full report part c.docx
TRANSCRIPT
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Summary
Regarding to this study, it was done to prepare immobilized enzyme by gel entrapment
method. In this experiment, sodium alginate was used as the medium for the gel entrapment due
to purification of enzyme. To achieve the objective, several steps have been done. First of all, the
sodium alginate was dissolved in water to make a 3% solution. When it is completely dissolved,
the solution was leaved undisturbed for 30 minutes to eliminate the air bubble. Then, it was
mixed with the 3% sodium alginate solution. In this step, it is essential to have the same ratio for
both enzyme and sodium alginate solution. The mixture was then being leaved for 15 minutes
after 5 minutes of stirring. In order to obtain the beads, the polymer solution was dripped from an
approximate high into an excess of stirred calcium chloride solution by using a syringe and a
needle at room temperature. The size of beads obtained was controlled by pump pressure and the
needle gauge. Based on the result, the size of beads obtained is in the range of 1.2 2 mm in
diameter. Then, it was being cured up to 16 hours. Before stored in pH 5.9 buffer solution at 4oC,
it was being rinsed with sterile distilled water. In this experiment, it is also to study the effect of
substrate concentration, pH and temperature on activity of free and immobilized enzyme. In term
of substrate concentration, the preparations was assayed with free and immobilized enzyme at
different soluble starch concentration in the range of 1 5 (%w/v) a 50oC. Enzyme assay
procedure has been used for this step and different enzyme activity has been determined at
different substrate concentration. The value of Km and Vmax for each enzyme has been
determined, which is the value of Km for both enzymes are 0.0088 and 0.4853 respectively. In a
meanwhile, the value of Vmax for both enzymes is 0.098and 0.149respectively. Based on the
result, the enzyme activity for both immobilized and free enzyme has shown an increasing
reading as the concentration is increasing. For determination of effect of pH on enzymes, the
preparations were assayed at different pH values in the range from 4.5 to 9.3 at 50oC. The result
has shown a fluctuated pattern of the enzyme activity, which is it was increasing and start to
decrease at optimum point. Apart from that, the effects of temperature on both types on enzymeswere determined at temperatures of 30
oC to 70
oC under assay conditions. As roughly, based on
the result, immobilized enzyme activity is increased, while free enzyme activity is decreased.
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Objectives
1. To determine the average size of beads, initial activity of free and immobilized enzyme.
2. To determine the values for Km, Vmax for both free and immobilize enzymes.
3.
To study the effect of substrate concentration, pH and temperature on activity of free and
immobilized.
Procedure
3g of Na (Alginate)was dissolved in
distilled water to makea 3% solution.
It is being leavedundisturbed for 30
minutes.
The enzyme wasmixed with the 3% Na
(Alginate) solution.
By dripping thepolymer solution, the
beads are formed.
The beads were leavedin the calcium solution
to be cured.
After that, the size of
beads were measured.
The immobilized enzymeswere rinsed with sterile
distilled water and was storedin pH 5.9 buffer solution at
4oC
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Effect of Substrate Concentration on Immobilized versus Free Enzyme
Effect of pH on immobilized versus enzyme
Effect of temperature on immobilized versus free enzyme
The preparations wereassayed with free andimmoblized enzyme atdifferent soluble starch
concentration..
Amount of enzymeused per assay is
o.25mL and 0.5g forfree and immobilizedenzyme respectively
Enzyme activity atdifferent substrateconcentration is
measured
The preparations was assayed atdifferent pH values at 50oC
Different temperature is used from 30oC to70oC under assay conditions (1% (w/v)
soluble starch as substrate solvated in 10mMcitrate buffer, pH 6)
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Result
In order to determine substrate utilization constant, Km and maximum velocity of
enzymatic reaction, Vmax, the rate of glucose produced are plotted against substrate
concentration according to LineWeaver Burksequation (Table 1, Figure 1, Figure 2 and
Figure 3). From the graphs plotted it was determined the Km and Vmax for free enzyme are
0.0088 and 0.098 respectively. Meanwhile for immobilized enzyme the Km and Vmax
values are 0.4853 and 0.0149 respectively. For the effect of substrate concentrations, pH
and temperature, OD575nm reading obtained were converted into amount of glucose
present by referring to glucose calibration curve prepared. Enzyme activities for every data
were then calculated using formula attached in the Appendices. The data for those
parameters were shown in their respective tables and figures; substrate concentration
(Table 2, Figure 4), pH (Table 3, Figure 5) and temperature (Table 4, Figure 6).
Determination of Kmand Vmax
Free Enzyme Immobilized Enzyme
Km 0.0088 0.4853
Vmax 0.098 0.149
Table 1 Reaction Kinetics for Free vs. Immobilized Enzyme under Various Substrate
Concentrations
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Substrate Concentration
Substrate
Concentration
Free Enzyme
(Enzyme Activity, U)
Immobilized Enzyme
(Enzyme Activity, U)
5% 0.354 0.1874% 0.347 0.12
3% 0.3 0.083
2% 0.2 0.07
1% 0.11 0.033
Table 2 Enzyme Activities for Free vs. Immobilized Enzyme under Various Substrate
Concentrations
pH
pHFree Enzyme
(Enzyme Activities, U)
Immobilized Enzyme
(Enzyme Activities, U)
5 0.234 0.227
6 0.2 0.2
7 0.41 0.51
8 0.183 0.384
9 0.207 0.04
Table 3 Enzyme Activities for Free vs. Immobilized Enzyme under Various pH
Temperature
TemperatureFree Enzyme
(Enzyme Activity, U)
Immobilized Enzyme
(Enzyme Activity, U)
30 0.294 0.19
40 0.484 0.19
50 0.183 0.25
60 0.19 0.173
70 0.107 0.033
Table 4 Enzyme Activities for Free vs. Immobilized Enzyme under Various Temperatures
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Discussion
In this part of study, titled Immobilizationof Amylase Enzyme by Gel Entrapment,
Enzyme kinetics differences between free suspending enzymes and immobilized enzymes
were studied. Enzymes purified from the previous part are entrapped in sodium alginate
forming beads which average at about ~1mm in diameter. Immobilization of enzymes or
cells was done to stabilize the enzymes, increasing its strength to withstand mechanical
forces usually used in the industry (Brena and Batista-Viera, 2006).
The first parameter that was studied in this part is the differences in V maxand Km
value between the two forms of enzymes. The Vmax and Km values were calculated usingHenris equation assuming that the appearance of P is linear with time during the assay
procedure, and no more than 5% of the substrates is utilized (Pandey, 2006). In Figure 1,
Vmax and Kmvalue for free suspending enzymes are 0.098 and 0.0088 respectively while for
immobilized enzymes (Figure 2), the Vmaxand Kmvalue are 0.149and 0.4853 respectively.
This indicates that from being free to being immobilized, the maximum rate achieved by
the enzyme at maximum substrate concentration increases by 52% and K m, substrate
concentration when V=Vmax/2 increases significantly
In Figure 3, where direct comparisons are being made between free suspending and
immobilized enzyme, it is observed that Vmaxvalue does not change significantly in contrast
to the Kmvalue. As Vmaxchanges, Kmare affected too and studies by Sung and Bae, (2003)
immobilization of the enzyme may alter the Km value. Vmaxvalue should be, according to
theory may decrease a little caused by improper location of enzyme in the matrix that may
block its active site from substrates.
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Figure 1 Free Enzyme Vmaxand KmDetermination
Figure 2 Immobilized Enzyme Vmaxand KmDetermination
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Figure 3 Comparisons of Vmaxand KmValues between Free and Immobilized Enzymes
In terms of enzyme activities, as shown in figure 4, free suspending enzymesrecorded higher activities than immobilized enzyme. The highest enzyme activities for free
enzyme are 0.354 U while for immobilized is only 0.187 U. Every substrates concentrations
recorded higher enzyme activities in free suspending enzymes than immobilized enzymes.
