Production of

proteins/enzymes
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 EXTRACTION OF ENZYMES

➢ The most important requirement would be to use a source which

enabled large amounts of a suitable enzyme to be extracted by
some convenient procedure. Any source which fulfilled this
requirement could be chosen.
➢ It is essential to know the location of the enzyme within the cell,
and whether the enzyme is present in free solution or bound to a
membrane.
 EXTRACTION OF SOLUBLE ENZYME
EXTRACTION
OF SOLUBLE
ENZYME

Animal Tissue Higher Plants Micro-organisms

Freezing the tissue

and grinding Mechanical
Homogenization disruption
(Small amount of
tissue)

1. Isotonic medium Homogenization in

blender (Large Lytic enzymes
2. Douce homogenizer amount of tissue)
3. Blender or
Polytron/Ultraturax disruptor
 Extraction of soluble enzyme
➢ Soluble cytoplasmic enzymes are the simplest to extract, for any disruption
of the plasma membrane will enable the enzyme to pass into the surrounding
medium.
➢ Soluble enzymes present in the organelles of eukaryotic cells are also easy
to liberate, but the disruption of intracellular membranes often requires
harsher conditions than disruption of the plasma membrane.
➢ To minimize the extraction of unwanted enzymes, cell fractionation may
be carried out prior to the disruption of organelles.
➢ The extraction procedures used depend on the type of organism acting as
source.
1. Animal tissue is removed as soon after death as possible and kept cold to
minimize autolysis. Obvious non-enzyme matter (e.g. fat deposits and
connective tissue) may be discarded.
• The remaining tissue is then treated to disrupt membranes using a
technique appropriate to the location of the enzyme being extracted.
• Homogenization in an isotonic medium disrupts the plasma membrane
but leaves most organelles intact.
- A Potter-Elvehjem or Douce homogenizer can be used for small amounts
of animal tissue.
- Blender or Polytron/Ultraturax disruptor may be required for larger
amounts of tissue.
2. Higher plants: Extraction of soluble enzymes from higher plants is
complicated by the presence of the cell wall surrounding the plant cell,
secondary metabolites (including tannins and phenols which can bind to
proteins, causing precipitation) and phenol oxidases (which convert the
phenols into reactive quinones which can bind to proteins).
• Freezing the tissue in liquid nitrogen and grinding to a powder with acid
washed sand in a mortar prior to the addition of extraction buffer can be
used for small amounts of tissue.
• Homogenization in a blender can be used for larger amounts of tissue.
• The fibrous material can then be removed by filtration through several
layers of muslin or cheesecloth.
• The extraction medium should be cold, set at a high pH, contain
high millimolar concentrations of a reducing agent (e.g. 10mM 2-
mercaptoethanol), include an anti-oxidant (e.g. ascorbic acid),
insoluble polyvinylpolypyrrolidone (which provides an alternative
substrate to proteins for reactive species) and a chelating agent such
as diethyldithiocarbamate to reduce the deleterious effect of phenol
oxidases.
3. Micro-organisms, also have a cell wall and so tend to be more resistant to
disruption than are animal cells.
➢ In general, methods of extraction fall into two main categories:
a) Mechanical disruption
b) Use of 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.
• Other mechanical procedures may involve grinding with carborundum or
glass beads, applying pressure (e.g. with Hughes or French presses) or
sonication.
• Lytic enzymes: A popular alternative to mechanical procedures is the use
of enzymes such as 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 or sonication.
• The enzymes can be also be used in combination with detergents to
weaken the bacterial cell wall and to solubilize the membrane.
 EXTRACTION OF MEMBRANE-BOUND
ENZYMES
• The Enzymes which are bound to plasma or intracellular membranes cannot
normally be extracted into the surrounding medium simply by the disruption
procedures.
• However, some enzymes are loosely bound and some tightly bound (many
actually forming an integral part of the membrane), so there can be no general rule
about their extraction.
• Membranes, besides containing enzymes and other proteins, are made up largely
of amphipathic lipids, i.e. lipids which contain hydrophilic and hydrophobic
regions.
• The main classes of membrane lipids are phospholipids (fatty acid/phosphoric
acid esters of glycerol and sphingosine) and glycolipids (fatty acid esters, mainly
of sphingosine, attached to a sugar). Some membranes in eukaryotic cells also
contain sterols.
• In each case the hydrophilic portion of the molecule is small compared with the
hydrophobic part the R-groups of the fatty acid residues are long hydrocarbon
chains).
• An amphipathic lipid may therefore be represented as a molecule with a
hydrophilic head and one or more hydrophobic tails, for example
• The model of membrane structure which best fits the available evidence is that
proposed by Jonathan Singer and Garth Nicolson (1972): this is termed the fluid-
mosaic model.
• According to this model, lipids and proteins can move laterally about the
membrane, but find greater difficulty in moving in a transverse direction, from one
membrane surface to the other. Proteins may be peripheral, when they are bound
