Measurement of Cellulase Activity

Since the viability of enzymes varies over time, the cellulase activity of
Novozyme NS-50013 (cellulase complex) was determined using filter paper assay. The
procedure conducted in the study is based on the method, following the IUPAC
guidelines, provided by NREL LAP (Adney and Baker, 2008).
The main reagents used for the cellulase assay were DNS reagent and citrate
buffer. For the preparation of the DNS reagent, the formulation is as follows. An amount
of 19.8 grams sodium hydroxide pellets must be dissolved in 1416 mL of distilled water.
This hydroxide solution would then be used to dissolve 10.6 g of DNS acid. After the
dissolution of DNS acid, 306 g of potassium acid tartrate, 8.3 g of sodium bisulfite, and
7.6 mL of phenol would be added to the solution. This preparation is based on 1416 mL
distilled water and since the total volume of the solution is too high, the amount of each
component was adjusted using ratio and proportion.
One molar citrate buffer solution was prepared by first dissolving 210 grams of
citric acid monohydrate in 750 mL of distilled water, and subsequently adding about 50 g
NaOH or more until pH 4.3 was reached. The solution was then transferred quantitatively
into a 1-L volumetric flask and then diluted to the mark. The pH level must be checked
and adjusted to pH 4.5 using NaOH pellets if necessary. From this stock solution, 1 L of
0.05 M of citrate buffer solution was prepared and the pH level was adjusted to 4.8 using
NaOH pellets.
To test the enzyme activity, a reagent blank, substrate and enzyme controls, and
enzyme assays was prepared. The reagent blank must contain 1.5 mL of the 0.05 M
citrate buffer, while the enzyme control should contain 1 mL of the buffer and 0.5 mL of
the enzyme. Separate controls were made for enzymes of different dilutions. To prepare
the filter paper assay, a Whatman No. 1 filter paper must be cut into 50 mg strips, which
is approximately 1 cm x 6 cm. Each strip was rolled then placed in the filter paper assay
tubes and the substrate control tube. The substrate control tube was added with 1.5 mL of
buffer solution, while the assay tubes was added with 1 mL of the buffer solution and 0.5
mL of the prepared enzyme dilutions.
For the construction of a standard calibration curve, four dilutions (1:1.5, 1:2, 1:3,
and 1:5) were prepared from a 10 mg/mL anhydrous glucose stock solution. From each
dilution, 0.5 mL was obtained and then placed in test tubes containing 1 mL of citrate
buffer.
The controls, blank, standard solutions and assays were eventually incubated in a
50°C water bath for an hour. To stop the activity, 3 mL of the DNS reagent was added to
each tube then boiled for five minutes in a vigorously boiling water bath. The test tubes
were then allowed to cool down in a cold-water bath. From the color- developed mixture,
0.2 mL aliquot: 2.5 mL distilled water solution was prepared for each sample and then
the absorbance at 540 nm was obtained using a UV-Vis spectrophotometer. To determine
the glucose concentration of the assay, the absorbance of the reagent blank and controls
must be subtracted to the absorbance of each assay sample, the difference of which was
then read against a glucose standard calibration curve.
Sample Calculation
Determination of Enzyme Activity

The enzyme activity measurement was carried out as outlined in Appendix A. The
experimental data are presented in Appendix Tables 17 and 18. For the calculations,
consider the data for 1:10 enzyme dilution shown in Appendix Table 17.
The final absorbance reading must be solved first and is given by the equation
𝐴𝐴 = 𝐴𝐴𝐴 − 𝐴𝐴𝐴 − 𝐴𝐴𝐴 – 𝐴𝐴𝐴

where
Af is the final absorbance reading
AEA is the enzyme assay absorbance
AEC is the enzyme control absorbance of the corresponding dilution ASC is the
substrate control absorbance
ARB is the reagent blank absorbance

Using the data mentioned previously,

𝐴𝐴 = 0.699 − 0.093 − 0.01 − 0.002 𝐴𝐴 = 0.594
Reading this absorbance against a standard curve shown in Appendix Figure 3,
𝐴 = 0.2193𝐴 + 0.0374
𝐴 = 0.594 − 0.0374 0.2193
𝐴 = 2.538075695 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴 0.5 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴
Upon determination of the glucose concentration of all enzyme dilutions used, the
two data points where 2 mg/ 0.5 mL concentration coincided were selected to solve for
the dilution that released this specific amount of glucose then an equation of the line was
obtained (shown in Appendix Figure 5) to mathematically solve for this enzyme dilution.
The calculation is as follows:
𝐴 = 0.2769 (2) − 0.3492 𝐴 = 0.2046
The enzyme dilution that produced 2 mg/ 0.5 mL of glucose was 0.2046. using
the equation from the NREL procedure, the enzyme activity was calculated as follows:
𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 = 0.37/𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 2 𝐴𝐴
𝐴𝐴𝐴𝐴𝐴𝐴𝐴
𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 = 0.37/0.2046
𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 = 1.8084066471 𝐴𝐴𝐴 𝐴𝐴
y = 0.4722x - 0.219
y = 0.4722(2) - 0.219
y (Abs) = 0.7254
concentration = 0.0116
FPA 31.89655172
Table 1.
CITRATE 1:10
DILUTION delta
BUFFER ENZYME CONCENTRATION Abs GLUCOSE
# Abs
(mL) (mL) (mg/0.5mL)
1 24 1 0.004 0.389 0.364 1.234646336
2 23 2 0.008 0.529 0.503 1.52901313
3 22 3 0.012 0.781 0.756 2.06480305
4 21 4 0.016 0.865 0.837 2.236340534
5 20 5 0.02 0.925 0.895 2.359169843

Table 2.
GLUCOSE CITRATE BUFFER CONCENTRATION
DILUTION Abs
(mL) (mL) (mg/0.5mL)
1 0.5 1:1.5 3.35 1.45
1 1 1:2 2.5 0.857
1 2 1:4 1.65 0.487
1 4 1:5 1 0.344

Table 3.
DILUTION
# Abs
1 0.009
2 0.01
3 0.009
4 0.012
5 0.014
REAGENT 0.014
SUBSTRATE 0.016
Figure 1.
1.6
y = 0.4722x - 0.219
1.4 R² = 0.9561
1.2
ABSORBANCE

1
0.8
GS
0.6
Linear (GS)
0.4
0.2
0
0 1 2 3 4
GLUCOSE CONCENTRATION (mg/0.5 mL)

Figure 2.
0.014
y = 0.0075x - 0.0034
0.012

0.01
DILUTION

0.008

0.006

0.004

0.002

0
0 0.5 1 1.5 2 2.5
GLUCOSE CONCENTRATION (mg/0.5mL)