DISCUSSION

Absolute Amounts of Glucose Vs A540

0.8
0.7
0.6 R
f(x)==0.98
5.69x + 0.04
0.5
Absolute Amounts of Glucose (mg/0.5 ml) 0.4
0.3
0.2
0.1
0
0 0.02 0.04 0.06 0.08 0.1 0.12
Spectrometer Reading

Figure 1: Graph of absolute amount of glucose Vs A540 spectrometer

In this experiment, cellulase reaction was determined by time to time. From analysing of
cellulose based reaction process, the amount of reducing sugar produced was recorded by
A450 spectrometer.The glucose concentration was analysed using the Glucose Analyser. The
graph of standard curve of solution (without enzyme) is plotted as shown in figure 1 which is
graph of absolute amount of glucose Vs A540 spectrometer graph. From the graph given, the
glucose concentration obviously decreasing as the amount of buffer solution increase. The
highest glucose concentration without enzyme reading is 0.67 mg/ml in sample 1 and its
absorbance is 0.098 while sample 4 shows the least concentration of glucose without enzyme
reading is 0.2 mg/ml with its absorbance which is 0.037 .
 Spectrometer reading of glucose concentration with enzyme Vs time
1.2

1
f(x) = 0.52x - 0.05
0.8 R = 0.95

0.6
Spectrometer reading
0.4

0.2

0
0.8 1 1.2 1.4 1.6 1.8 2 2.2

Time, hour

Figure 2: Graph spectrometer reading of glucose concentration with enzyme Vs time.

Figure 2 shows the graph of spectrometer reading of glucose concentration with enzyme Vs
time of incubation from 1 hour untill 2 hour. The spectrometer reading shows highest reading
at time 2 hours and shows the least at time 1 hour. The filter paper activity occurred as the
time increase. From the graph given for figure 2, it can be concluded that the longer the time
of reaction, the higher the cellulose will be graded by the enzyme. In fact, time is one of the
factor in enzyme reaction. In Several requirements are necessary for automate cellulose
assays, including the following:
1. Simultaneous reagent addition/sampling in the assay plates 2. Creation of substrate plates
3. Prevention of evaporative losses at low-volume scale
4. Automated absorbance reading of samples
5. Scale-down of assay volume/substrate
6. Correct dilution of enzyme stock
7. Consistent and rapid heating during hydrolysis and color development
8. Data analysis
From this experiment whish filter paper assay, the objective is achieved. Based on the
experiment,the factor that influences the enzymatic activity is the temperature of the culture.
During the experiment, the sample was place in an incubator at optimum temperature for
cellulose enzyme activity at 50C. The enzyme will be denatured and the OD will remain
constant if the temperature exceed the optimum temperature as that condition temperature is
not favourable for cellulose enzyme activity.
The precaution steps that we should consider in this experiment are:
1. To avoid any error in OD reading, the filter paper should be make sure is free
contaminant before using it.
2. The filter should be fully immersed to get best result.
3. The concentration of glucose also should be make sure at 2mg/ml before the
preparation of sample.
4. The solution should be shake evenly to make sure all the the cellulose enyme react
completely.

CONCLUSION
In conclusion, the enzyme activity is affected by the temperature, substrate concentration and
time. The glucose concentration increases at different time interval. At the end of this
experiment, it can be concluded that the higher the time,the higher the cellulose will be
graded by the enzyme and it has been proven by the spectrometer reading given in figure 2.It
is shown that the cellulose in the filter paper degraded to produce glucose.