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CHAPTER 4

RESULTS AND DISCUSSION ON CO-IMMOBILIZATION

OF CHOLESTEROL ESTERASE

4.1 CO-IMMOBILIZATION OF CHOLESTEROL ESERASE

This chapter presents the results and discussion on the

co-immobilization of cholesterol esterase, cholesterol oxidase and peroxidase.

These enzymes obtained from commercially available cholesterol diagnostic

kit, were co-immobilized on to different matrices by different coupling

methods. The stability, activity of the co-immobilized enzymes and their

applications to clinical samples were evaluated. The different matrices used

for co-immobilization include (i) nylon-6,6 beads (ii) gelatin film and

(iii) porcelain beads. The coupling reagents employed for different matrices

include (i) glutaraldehyde and (ii) ascorbic acid.

4.2 ASCORBIC ACID MEDIATED CO-IMMOBILIZATION OF

CHOLESTEROL ESTERASE ON POLYAMINO MATRICES

Co-immobilization of enzymes on polyamino matrices of nylon-6,6

beads and gelatin film coated on cellulose acetate polymeric membrane was

carried out using ascorbic acid coupling as per the procedure described in

Chapter 2 (2.6.1). The co-immobilization of enzymes was found to be

successful on both the matrices. The reaction that occur on treating the amino

matrix with ascorbic acid solution may be represented as the

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bifunctional reaction of the ascorbic acid with amino compounds can be

transferred to enzyme immobilization on support materials containing NH2

groups.

The pathway of enzyme immobilization by ascorbic acid is

discussed in chapter 3.2. The possible mechanism was based on the

assumption that the bifunctional reaction of the ascorbic acid with amino

compound can be utilized for the co-immobilization of enzymes on support

materials containing NH2 groups.

4.3 GLUTARALDEHYDE MEDIATED CO-IMMOBILIZATION

OF CHOLESTEROL ESTERASE ON POLY AMINO

MATRICES

The cross-linking of enzymes with glutaraldehyde involves the

reaction between the bifunctional reagent and the residual free amino groups

of the enzymes are discussed in chapter 3.3. The linkages formed between the

glutaraldehyde and amino groups are irreversible and can survive extreme pH

and temperature conditions.

4.4 PROTEIN CONTENT OF REAGENT 1 OF CHOLESTEROL

DETERMINATION KIT

Protein estimation from the source Reagent 1, cholesterol diagnostic

kit was carried out by Lowry method as described in section in section 2.2.

The experimental data obtained is given in Table 3.3 and the Beer –

Lambert’s plot is shown in Figure 3.5. The protein content of cholesterol

diagnostic kit containing enzymes was found to be 9 mg / mL.

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4.5 CO-IMMOBILZATION OF ENZYMES ONTO VARIOUS

MATRICES

Cholesterol esterase, cholesterol oxidase and peroxidase from

Commercially available Reagent 1 of cholesterol diagnostic kit was

co-immobilized on to nylon-6,6 beads, gelatin coated photographic film

through glutaraldehyde and ascorbic acid coupling and porcelain beads by

physical adsorption showed yield of 1.3, 1.2, 1.5, 1.4 and 1.7 mg protein / gm

support which is comparable with that of data reported for co-immobilization

of cholesterol esterase, cholesterol oxidase onto alkylamine glass (Suman and

Pundir 2003).

These methods has the advantage over other methods employing

individually immobilized and co-immobilized cholesterol esterase and

cholesterol oxidase enzymes on matrices (Tabata et al 1981, Malik and Pundir

2002) that it is economical, less time consuming and more effective, as the

method require less amount of support, chemicals used for immobilization

and saves the time required for immobilization of the enzymes individually.

Further all the three enzymes have been co-immobilized on to the same

support, which avoids the transport of substrate between the support

molecules. The product of first enzyme (Cholesterol esterase) act as the

substrate of second enzyme (Cholesterol oxidase) and similarly the product of

second enzyme acts as the substrate of third enzyme (Peroxidase) on the same

support molecule. This would increase the efficiency of co-immobilized

system compared to that employing individually immobilized enzyme (Suman

and Pundir 2003) (Table 4.1).

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The co-immobilization of cholesterol esterase onto gelatin coated

photographic film resulted in a higher conjugation yield (1.2 mg/g of support)

with a higher retention of initial activity (62 %) than that of nylon-6,6 bead by

glutaradehyde coupling was found to be (1.2 mg/g of support) with retention

of (50 %) initial activity. But Porcelain bead was found to have higher

retention of initial activity (70 %) than the other immobilization methods.

