Extraction of enzymes using various methods

1. UNIT 4
EXTRACTION OF ENZYMES FROM
VARIOUS SOURCES
PRESENTATION BY
The students of batch 1
II-IBT

2. Gist in various sources used for enzyme
extraction
SOLUBLE
ENZYMES
Microorganisms
Plant tissues
Animals
MEMBRANE-
BOUND
ENZYMES
Detergents
Solvents and
other enzymes

3. 1) Microorganisms as source
•Microorganisms have various enzymes which
can be easily extracted when expelled out of their
cells.
•They have cell wall – Resistant to disruption.
•Thus the cell wall must be disrupted.Generally 2
methods area used
1.Mechanical disruption
2. use of lytic enzymes

4. • Mechanical Disruption:
Mechanical procedures may involve grinding with carborundum or
glass beads, applying pressure.
Carborundum is silicon carbide which is a hard metal which can be
used to crush the microorganism to propel the enzyme out.
Applying pressure involves various processes like Hughes Pressure and
French Pressure or Sonication
Hughes Pressure is the process of applying pressure to disrupt the cell
wall of thee microorganism and French Pressure is the process of
passing the cells in a narrow valve under high pressure to disrupt the
plasma membrane and other organelles.
Sonication is the process of applying sound energy to agitate the
particles and disrupt the cellular organisation.

5. • Using Lytic Enzymes
• Cell pastes collected by centrifugation or filtration can be frozen in
liquid nitrogen and ground in a mortar before the addition of
extraction medium.
Why frozen?(to avoid cell death)
Why liquid nitrogen and not freezer? (to avoid contamination and
enable re working of the cells)
• Lysozyme and/or β-1,3- glucanases, particularly where these are
available in an insolubilized form and can be recovered. This treatment
makes the cell more susceptible to disruption by relatively mild
techniques, e.g. osmotic shock.

6. Can simply all microorganisms used for
extraction of enxymes?
Characteristics to be considered:
• Which subcellular level aids better outcome
• Which microorganism promotes good yield
• Stable conditions under extraction process
• Suitable to mankind or not
• Easy process

7. Extracting medium and its nature
• Extracting medium is the medium added on after cell disruption to
isolate the enzymes. This can be done through various methods like
liquid extraction, solid extraction etc.
What is a need of extracting medium:
Proteins exist within the cell at high concentrations in a reducing
environment. Once cells have been disrupted, by whatever method, the
environment is now oxidising and the contents are diluted with several
volumes of aqueous medium.
To minimise the possible damage to the enzyme, several factors have to
be considered.

8. • A considerable amount of debris (mainly membrane
fragments) is likely to be present, so it is essential that the
enzyme of interest can be easily separated from this by being
soluble in the extraction medium.
Based on this solubility factors four main factors govern the
extraction process, They are:
1. Temperature
2. pH
3. Salt concentration
4. Organic solvent content

9. • Temperature:
The temperature of the medium is usually kept below 4°C, despite the
reductions in solubility that this entails, in order to minimize loss of
activity of the enzyme
• pH:
Proteins are least soluble at their isoelectric point, so the extraction of
an enzyme must be carried out at a pH value far from its isoelectric
point, but nevertheless at a value where the enzyme is stable.
. Salt and organic solvent concentrations are also noted precisely as
they play vital role in enzyme solubility and thus its purification.

10. • In general solubility is directly proportional to the substrate.
Thus, addition of substrate increases solubility.
Sometimes to increase solubility chelating agents are also used.
For example:
Alkaline phosphatase is more soluble in the presence of
glycerophosphate than otherwise. Hence the extraction of an enzyme
can be facilitated by adding its substrate to the medium.
To prevent the loss of enzyme activity and to prevent the oxidation of
sulphydral groups
Example: Cleland’s Reagent.

11. Extraction of soluble enzymes
from higher plants
1)Plant source:
Extraction of enzymes from plants are complicated due to the presence of
cell wall,secondary metabolites ( tannins and phenols) and phenol
oxidases.
 Different types of raw materials include:
1.plants
2.algae
3. mushrooms & etc..
Four important enzymes often found in plants are protease,
amylases,lipases and cellulose

13. The extraction process consists of three parts:
Grinding, to reduce the size of the plant and increase the contact
surface available for the enzymes.
 The enzymatic reaction takes place in a tank stirred and
thermostated in the presence of water.
 Centrifugal separation is the last step of the extraction process: it
allows to obtain an aqueous extract, a vegetable oil and a cake.These
three fractions are then worked separately to ensure the
microbiological quality and stability and to increase the quality, purity
or concentration of certain elements (oligosaccharides, peptides,
amino acids, minerals...).

14. PROCESS FOR EXTRACTION OF ENZYMES FROM PLANT SOURCE:
In general,the plant material has to be crushed,usually by
milling,grinding with sand with or without an extraction medium or by
Homgenization in aqueous buffered solutions.
1. A.For small amount of tissue:
a.Freezing the tissues in liquid nitrogen and grinding to a powder
with acid-washed sand in a mortar
b.Addition of extraction buffer.
B.For large amount of tissue:
Homogenization in blender.
[Homogenization: The process used to reduce the particle size of fluid
products under conditions of high pressure,shear,turbulence,accleration.It
creates very stable and uniform products]

15. 2. Then fibrous material is removed by filtration using several
layers of muslin or cheese cloth.
3.The extraction medium should be cold.
4.Conditions for extraction medium are
a. high pH
b. should contain high millimolar concentrations of reducing
agent ( 10mm of 2-mercaphoethanol)
c. anti-oxidant (ascorbic acid)

16. d.Insoluble polyvinylpolypyrrolidone ( provide alternative
subrate to proteins)
e.Chelating agent ( eg: Diethyldithiocarbonate to reduce
harmful effect of phenol oxidases)
[chelating agent – used to minimize metal ion contamination and
prevent enzymatic activity]

17. OVERALL PROCESS FOR EXTRACTION OF ENZYMES FROM
PLANTS: