Full Report Part C.docx

8/10/2019 Full Report Part C.docx

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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


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


Immobilized Enzyme


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


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


Immobilized Enzyme


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


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

Documents

Full Report Part C.docx