As immobilized enzymes sediment at the bottom of the test tubes, it decreases the amount
of interactions between the enzymes and the substrates. Similarities in the two graphs
show that increment of the substrates concentration, increases enzyme activities. For the
free suspending enzymes, the slope declines at the end, indicating that almost all of the
enzymes are occupied with substrates and increasing the substrate concentrations would
not speed up the process. Immobilized enzymes at the other hand, does not indicates
declination at 5% substrate concentrations. It can be said that the enzymes does not
working at maximum rate because in theory, Vmaxbetween free and immobilized enzyme
would not have a large difference (Li et al, 2010).
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Figure 4 Effect of Substrate Concentration on Free vs. Immobilized Enzyme Activities
In Figure 5, it shows that both free enzyme and immobilized enzyme shares the
same optimum working pH at 6.9. The optimum pH for -amylase is between neutral
region of 6.5 to 7.5 which is the same as body pH. Both enzyme shows low activity under
acidic and basic environment. At basic pH, immobilized enzyme recorded higher activity
than free suspending enzyme possibly because the basic properties of sodium alginate.
Acidic environment may interrupt the charge in the gel making it burst and releasing the
enzymes thus denaturing it respectively. Under acidic conditions, properties of
immobilized enzymes are similar to free suspended enzymes.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0% 1% 2% 3% 4% 5% 6%
EnzymeActivity,U
Substrate Concentration
Effect of Substrate Concentration on Free
vs Immobilized Enzyme Activity
Free Enzyme
Immobilized Enzyme
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Figure 5 Effect of pH on Free vs. Immobilized Enzyme Activities
In Figure 6, it can be seen that by immobilizing the enzyme, the optimum
temperature of the can be increased. Optimum temperature of immobilized enzymes is
50C while for free suspending enzymes is 40C. The gel surrounding the enzyme may
provide stability to the enzyme from denaturation at high temperature. Under extremely
high temperature for enzymes (60C and 70C), both forms of enzymes recorded low
enzymatic activities. This conforms to previous studies that state immobilization can alter
not only stability but also the pH optimum, temperature optimum, energy of activation,
linear range and Kmvalue.
0
0.1
0.2
0.3
0.4
0.5
0.6
4.5 5.7 6.9 8.1 9.3
EnzymeActivity,U
pH
Effect of pH on Free vs Immobilized
Enzyme Activity
Free Enzyme
Immobilized Enzyme
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Figure 6 Effect of Temperature on Free vs. Immobilized Enzyme Activities
0
0.1
0.2
0.3
0.4
0.5
0.6
30 40 50 60 70
EnzymeActivity,U
Temperature (C)
Effect of Temperature on Free vs
Immobilized Enzyme Activity
Free Enzyme
Immobilized Enzyme
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Conclusion
This experiment, which is lie under three different objectives has been successfully
conducted. Immobilization that has been applied in this experiment was gel entrapment. While
preparing immobilized enzyme, calcium alginate has been used as the gel media. The sizes of
beads obtained were in the range of 1.2-2.2 mm. This experiment has been conducted due to
determination of the effect of various parameters (pH, substrate concentration and temperature)
on the enzyme activity for free and immobilized enzyme. In term of substrate concentration, the
enzyme kinetic on the value of Km and Vm has been calculated. Immobilized enzyme has higher
value of Km and Vm. The result has shown that, the activity of immobilized enzyme was rapidly
increased as the product concentration increased compared to free enzyme. Enzyme activity
increases with an increase in product concentration as there are more random collisions between
the substrate and the active site. On the other hand, for pH effect, the highest point is where the
optimum pH to have highest enzyme activity, which is at pH 6.9 for both enzymes. However,
free enzyme has shown it was denatured much faster than immobilized enzyme. Immobilized
enzyme can stand longer as higher pH. In term of temperature effect, immobilized has higher
optimum temperature compared to free enzyme. This is because the immobilized enzyme has
much stable molecule and can resist higher temperature. From this experiment, it can be
concluded that immobilized enzyme has more advantages on the efficiency of the reaction.
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References
Brena, B. M., & Batista-Viera, F. (2006).Immobilization of enzymes.InImmobilization of
enzymes and cells(pp. 15-30). Humana Press.
