7.

0 RESULTS

GLUCOSE STANDARD CURVE:

Table 7.1: The values of absorbance Optical Density at different glucose concentration.

Glucose concentration (g/L) Absorbance Optical Density (OD) (nm)

0 0
600 3.57
700 2.98
900 4.87

6
Absorbance Optical Density (OD)(nm)

5
f(x) = 0.01 x + 0.01
4 R² = 0.95

0
0 100 200 300 400 500 600 700 800 900 1000

Glucose concentration (g/L)

Figure 7.1: The standard curve of absorbance Optical Density (OD) (nm) against glucose
concentration (g/L)
EFFECT OF TEMPERATURE:

Table 7.2: The values of Absorbance Optical Density (OD) (nm) at different temperature.

Temperature (oC) Absorbance Optical Density (OD)

30 8.72

40 7.46

50 9.05

60 8.65

70 9.78

12

10
Absorbance Optical Density (OD) (nm)

0
25 30 35 40 45 50 55 60 65 70 75

Temperature (oC)

Figure 7.2: The values of Absorbance Optical Density (OD) (nm) against temperature ( oC)
Table 7.3: The values of enzyme activity at different temperatures.

Temperature Absorbance Optical Glucose concentration, Glucose Enzyme activity,

(oC) Density (OD) X (g/mL) released (mol) V (mol/min)

30 8.72 0.00168 9.33 × 10-6 9.33 × 10-7

40 7.46 0.00143 7.94 × 10-6 7.94 × 10-7
50 9.05 0.00174 9.66 × 10-6 9.66 × 10-7

60 8.65 0.00166 9.21 × 10-6 9.21 × 10-7

70 9.78 0.00188 1.04 × 10-5 1.04 × 10-6

0
Enzyme activity, V (mol/min)

0
25 30 35 40 45 50 55 60 65 70 75

Temperature (oC)

Figure 7.3: The values of enzyme activity against temperature ( oC)

EFFECT OF pH:

Table 7.4: The values of Absorbance Optical Density (OD) (nm) at different pH values.

pH value Absorbance Optical Density (OD)

5 10.58

6 8.13

7 9.47

8 9.90

12

10
Absorbance Optical Density (OD)(nm)

0
4.5 5 5.5 6 6.5 7 7.5 8 8.5

pH Values

Figure 7.4: The values of Absorbance Optical Density (OD) (nm) against pH values
Table 7.5: The values of enzyme activity at different pH values.

pH Absorbance Optical Glucose concentration, Glucose Enzyme activity,

Value Density (OD) X (g/mL) released (mol) V (mol/min)

5 10.58 0.00203 1.13 × 10-5 1.13 × 10-6

6 8.13 0.00156 8.66 × 10-6 8.66 × 10-7

7 9.47 0.00182 1.01 × 10-5 1.01 × 10-6

8 9.90 0.00190 1.05 × 10-5 1.05 × 10-6

0
Enzyme activity, V (mol/min)

0
4.5 5 5.5 6 6.5 7 7.5 8 8.5

pH Values

Figure 7.5: The values of enzyme activity against pH values

EFFECT OF SUBSTRATE CONCENTRATION:

Table 7.6: The values of Absorbance Optical Density (OD) (nm) at different substrate
concentration.
Substrate Concentration (%) Absorbance Optical Density (OD)

0.5 10.57

1.5 7.80

2.0 9.32

2.5 8.88

3.0 7.92

12
Absorbance Optical Density (OD) (nm)

10

0
0 0.5 1 1.5 2 2.5 3 3.5

Substrate Concentration (%)

Figure 7.6: The values of Absorbance Optical Density (OD) (nm) against substrate concentration
 Table 7.7: The values of enzyme activity at different substrate concentration and the data for
Michaelis – Menton

Substrate Absorbanc Glucose Glucose Enzyme 1/V 1/S

Concentratio e Optical concentration, released activity, V
n (%) Density X (g/mL) (mol) (mol/min)
(OD)
0.5 10.57 0.00203 1.13 × 10-5 1.13 × 10-6 8.85 × 105 2.00

1.5 7.80 0.00150 8.33 × 10-6 8.33 × 10-7 1.20 × 106 0.67

2.0 9.32 0.00179 9.94 × 10-6 9.94 × 10-7 1.01 × 106 0.50

2.5 8.88 0.00171 9.49 × 10-6 9.49 × 10-7 1.05 × 106 0.40

3.0 7.92 0.00152 8.44× 10-6 8.44× 10-7 1.18 × 106 0.33

0
Enzyme activity, V (mol/min)

0
0 0.5 1 1.5 2 2.5 3 3.5

Substrate Concentration (%)

Figure 7.7: The values of enzyme activity against substrate concentration

 1.40E+06

1.20E+06

f(x) = − 137657.08 x + 1172372.52

1.00E+06 R² = 0.54

8.00E+05
1/V

6.00E+05

4.00E+05

2.00E+05

0.00E+00
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2

1/S

Figure 7.8: 1/V against 1/S for Michaelis-menten

8.0 SAMPLE CALCULATION

1. Determination of glucose concentration, X (g/mL)

From the standard curve of glucose that had been plotted as shown in Figure 7.1, the linear
equation of the curve is presented as:

Y = 0.0052x

Where:
X = protein concentration
Y= Absorbance reading

Calculate glucose concentration for effect of temperature at 30oC:

Y
X=
0.0052
8.72
X=
0.0052
1676.92mg 1L 0.001
X= × ×
L 1000 mL mm
X = 0.00168 g/Ml

2. Determination of glucose released (mol)

Calculate glucose released for effect of temperature at 30oC:

MW of glucose = 180.1559 g/mol

Volume of enzyme = 1 mL
Concentration of glucose (g /mL)
Moles of glucose released (mol) = × Volume of enzyme
MW of glucose
(mL)
0.00168 g /mL
Moles of glucose released (mol) = × 1 mL
180.1559 g /mol
Moles of glucose released (mol) = 9.33 × 10-6 mol
3. Determination of enzyme activity, V (mol/min)

Calculate glucose released for effect of temperature at 30oC:

Duration of hydrolysis reaction: 10 minutes
mol of glucose released
Enzyme Activity (mol/min) =
Hydrolysisreaction time
9.33 ×10−6 mol
Enzyme Activity (mol/min) =
10 min
Enzyme Activity (mol/min) = 9.33 × 10-7

4. Equation of Michaelis – Menten

V max [ S]
V=
K m+S
Double reciprocal:
1 Km 1 1
V
= V max S
+ V max

1
y - axis =
V

1
x - axis =
S

1
Intercept =
V max

Km
Slope =
V max

From the graph 1/V vs 1/S, the linear equation obtain:

Y = -137657x + (1×106)

Finding the value of Vmax, maximum enzyme activity:

 1
= 1×106
V max

Vmax = 1 × 10-6 mol/min

Finding the value of Km, Michaelis constant

Km
= -137657
V max

Km = (-137657) × (1 × 10-6)

Km = -0.1377