chapter 4 results and discussion on co-immobilization...
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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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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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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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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.