4.6 CHOLESTEROL ESTERASE ASSAY

The commercially available cholesterol diagnostic kit was used as a

source for the estimation of cholesterol by Alliain’s method, using cholesterol

acetate as substrate as described in section 2.4. The combined cholesterol

esterase activity value was found to be 3.5 mg/mL.

4.7 COMPARISON OF EFFICIENCY OF CO-IMMOBILIZATION

The percentage immobilization varied with the matrix. Physical

adsorption of enzyme on porcelain beads was able to retain 72% of the native

enzyme activity on co-immobilization; while glutarladehyde coupled

nylon-6,6 beads retained 58 % of the activity. Ascorbic acid activated of

gelatin film was able to retain 67% of the native enzyme activity on

co-immobilization while glutaraldehyde coupled gelatin film retained 65 % of

its activity. The percentage of co-immobilization of enzymes by different

methods on various matrices is shown in Table 4.2.

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77

4.8 STORAGE STABILITY OF CO-IMMOBILIZED ENZYMES

ON DIFFERENT MATRICES

The long-term stability of co-immobilized enzymes on various

matrices were investigated. The enzymes activities were found almost to be

same as the initial activities up to 10 days. After 10 days the activity of

enzyme stored at 4°C started to decrease with time, and almost no activity

was observed on the 50th day. In a different condition, at room temperature,

the co-immobilized enzymes were found to be stable up to 10 days and

retained almost 50% activity even after 30 days, when compared to native

enzymes. Co-immobilized enzymes on nylon-6,6 beads stored in 50 mM

phosphate buffer (pH 7) at 4°C showed practically no leaching of the enzymes

for both the matrices retaining 70% activity over a period of 30 days for

nylon-6,6 beads, while in the case of gelatin film the co-immobilized enzymes

were able to retain 90% of its activity under the same conditions for a period

of 15 days (Table 4.3).

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4.9 EFFECT OF pH ON CO-IMMOBILIZED CHOLESTEROL

ESTERASE ACTIVITY

An optimum pH range for free enzyme activity was found between

pH 6 and 7, while co-immobilized enzymes was found to display activity over

a broad pH range of pH 5 and 8 for most of the matrices. In the case of

cholesterol esterase co-immobilized on nylon-6,6 beads and gelatin film by

glutaraldehyde coupling the maximum activity was found to occur between

pH 6 and 8 while the co-immobilized enzymes on ascorbic acid activated

nylon-6,6 beads and gelatin film exhibited maximum activity over a pH range

of 5 to 8, as shown in Table 4.4 and Figure 4.1. The activity of the free

enzymes was found to decrease by about 50% at pH 5 and 8, while its

maximum activity was exhibited at optimum pH range of 6-7. In contrast the

co-immobilized enzymes showed maximum activity over a wide pH range

investigated (5-10) and the activity was found to decrease only by 20% at low

or higher pH values. Similar enhanced pH stability has been reported for

several covalently bound co-immobilized cholesterol esterase (Suman et al

2006).

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81

0

1.5

3

4.5

5 6 7 8 9 10pH

1 2 3 4 5 6

Figure 4.1 Effect of pH for free and co-immobilized enzymes activity,

(mg / mL)

1. Free enzymes 2. Glutaraldehyde coupling on nylon-6,6 beads 3. Ascorbic

acid coupling on nylon-6,6 beads 4. Glutaraldehyde coupling on gelatin film

5. Ascorbic acid coupling on gelatin film 6. Porcelain beads.

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4.10 EFFECT OF TEMPERATURE ON CO-IMMOBILIZED

CHOLESTEROL ESTERASE

The co-immobilized enzymes was found to be relatively more stable

than the free enzymes over a wider range of temperature as shown in

Table 4.5 and Figure 4.2. The optimum temperature range for free enzymes

was found to be 30-40 oC compared to that of co-immobilized enzymes whose

temperature optimum is 20 to 50 oC. The free enzymes activity was found to

decrease by about 70% at 10oC and at 60oC from its maximum activity at the

optimum temperature of 30 oC. The loss in activity of the co-immobilized

enzymes was only 20%. Similar enhanced thermal stability has been reported

for several covalently bound co-immobilized cholesterol esterase (Suman et al

2006).

