Preparation of Cellular Fraction

3.2.1. Extracellular Fraction

1. The supernatant of the growth medium is considered the extracellular fraction.

2. Neutralize the supernatant with 5 M NaOH.

3. Sterilize by filtration using a 0.22 ^m-pore filter, white GSWP, 25 mm (Millipore).

4. Keep on ice to preserve the enzyme activity.

3.2.2. Intracellular Fraction

1. Add 1-2 g of glass beads to the resuspended cells.

2. Disrupt the cells five times for 1 min under CO2 atmosphere (see Note 3).

3. Cool the samples in ice for 5 min between each mixing sequence.

4. Centrifuge the sample at 20,000g for 30 min at 4°C to remove glass beads and unbroken cells.

5. Transfer the supernatant (intracellular fraction) to a clean tube by carefully decanting from the pellet.

6. Keep on ice to preserve the enzyme activity.

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