11.5 Extraction of Enzymes from Aquatic Organisms
245
This results in the solution separating into two phases (indicating the presence of
globular proteins). The phases can be separated by pipetting out each phase.
In a third method, the protein can be separated using acetone–ether precipitation.
An oil–water emulsion is first formed using castor oil and sunflower oil in a bile salt
solution at a neutral pH and gum acacia as emulsifier. The ground algae biomass
is homogenized with acetone at ice-cold temperature. This is followed by filtration
with the solid residue retained and washed with acetone, an equal volume of acetone
and ether and then with ether. This process removes the lipids, polysaccharides and
other compounds which dissolve in acetone and ether from the algae leaving behind
the protein as the solid mass. The water-soluble protein is then obtained from the
dry mass by dissolving in cold water followed by centrifugation at 15,000 rpm for
10 min. The supernatant is obtained as the enzyme containing fraction (Bele et al.
2014a, b).
The salts used in precipitation can be removed by methods such as ion-exchange
resin or gel filtration using Sephadex G-25. The method of precipitation significantly
affects the yield of protein and the enzyme activity. For example, using the ammonium
sulfate precipitation method, protein concentration in the extract from the alga E.
intestinalis was 117 μg/ml and the enzyme activity was 0.123 meq/min/g using
castor oil as the fat substrate. Protein concentration from U. lactuca was lowest of
all the algae tested in the said study. It had a protein concentration of 87 μg/ml and
an enzyme activity of 0.109 meq/min/g (Bele et al. 2014a, b).
Upon purification using methods like gel filtration, the purity of the enzyme is
confirmed by SDS-PAGE (Jayapriya et al. 2014). This confirms the molecular weight
of the polypeptide extracted from the sample. A single band confirms that the protein extracted has a single molecular weight value. For example, purified protease
extracted from great barracuda viscera shows a single band at 34 kDa (Jayapriya
et al. 2014), while crude lipase extracted from algae shows a band ranging between
45 and 60 kDa (Bele et al. 2014a, b), indicating a less pure sample.
Since in both cases the enzyme is purified mainly of the bases of molecular
weight, the extract contains all proteins within a specified molecular weight range.
Some of these might not be enzymes and will also contain a combination of different
enzymes. The distinction is made by introducing the enzyme into a reaction it catalyzes following incubation at the appropriate conditions. For example, the extract
from algae is mixed with castor oil formulation following incubation at 37 °C and
shaking at 200 rpm (Bele et al. 2014a, b). The amount of fatty acid produced is then
determined by titration against alkali (sodium hydroxide) to obtain a measure for the
lipase activity present within the sample.
11.5.3 Enzyme from Marine Microorganisms
Several marine organisms isolated from the waters and from aquatic organisms have
proven to be reliable sources of enzymes such as chitinase, cellulase, lipase and
245
This results in the solution separating into two phases (indicating the presence of
globular proteins). The phases can be separated by pipetting out each phase.
In a third method, the protein can be separated using acetone–ether precipitation.
An oil–water emulsion is first formed using castor oil and sunflower oil in a bile salt
solution at a neutral pH and gum acacia as emulsifier. The ground algae biomass
is homogenized with acetone at ice-cold temperature. This is followed by filtration
with the solid residue retained and washed with acetone, an equal volume of acetone
and ether and then with ether. This process removes the lipids, polysaccharides and
other compounds which dissolve in acetone and ether from the algae leaving behind
the protein as the solid mass. The water-soluble protein is then obtained from the
dry mass by dissolving in cold water followed by centrifugation at 15,000 rpm for
10 min. The supernatant is obtained as the enzyme containing fraction (Bele et al.
2014a, b).
The salts used in precipitation can be removed by methods such as ion-exchange
resin or gel filtration using Sephadex G-25. The method of precipitation significantly
affects the yield of protein and the enzyme activity. For example, using the ammonium
sulfate precipitation method, protein concentration in the extract from the alga E.
intestinalis was 117 μg/ml and the enzyme activity was 0.123 meq/min/g using
castor oil as the fat substrate. Protein concentration from U. lactuca was lowest of
all the algae tested in the said study. It had a protein concentration of 87 μg/ml and
an enzyme activity of 0.109 meq/min/g (Bele et al. 2014a, b).
Upon purification using methods like gel filtration, the purity of the enzyme is
confirmed by SDS-PAGE (Jayapriya et al. 2014). This confirms the molecular weight
of the polypeptide extracted from the sample. A single band confirms that the protein extracted has a single molecular weight value. For example, purified protease
extracted from great barracuda viscera shows a single band at 34 kDa (Jayapriya
et al. 2014), while crude lipase extracted from algae shows a band ranging between
45 and 60 kDa (Bele et al. 2014a, b), indicating a less pure sample.
Since in both cases the enzyme is purified mainly of the bases of molecular
weight, the extract contains all proteins within a specified molecular weight range.
Some of these might not be enzymes and will also contain a combination of different
enzymes. The distinction is made by introducing the enzyme into a reaction it catalyzes following incubation at the appropriate conditions. For example, the extract
from algae is mixed with castor oil formulation following incubation at 37 °C and
shaking at 200 rpm (Bele et al. 2014a, b). The amount of fatty acid produced is then
determined by titration against alkali (sodium hydroxide) to obtain a measure for the
lipase activity present within the sample.
11.5.3 Enzyme from Marine Microorganisms
Several marine organisms isolated from the waters and from aquatic organisms have
proven to be reliable sources of enzymes such as chitinase, cellulase, lipase and