten-3-one according to the method as described by Allain et.al., (1974). The buffer mixture contained [KH.sub.2] P[O.sub.4] 400mM, KOH 360mM, EDTA disodium salt 2.7mM, 4-hydroxybenzoic acid 24mM, 4-amino anti pyrine 0.7mM, triton X-100 2g/l, methanol 5ml/l and peroxidase 20 unit/100ml. The substrate (cholesterol) was prepared in iso-propanol and tritonX-100. The reaction mixture was prepared by adding 2.5 ml of assay buffer and 0.125 ml of substrate solution. The reaction was started by adding 50 ul of enzyme solution. The red color of 4-cholestene-3-one was measured spectrophotometrically at 500 nm for 5 min. One unit of cholesterol oxidase is defined as the amount of enzyme that converts 1 umol of cholesterol/min at 37[degrees]C.

Cholesterol esterase

Cholesterol esterase activity was measured as described below. In the 3 ml reaction mixture, potassium phosphate 287mM, taurocholic acid 0.05%, peroxidase 4.0 units, cholesteryl oleate 1.4 mM, polyoxyethylene 9 lauryl ether 1.7% (v/v),) 4-amino anti pyrine 0.14% (w/v) and cholesterol oxidase 0.03mg were added. The reaction was started by adding 50ul of cholesterol esterase solution to the reaction mixture and the mixture was incubated at 37[degrees]C. The activity of the enzyme was estimated from the rate of absorbance increase at 500nm. The activity measurement of enzyme membrane was performed under the same condition as the free enzymes.

Preparation of cholesterol solution

Cholesterol was used as a substrate for Cholesterol oxidase. 21mg cholesterol was dissolved in 10ml iso-proponal and 0.4% solution of tritonX-100 in distilled water. The surfactant solution was used because of the poor solubility of cholesterol in water. The cholesterol solution was prepared fresh every day by first dissolving the cholesterol in iso-propanol then adding the detergent. This cholesterol solution was stable for one day, after which the cholesterol started to precipitate.

Preparation of cholestryl acetate solution

Cholestryl acetate was used as a substrate for cholesterol esterase. Cholestryl acetate (8.6mm) was dissolved in one ml of polyoxyethylene 9 lauryl ether by gentle stirring until the solution was clear. Then 9 ml of hot 0.9% (w/v) sodium chloride solution was added to it with continuous stirring and heating gently till the solution was clear and colorless. Different concentrations of known Cholestryl acetate (50mg/dl--400mg /dl) were prepared to study the linearity of this method and stored at 4[degrees]C.

Assay of co-immobilized enzyme membrane

The combined assay of cholesterol esterase and cholesterol oxidase were done by as described by Allain et. al., (1974) with slight modifications. In the reaction mixture instead of free enzyme the immobilized membrane 1x1 Cm was used.

Determination of total cholesterol in serum using enzyme membrane

The total cholesterol in serum was measured using enzyme membrane instead of free enzymes. The serum was pretreated with triton X-100, isopropanol and sodium phosphate buffer and used as a substrate. The same batch of the serum was further analyze by the commercially available Enzo kit.

Results

Properties of Enzyme Membrane; Effect of glutaraldehyde on Enzyme Membrane

The effect of glutaraldehyde concentration on the response of the enzyme membrane was investigated (Figure-1). Due to porous structure of the membrane and long absorption time enzyme membrane responds well without glutaraldehyde but enzyme leaching occur during reaction. It is in fact that, when glutaraldehyde concentration lower than 0.25 % was applied, cross linker amount could be in sufficient to bind all the enzyme molecules on the membrane. While when glutaraldehyde concentration was more than 0.25 %, the excess glutaraldehyde may cause denaturation of the enzyme and the response time may also be increased. We used different concentrations of glutaraldehyde ranging from 0.25 to 2.5 % for the preparation of enzyme membrane. Our results suggested that the optimal concentration was found 0.25 % in the reaction mixture.

[FIGURE 1 OMITTED]

Effect of Temperature on Enzyme Membrane

The influence of temperature on the enzyme membrane was investigated by storing the membrane at different temperatures ranging from 10-80[degrees]C for four hour. The enzymes entrapped in membranes were found to be more thermal stable than native enzymes as shown in the figure-2. It was found that native enzyme lost their complete activity (relative activity) at 40[degrees]C while membrane bound enzyme retained their enzyme activity (relative activity) approximately 60 % at 40[degrees]C and lost all enzyme activity at 70[degrees]C.

