Chapter 3 METHODOLOGY

3.1 Sampling

Samples of water and sediments are collected from Matang’s drains and Paku Hot
Springs. The samples are kept in plastic bottles and covered with aluminium foil paper
prior being cultured. The pH is checked using pH paper.

3.2 Culturing of the Sample

The samples’ pH is checked and pH adjustment to pH7.5 is done using 1M NaOH. The
amount of water, sediments and shredded MOW are 150 mL, 50 mL and 1.0 gram
respectively. The samples are cultured in 250 mL flask for 1-2 weeks at 55oC at 120 rpm.

3.3 Isolation

The cultures are filtered using filter paper under sterile condition and each filtrate are
subjected to standard serial dilution prior plated onto Luria Broth (LB) agar containing
1% (w/v) carboxymethylcellulose (CMC) which will be incubated at 55oC for an
overnight. Serial dilution plate that gave best colony observation is observed Each
colony identified is streaked two times in order to obtain single colony.

3.4 Congo Red Screening

First, single colonies are transferred onto MSM + 1% CMC agar plates using sterile

needles and grown at 50oC for 3 days to allow endoglucanase secretion. Then, the plates

is flooded with 0.2 % Congo Red solution and left for 1 hour. Lastly, the Congo Red

solution is poured off and washed with 1.0 M NaCl for 15 minutes. Yellow halos

indicated colonies that hydrolyzed CMC where Congo red is absent.

3.4 Enzyme Assay

Crude supernatant of the enzyme is obtained by centrifuging the bacterial culture at 3000
rpm for 5 minutes at 4oC. Endoglucanase activity is determined via dinitrosalicylic acid
(DNS) method using glucose as standards. 400 µl of culture supernatant and 400 µl of
MSM + 1% CMC are pipetted into Falcon tube. The mixture is vortexed and incubated at
55oC for 30 minutes. Then, 800 µl of dinitrosalicylic acid is pipetted into the Falcon tube
and mixed by vortexing. The mixture is then boiled for 20 minutes for colour
development. Subsequently, 400 µl of Rochelle Salt is added immediately into the
Falcon tube.

Ezyme control, substrate control, blank.

3.5 Optimization of Endoglucanase Production (plicates!!!!!!!!)

In order to determine the best condition for enzyme production that gives highest
endoglucanase activity, bacteria incubation/fermention assay is done by optimizing 3
parameters; pH, temperature and time course assay. Endoglucanase activity is determined
via DNS method as in 3.2.1.

3.5.1 pH

3.5.2 Temperature

3.5.3 Time Course of Incubation

3.6 Kinetic Studies of Crude Endoglucanase Enzyme

To

3.6.1 pH

3.6.2 Temperature

3.6.3 Time Course Assay

CHAPTER 3 METHODOLOGY

Screening and Isolation

Biochemical and Molecular Characterization of Isolates

Optimization of Enzyme Endoglucanase Production and Kinetic Studies of Crude

Endoglucanase Enzyme

Biodeinking Trial on MOW

CHAPTER 4 RESULTS

Screening and Isolation

Biochemical and Molecular Characterization of Isolates

Optimization of Enzyme Endoglucanase Production and Kinetic Studies of Crude

Endoglucanase Enzyme

Biodeinking Trial on MOW