to the surface of the membrane, or they may be integral, when they are totally or
partly embedded in the lipid layers.
• Peripheral proteins are apparently linked by electrostatic or hydrogen bonds to
the polar heads of lipids or to integral proteins and can easily be dissociated from
the membrane, e.g. by washing with a buffer of high ionic strength (1.0M NaCl)
or by changing the pH of the buffer.
• Integral proteins, on the other hand, can only be extracted by breaking the
hydrophobic interactions between lipid and protein (i.e. by dissociating a
lipoprotein complex). This means either the use of a solvent or a detergent.
Furthermore, for the extraction of an integral protein which is an enzyme, this
must be done in such a way that enzyme activity is not lost during the process.
• The most common method for the extraction of membrane proteins is the use of
detergents, which disrupt membranes and extract lipoprotein fragments into
aqueous media as components of micelles (aggregates of amphipathic molecules
arranged with their hydrophobic parts in the interior of the micelle and their
hydrophilic parts on the surface).
• These detergents include natural bile salts such as cholate; non-ionic synthetic
detergents like Triton X-100; and ionic synthetic detergents which may be
zwitterionic, as in the case of CHAPS, anionic like sodium dodecyl sulphate
(SDS) or cationic, e.g. CTAB.
• In addition to the protein-detergent micelles, molecules of a detergent will themselves form
micelles if present at a concentration above the critical micelle concentration (CMC), which is
dependent on the temperature and, if the detergent is ionic, also on the ions present in solution.
• The choice of detergent will be influenced by whether or not the enzyme is
required in an active form, and what further purification procedures are intended.
• A detergent widely used in membrane studies is sodium dodecyl sulphate (SDS),
which will sever most protein-lipid (and protein-protein) interactions.
• However, for extraction of an active enzyme, non-ionic detergents are usually
preferred.
• To maintain the solubility of the membrane protein during a purification process,
low concentrations of detergent will be required in all buffers. This can present
problems as some detergents can interfere with protein assays and may also absorb
light in the UV at the same wavelength as proteins (the amino acids tyrosine and
tryptophan present in a proteins structure absorb light at 280nm) making detection
of protein-rich fractions from chromatographic runs difficult.
• The presence of a detergent requires careful thought about the choice of the
chromatographic procedure.
• Size-exclusion chromatography (SEC) is compatible with detergents and is a
popular choice as the first chromatographic procedure in the purification of
membrane proteins.
• To avoid detergents binding to an ion exchange (IEX) resin, neutrally-charged or
oppositely-charged detergents are required.
• Many membrane proteins are also glycosylated, so the use of a detergent-
tolerant lectin affinity resin provides a highly selective tool for the purification of
membrane proteins.
• Lipoprotein fragments may also be extracted by the use of organic solvents,
and some free proteins may be liberated on subsequent dispersion in aqueous
media.
• However, the use of solvents to extract proteins usually results in the denaturation
of the protein and loss of biological activity, although solvent extraction can be
useful to concentrate membrane proteins prior to an electrophoretic procedure e.g.
isoelectric focusing or SDS-PAGE.
• Alternatively, the lipid-protein interactions may be broken by the action of
specific enzymes: for example, phospholipase and triacylglycerol lipase have
been used to extract alkaline phosphatase.
 The nature of the extraction 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
oxidizing and the contents are diluted with several volumes of aqueous medium.
• To minimize the possible damage to the enzyme, several factors have to be
considered.
• In general, 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.
• Four main factors govern the solubility of proteins: salt concentration, pH, the
organic solvent content and 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.
• 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.
• The salt and organic contents of the medium are chosen, largely on an empirical
basis, to ensure that the enzyme is soluble.
• The solubility of an enzyme can sometimes be increased by the addition of its
substrate. For example, alkaline phosphatase is more soluble in the presence β-
glycerophosphate than otherwise. Hence the extraction of an enzyme can be
facilitated by adding its substrate to the medium.
• Stabilizers, such as dithiothreitol (Cleland's reagent) or 2-mercaptoethanol (2-
ME) are sometimes added to extraction media to prevent oxidation of sulphydryl
groups and thus loss of enzyme activity.
• Inhibitors of proteolytic enzymes, e.g. DFP, PMSF
(phenylmethylsulphonylfluoride), leupeptin, pepstatin, bestatin, E-64 or
antipain, may also be added to prevent these attacking the enzyme of interest.
Polyols (e.g. glycerol) or chelating agents (e.g. EDTA) also have a use in
stabilizing solubilized enzymes.
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