[FIGURE 2 OMITTED]

Effect of pH on Enzyme Membrane

To investigate the enzyme membrane response at different pH levels, the enzyme membrane was kept at different pH buffers ranging from pH 3 to pH 10.0 The results suggested that enzymes membrane retained good enzyme activities in between pH 5.0 to pH 9.0 as compared to native enzymes (pH 6-7) as shown in figure-3. The 80-100 % relative activity was observed in membrane bound enzyme at pH 5-9 while in case of native enzymes the 80-100% relative activity was observed at pH 6-7 only.

[FIGURE 3 OMITTED]

Linearity and Stability of Enzyme Membrane

There was a linear relationship was observed when different concentration of cholesteryl acetate was used in the reaction mixture and the color was observe at 520nm (data not shown). The stability of enzyme membrane was investigated by keeping it at room temperature (25[degrees]C) for 60 days. The stability of enzyme membrane was determined in terms of the retention of the enzyme activities. It was found that the COE and COD activities start decreasing after 15 days and retained 50% activities after 60 days at room temperature (Figure-4). This membrane can be reused 50 times showed good application of enzyme membrane.

It is well known that detergents like TritonX-100 play an important role on the enzyme activities. Keeping in view the effect of TritonX-100 was investigated against enzyme activities. It was found that 2% TritonX-100 is sufficient to inactivate the enzyme activities in native form. However, the enzyme membrane was found to be stable up to 5% TritonX-100 concentration (data not shown).

[FIGURE 4 OMITTED]

Applications of enzyme membrane

i. The total cholesterol was estimate in different serum samples by enzyme membrane method and it was compare with commercially available Enzo-kit method using same serum samples (Figure- 5).

ii. The enzyme-membrane prepared in above manner was attached to the oxygen electrode in dissolved oxygen analyzer. When different concentrations of cholesterol were used the current started decreasing as a result of oxygen consumption and finally achieved the stationary phase. Increasing oxygen consuption ([O.sub.2] mg/dl) levels observed with different cholesterol concentration using [O.sub.2] electrode and enzyme membrane. The standard curve showed a good linearity up to 700 mg/dl in cholesterol solution (Figure-6). The minimum detection limit was 50 mg/dl.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Discussion

The method of enzyme immobilization on polymers have been well documented (19, 20 & 21 ). Such type of enzyme membranes have been proposed for the estimation of cholesterol, glucose, urea and uric acid etc. in blood and urine.(22). These enzyme membranes are also useful in long term operation in bioconversions. However, suitable methods for effective enzyme immobilization are lacking. To explore the possibility of a suitable method, it is a necessary that a random screening of matrices and optimized conditions for enzyme immobilization should be carried out. We have selected the strategy that enzymes behaves totally different in organic medium and once they are immobilized they remain active for long times (. Hence, we have selected a polyvinyl formal resin for the immobilization of COE and COD in the presence of organic solvent. The structure of poly vinyl formal resin is R=[C.sub.3][H.sub.7] and having a property of excellent insulation. The individual enzyme activities were increased several folds when they were co-immobilized. On the basis of earlier work (23) it was suggested that cross-linking agent should be used for preparation of enzyme to avoid leaching of enzyme from the membrane. Glutaraldehyde is widely used as a cross linking agent in the immobilization procedures. It is known to be bifunctional compounds which is mainly used in the chemical modifications of proteins and polymers. In the reaction mixture these glutaraldehyde links covalently to the amine groups of lysine or hydrolysine of protein molecules (COD and COE) and creating a more stable structure in the present study an enzyme membrane was prepared COE and COD immobilized on polyvinyl formal resin (Commercial name Formvar) has been developed successfully. The membrane was used for the determination of total cholesterol from serum and results were comparable with commercially available kits. The structure and porosity of the membrane permits easy diffusion between the reaction mixture and electrode surface and vice versa without forming a barrier to the product of enzyme reaction. The enzyme membrane was thin and retained their enzymatic activities without leaching out. The stability of enzyme-membrane at different pH values and temperature was found to be improved to great extent when compared with native enzymes. This enzyme membrane has longer shelf life which may due to its hydrophobic nature. The enzyme-membrane was successfully used in the determination of different cholesterol concentrations and has potential to estimate of total cholesterol in serum/blood. This enzyme membrane has also potential to be used in biosensors and bioreactors.

Acknowledgement

The authors are thankful to Director, Institute of Genomics and Integrative Biology for encouragement and providing necessary facilities to carry out the work.

References

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[2] Clark D.S., 2004. Characteristics of nearly dry enzyme in organic solvents: Implications for biocatalyst in absence of water. Phill. Trans. R. Soc. Lond. B, 359, 1299.

