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)

y = 0.0052x + 0.0072
5 R² = 0.9471

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
Absorbance Optical Density (OD) (nm)

10

0
0 10 20 30 40 50 60 70 80
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

0.0000012

0.000001
Enzyme activity, V (mol/min)

0.0000008

0.0000006

0.0000004

0.0000002

0
0 10 20 30 40 50 60 70 80
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
0 1 2 3 4 5 6 7 8 9
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

0.0000012

0.000001
Enzyme activity, V (mol/min)

0.0000008

0.0000006

0.0000004

0.0000002

0
0 1 2 3 4 5 6 7 8 9
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

10
Absorbance Optical Density (OD) (nm)

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 Absorbance Glucose Glucose Enzyme 1/V 1/S

Concentration Optical concentration, released activity, V
(%) 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

0.0000012

0.000001
Enzyme activity, V (mol/min)

0.0000008

0.0000006

0.0000004

0.0000002

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
y = -137657x + 1E+06
R² = 0.5439
1.00E+06

8.00E+05
1/V

6.00E+05

4.00E+05

2.00E+05

0.00E+00
0 0.5 1 1.5 2 2.5
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
1676.92 𝑚𝑔 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 (mL)
𝑀𝑊 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒

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) =
𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 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

𝑉𝑚𝑎𝑥 [𝑆]
V=
𝐾𝑚+𝑆

Double reciprocal:
1 𝐾𝑚 1 1
= +
𝑉 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥

1
y - axis =
𝑉

1
x - axis =
𝑆

1
Intercept =
𝑉𝑚𝑎𝑥

𝐾𝑚
Slope =
𝑉𝑚𝑎𝑥

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
𝑉𝑚𝑎𝑥

Vmax = 1 × 10-6 mol/min

Finding the value of Km, Michaelis constant

𝐾𝑚
= -137657
𝑉𝑚𝑎𝑥

Km = (-137657) × (1 × 10-6)

Km = -0.1377