Pandey, A. (Ed.). (2006). Enzyme technology.Springer.
Sung, W. J., &Bae, Y. H. (2003). A glucose oxidase electrode based on polypyrrole with
polyanion/PEG/enzyme conjugate dopant. Biosensors and Bioelectronics, 18(10), 1231-
1239.
Li, Y., Gao, F., Wei, W., Qu, J. B., Ma, G. H., & Zhou, W. Q. (2010). Pore size of macroporous
polystyrene microspheres affects lipase immobilization.Journal of Molecular Catalysis B:
Enzymatic, 66(1), 182-189.
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Appendices
1.
Amylase Assay
a.
Glucose Solution Standard Curve for Amylase Assay
Glucose Concentration (mg/L) Optical Density (OD575nm)
0 0.000
50 0.010100 0.011
150 0.018
200 0.023
300 0.031
400 0.045
y = 0.1114x
0
0.01
0.02
0.03
0.04
0.05
0 0.1 0.2 0.3 0.4 0.5
OpticalDensity(575nm)
Concentration (g/L)
Glucose Solution Standard Curve
Optical Density
(575nm)
Linear (Optical Density
(575nm))
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b.
Kmand VmaxDetermination
Substrate [S]
Free Enzyme
Velocity (v),
g.l-1min-1
Immobilized
Enzyme Velocity
(v) g.l-1min-1
1/[S]
1/v
Free
Enzyme
1/v
Immobilized
Enzyme
5%0.955 30
= 0.0318
0.505 30
= 0.0168
10.05
= 20
10.0318
= 31.45
10.0168
= 59.53
4%0.937 30
= 0.0312
0.324 30
= 0.0108
10.04
= 25
10.0312
= 32.05
10.0108
= 92.59
3%0.811 30
= 0.0270
0.225 30
= 0.0075
10.03
= 33.33
10.0270
= 37.04
10.0075
= 133.33
2% 0.541 30
= 0.0180
0.189 30
= 0.0063
10.02
= 50
10.0180
= 55.56
10.0063
= 158.73
1%0.297 30
=0.0099
0.090 30
= 0.0030
10.01
= 100
10.0099
= 101.01
10.0030
= 333.33
Free Enzyme Immobilized Enzyme
y = 0.0903x + 10.16
1/Vmax= 10.16
Vmax= 0.098
Km/Vmax= 0.0903
Km= 0.0088
y = 3.258x + 6.714
1/Vmax= 6.714
Vmax= 0.149
Km/Vmax= 3.258
Km= 0.4853
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c.
Glucose Produced in Samples in the Effect of Substrate Concentrations on
Free vs. Immobilized Enzyme
Substrate Concentration Free Enzyme
(Glucose Concentration)
Immobilized Enzyme
(Glucose Concentration)
5% 0.955 0.505
4% 0.937 0.324
3% 0.811 0.225
2% 0.541 0.189
1% 0.297 0.090
d.
Glucose Produced in Samples in the Effect of pH on Free vs. Immobilized
Enzyme
pH Free Enzyme
(Glucose Concentration)
Immobilized Enzyme
(Glucose Concentration)
4.5 0.631 0.613
5.7 0.541 0.541
6.9 1.108 1.378
8.1 0.495 1.036
9.3 0.559 0.108
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e.
Glucose Produced in Samples in the Effect of Temperature on Free vs.
Immobilized Enzyme
Temperature Free Enzyme (Glucose
Concentration)
Immobilized Enzyme
(Glucose Concentration)
30C 0.793 0.514
40C 1.306 0.514
50C 0.495 0.676
60C 0.514 0.468
70C 0.288 0.090
f.
Enzyme Activity Calculation Example (Free Enzyme, 5%)
Glucose Concentration = 0.955 g/L 180 g/gmol = 0.0053 mol/L = 5306 mol/L
Enzyme Activity, U =oncentration of Product (molL olume of ssay (L
ssay Incubation Period (min
Enzyme Activity, U =molL L
min= 0.354 U