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84

0

1.5

3

4.5

10 20 30 40 50 60 70Temperature, oC

1 2 3 4 5 6

Figure 4.2 Effect of temperature on free and co-immobilized enzymes

activity, mg / mL

1. Free enzymes 2. Glutaraldehyde coupling on nylon-6,6 beads 3. Ascorbic

acid coupling on nylon-6,6 beads 4. Glutaraldehyde coupling on gelatin film

5. Ascorbic acid coupling on gelatin film 6. Porcelain beads.

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4.11 EFFECT OF CO-IMMOBILIZATION ON KM

Enzymes co-immobilized on porcelain beads showed an apparent

Km value of 30 mM, which is higher than the Km value of soluble enzyme

(20 mM). This may be explained on the basis of a concentration gradient of

substrate established across the ‘Nernst layer’, an unstirred layer of solvent

surrounding the suspended matrix particles. Consequently, saturation of an

immobilized enzyme molecule occurs at a higher substrate concentration than

normally required for the saturation of the freely soluble enzyme and hence a

greater Km value, these are discussed in chapter 3.13 (Table 4.6).

Table 4.6 Effect of cholesteryl acetate concentration on enzymes

co-immobilized on porcelain beads

S.No Concentration of cholesteryl acetate,mM Activity, mM

1 2.5 2.4

2 5.0 4.6

3 7.5 7.1

4 10.0 9.2

5 12.5 10.9

6 15.0 11.2

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The apparent Michaelis - Menten constant (Km) and maximal

velocity (Vmax) for co-immobilized enzymes were calculated using the initial

activity data according to the Lineweaver-Burk plot Figures 4.3-4.4 and

Table 4.7. The Km values revealed that co-immobilized enzymes needed

higher cholesteryl acetate concentration to reach the maximal activity than the

free enzyme.

0

4

8

12

0 2.5 5 7.5 10 12.5 15Conc. of Cholestryl acetate, mM

Figure 4.3 Effect of substrate concentration on enzymes

co-immobilized on to porcelain beads

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y = 1.0084x + 0.0155

-0.1

0

0.1

0.2

0.3

0.4

-0.05 0.05 0.15 0.25 0.35 0.45

1 /S

Figure 4.4 Line weaver-Burk plot for co-immobilized enzymes on porcelain beads

Table 4.7 Data for transformation of Michaelis – Menten curve

S.No 1 / Conc. of cholesteryl acetate in mM

1 / Activity in mM

1 0.4 0.42

2 0.2 0.22

3 0.13 0.14

4 0.1 0.11

5 0.08 0.092

6 0.06 0.089

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4.12 ESTIMATION OF BLOOD CHOLESTEROL

Blood cholesterol levels in healthy male and female individuals of

different age groups (10-20, 20-30, 30-40 and 40-50) were measured using

co-immobilized enzymes which was ranged from 142 to 275 mg/dL with a

mean of mg/dL (n = 30). The established normal range was 150 - 250 mg/dL

with a mean of 189 mg/dL (n = 30). The mean and standard deviations for

enzymes in the total group screened were 189±40 mg / dL (Figure 4.5).

0

0.05

0.1

0.15

0.2

Cholestryl acetate (mg / dL)

Figure 4.5 Standard curve for the total cholesterol concentrations on

co-immobilized cholesterol esterase

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4.12.1 Linearity and Detection limit

Linearity between cholesterol acetate concentration and absorbance

at 520 nm was obtained between 60 and 370 mg/dL for the co-immobilized

enzymes. The minimum detection limit of the present method is 60 mg/dL,

which is comparable with that of those reported earlier using co-immobilized

enzymes 50 mg/dL (Suman and Pundir 2003).

4.12.2 Recovery

The percentage of added cholesterol acetate in serum to imitate

hyper cholesterolaemia (50 and 100 mg /dL) by the present method was

94.2 ± 6.1 and 96.2± 4.3 (Mean ± Standard deviation) (n = 5) (Table 4.8),

which is comparable with that of GLC method where 98-102 % was observed

for the added cholesterol concentration of 50 and 100 mg/ dL (Blomhoff

1973) and 88.68 – 88.97% for added cholesterol concentration of 100 and

200 mg/ dL (Suman et al 2003).