[3] Giorno L. and Drioli E., 2000. Biocatalytic membrane reactor : application and prespective. TIBTECH 18, 339-348.

[4] Sandeep K.D., and Baltus R.E.,1992. Distribution of immobilized enzymes on porous Membranes, Biotechnol Bioeng. 40 1173-1180

[5] Lin C.C. and Yang M.C., 2003. Cholesterol oxidation using hollow fiber dialyzer immobilized with cholesterol oxidase: preparation and properties, Biotechnol. Prog. 19: 361--364

[6] Jung B., 2004. Preparation of hydrophilic polyacrylonitrile dialysis blend membranes for ultrafiltration, J. Membr. Sci. 229:129--136

[7] Fernandez-Romero J.M., MDL de Castro, Valcarcel, 1987. Determination of total cholesterol in serum by flow injection analysis. Clin. Chem. Acta 167: 97-104

[8] Kumar Hemant, Ashok Kumar, Poonam Kumari, Jyotirmai Savita, Tulsani N.B., 1999. Immobilization of cholesterol oxidase on formvar using organic solvents. Biotechnol. Appl. Biochem. 30: 231-233.

[9] Levy R.I., 1981. Cholesterol, lipoproteins , apoproteins and heart disease present status and future prospectus. Clin. Chem. 27: 5, 653-662.

[10] Millar G.J., 1980. High density lipoprotein and atherosclerosis. Ann. Rev. med. 31: 97-108.

[11] Admirand W. H. and Small D.M., 1968. Physiochemical basis of cholesterol gall stone formation in Man . J.Clin.Invest. 47: 1043-1052

[12] Peter F. and Reynolds R.G., 1977. Quantitative analysis of human serum cholesterol by thin layer chromatographic spot test, J. Chromatogr. 143 577-97. 285

[13] Kinter M., D.A. Herold, J. Hundley, M.R. Wills and Savory J., 1988.Measurement of cholesterol in serum by gas chromatography / mass spectrometry at moderate mass resolution with a nonendogenous cholesterol isomer as internal standard, Clin. Chem 34: 531-534.

[14] Takatsu W, and Nishi A.D.S., 1987. Total cholesterol in serum determined by isotope dilutions / mass spectrometry with liquid chromatographic separation. Clin. Chem 33: 124-127.

[15] Allain C,C., L.S. Poon, C.S.G.Chan, and Richmond W., 1974. Enzymatic determination of total serum cholesterol, Clin. Chem. 20: 470-475.

[16] Brahim S., D. Nariresingh, A. and Elie G, 2001. Amperometric determination of cholesterol in serum using a biosensor of cholesterol oxidase contained within a polypyrrole--hydrogel membrane, Anal. Chim. Acta 448: 27--36.

[17] Singh S., A. Chaubey, and Malhotra B.D., 2004. Amperometric cholesterol biosenor based on immobilized cholesterol esterase and cholesterol oxidase on conducting polypyrrole film, Anal. Chim. Acta 502: 229--234

[18] Tan X ,M. Li, P. Cai, L. Luo, and Zou X., 2005. An amperometric cholesterol biosensor based on multi walled carbon nanotubes and organically modified sol--gel/ chitosan hybrid composite film, Anal. Biochem. 337: 111--120.

[19] Chang TMS, (ed) 1977. Biomedical application of immobilized enzymes and proteins. Plenum Press new Plenum Press New York

[20] Karube I, Hara H, Mastsuoka and Suzuki. 1982. Amperomatric determination of total cholesterol in serum with use of immobilized cholesterol ester hydrolase and cholesterol oxidase. Clin. Chem. Acta 139: 671-76.

[21] Murachi T and Sakaquchiy Y., 1980. Application of immobilized enzymes to clinical analysis ; use of coimmobilized glucose oxidase and peroxidase in column. Biochimita. 62 (8-9): 581-585.

[22] Trevan MD., 1980. Immobilized enzymes : an introduction and applications in biotechnology (ed) 14-53. John Willey & Sons. 287

[23] St. Clair NL, and Navia MA, 1992. Cross-linked enzyme crystals as robust biocatalysts. J.Am.Chem. Soc. 114: 7314-7317.

Hemant K. Gautam * and Ashok Kumar

Institute of Genomics and Integrative Biology (CSIR) Mall Road, Delhi University Campus Delhi- 110007 India * Email: hkumar65@yahoo.com
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Author:Gautam, Hemant K.; Kumar, Ashok
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Date:Sep 1, 2008
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