Table 4.8 Analytical recovery of added cholesterol in serum using

co-immobilized enzymes

Cholesterol added (mg/dL)

Cholesterol found (mg/dL)

Recovery (%) (Mean ± SD) (n = 5)

None 220 --

50 263 94.2 ± 6.1

100 312 96.2± 4.3

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4.12.3 Precision and reproducibility

In order to assess the reproducibility and reliability of the method,

the total cholesterol level of the same serum sample in one run (within a day

variation) and after one week of storage (in between day variation) were

determined (Table 4.9). The C.V. for total cholesterol determination in serum

for the above by the present method were <1.5% and <3.0% respectively

which is much comparable to those of immobilization of enzymes employing

nylon mesh (Mascini et al 1983).

Table 4.9 Precision data of total serum cholesterol by co-immobilized

enzymes

Parameter studied Total cholesterol by co-immobilized enzymes

Within a day (n =5)Mean (mg / dL) C.V. (%) S.D. (mg / dL)

184.51.080.02

Between days (n =5) Mean (mg / dL) C.V. (%) S.D. (mg / dL)

226.62.65.9

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4.12.4 Correlation

To evaluate the accuracy of the present method, total cholesterol

levels of 15 serum samples, were determined by the nylon-6,6 beads

employing co-immobilized enzymes (y) was compared with commercial

enzymes kit (x) employing free cholesterol esterase, cholesterol oxidase and

peroxidase. Total cholesterol values in serum obtained by other methods

showed good correlation (r = 0.99) (Figure 4.6), which is comparable with

the method of Suman and Pundir (2003). The correlation coefficient ‘r’ was

found to be 0.83 which is comparable to the method of Abell et al (1952) (x),

the correlation coefficient ‘r’ being 0.99 and with Boehringer Mannheim

enzymatic method (x) ‘r’ being 0.98 (Majkic and Berkes1977).

4.12.5 Interference study

To test the possible interference by various metabolites found in

serum, the following compounds were added in above normal serum level in

the reaction mixture and at their respective physiological concentrations:

glucose, citrate, vitamin C, vitamin D, hemoglobin, albumin, sodium

bicarbonate, urea, uric acid and creatinine. Of these above mentioned

compounds, none of these metabolites had much effect, except vitamin C,

vitamin D and albumin caused inhibition on the co-immobilized enzyme

system, and was to be following similar with the reports earlier (Suman and

Pundir 2003). The inhibition of these metabolites on the co-immobilized

enzyme system was found to be 38 %, 40 % and 30 %.

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y = 0.9853x - 2.6912R2 = 0.9857

0

70

140

210

280

350

0 70 140 210 280 350

Total cholesterol (mg/dl) by kit method

Figure 4.6 Correlation between blood total cholesterol values

determined by readymade cholesterol kit and

co-immobilized enzymes

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4.13 CONCLUSION

The co-immobilized enzymes described in the present study have

been shown to retain its activity for various matrices. The study on the

co-immobilization of enzymes on different coupling methods ascorbic acid or

glutaraldehyde and physical adsorption for porcelain beads matrix were

evaluated. This result showed higher coupling efficiency of enzymes on

nylon-6,6 beads and gelatin coated photographic film than the glutaraldehyde

coupling. The percentage immobilization varied with the method / matrix.

Porcelain bead by physical adsorption found to show higher retention of

initial activity 72% than the other co-immobilized cholesterol esterase

methods.

Ascorbic acid coupling of gelatin film was able to retain higher

enzyme activity on immobilization than glutaraldehyde coupling.

Co-immobilized enzyme on porcelain beads stored in 50 mM phosphate

buffer (pH 7) at 4°C showed very less leaching of the enzymes retaining the

activity over a period of 50 days, while co-immobilized enzymes coupled to

nylon-6, 6 beads and gelatin film stored under the same conditions was stable

over a period of 30 days. The co-immobilized enzymes were more stable than

the free enzymes over wide pH range. The co-immobilized enzymes were also

more stable than the free enzyme over a wide temperature range. Porcelain

beads co-immobilized enzymes had a higher Km (36 mM) than the soluble

enzyme (29 mM). Coefficients of variation within day and between

successive days were <1.5% and <3%, respectively. A good correlation

(r=0.99) was found between the total serum cholesterol obtained by the

present method and commercial available kit method employing free

enzymes. Among the various serum substances tested at their physiological

concentrations had much no effect, except vitamin C, vitamin D and albumin

which caused 38%, 40% and 30% inhibition of the co-immobilized enzyme

system respectively.