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Enzyme purification
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It's all about the methods of purifying the enzymes
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ENZYMOLOGYEnzyme purification
Anil Kumar and Neha Garg
School of Biotechnology
Devi Ahilya University
Khandwa Rd. Campus
Indore-452017
12-Jan-2006 (Revised 09-Jul-2006)
CONTENTS
Introduction
Criteria for selection of tissue/ organism
Enzyme solubilization techniques
Techniques used for enzyme isolation
Methods of enzyme purification
Fractionation of the proteins on the basis of solubility in aqueous solutions of salts
or organic solvents
Chromatographic separation of the enzyme proteins
Ion exchange chromatography
Adsorption chromatography
Gel filtration (Molecular sieve) chromatography
Affinity chromatography
Chromatofocusing
Electrophoretic techniques
Isoelectrofocusing
Miscelleneous
Ultrafiltration
Dialysis
Crystallization
Criteria of purity of the enzyme protein
Preparation of purification table
Characterization of enzymes
Determination of molecular weight of the enzyme protein
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis for subunit
molecular weight determination
Sulfhydryl groups determination
Absorption spectrum
Amino acid composition
End groups determination
Chemical modification studies
Keywords
Pestle & mortar; Waring blender; Potter Elvejm homogenizer; Ultrasonicator; Ammonium sulfate fractionation;
Ion-exchange chromatography; Gel filtration chromatography; Chromatofocusing; Affinity chromatography;
Polyacrylamide gel electrophoresis; Isoelectrofocusing; Fold purification; Molecular weight; Sulfhydryl groups,
N- & C-terminal groups
,Introduction
Enzymes are biological catalysts and are protein in nature with an exception of ribozyme, an
RNA catalyzing the RNA splicing in eukaryotes. Almost, all the metabolic reactions, in the
living systems, are catalyzed by the enzymes. Enzymes are catalytically active in isolated
form too. In other words, living cells are not essential to get the enzyme activity. This
property of enzymes has been exploited by the biochemists to study enzymes in vitro. While
studying enzymes in vitro, it is believed that whatever we are getting in vitro, the same is in
vivo. Although in almost all the cases, it is true but still there may be exceptions.
Although, mostly enzymes are found within the cells, there are few enzymes especially in
microbes which are secreted in the medium by the microbe and are called extra-cellular
enzymes. In order to study an enzyme in vitro, it is essential to isolate it from the cell. It is
preferable to purify too at least up to some extent before studying its characteristics. It is
pertinent to mention that some times, there is change in the conformation of the enzyme
molecule after isolation and due to that, it is not necessary, whatever is studied in vitro, the
same property is exhibited in vivo too. For isolation of the enzyme, cell has to be ruptured
using a suitable isolation medium and suitable rupturing technique. However, it is also
important to select tissue, organism for isolating the enzyme.
Criteria for selection of tissue/ organism
Although, there is no hard and fast rule for selecting tissue and/ or organism for the isolation
of an enzyme, it is always preferable to select a source enriched in that particular enzyme.
First, the worker should check from the literature whether the enzyme occurs universally (in
animals, plants as well as microbes) or confined to a particular Kingdom. It is believed that
working with microbial and animal enzymes is easier compared to plant enzymes since plants
are generally rich in phenolics, which on exposure with air get converted into quinones and
quinones bind with enzyme protein and makes it inactive. Special care has to be taken while
working with plants. On the other hand, it is easier to get a plant tissue provided plants are
grown in plenty in the surrounding compared to get animal tissue or a pure microbe. For
animal tissue, either one will have to sacrifice the animal in the laboratory or will have to
bring the tissue from a slaughterhouse. In case of microbes, one will have to grow microbe in
pure form on a suitable growth medium under aseptic conditions after getting inoculum of the
microbe. Upto 1980s, there were comparatively fewer reports of enzyme studies from plant
sources and especially purification was being considered to be very difficult job. However,
considering the fact that only few studies have been done from plant sources compared to
their abundance in nature, many scientists preferred working with plant tissues and also
became successful in purifying plant enzymes upto homogeneity (free from other protein(s)).
During this period, there became drastic development in the techniques. Nowadays, enzyme
purification is not considered to be a difficult job. After deciding whether one wishes to work
with the enzyme from animal, plant or microbial source, one tries to select enriched source in
that category. Some people also keep in mind about the availability of the tissue throughout
the year otherwise the tissue will have to be stored in a deep freezer (generally at –80oC).
Enzyme solubilization techniques
Since almost all the enzymes (with few exceptions) are heat labile and not much stable at
room temperature, the entire process of enzyme isolation, purification is carried out at 0-4oC
using a cold room. However, enzyme work can also be done without a cold room if
2
, precautions of cold conditions are followed. All the isolation medium components should be
in chilled condition (which is done by putting them in a refrigerator overnight). The
component of the homogenization technique like pestle and mortar, bowl of the Waring
blender should also be in chilled condition. While homogenizing in a pestle and mortar, it
should be surrounded by the ice flakes. In case of Waring blender bowl, many people also
wrap a cloth wet with chilled water.
Enzyme may be present in the cytoplasm or may be localized in an organelle (applicable only
in eukaryotic cells, there are no distinct organelles in prokaryotes). Even in the organelle, the
enzyme may be present in the matrix of the organelle or may be bound with the membrane.
If the enzyme is localized in a particular organelle, it is preferable first to isolate the organelle
in an intact form and afterwards conditions may be applied to rupture the organelle. In this
process, the enzyme protein will not get contaminated with the proteins present in other
organelles. If it is a soluble enzyme present in cytoplasm, then generally the complete cell is
ruptured without taking care of getting organelles in intact form.
If one has to isolate the organelle in an intact form, one will have to use an isotonic isolation
medium and there should not be any detergent in the medium capable of rupturing the
organelles. It is important to select a suitable cell rupturing technique and an optimal isolation
medium.
Selection of the isolating medium
In case of animal tissues and microbes, many people just use distilled water as isolating
(homogenizing) medium. However, generally a buffer of a suitable ionic concentration and
pH is preferred in order to maintain the pH and ionic concentration in the medium. A certain
ionic strength of the buffer is essential for maintaining the buffering capacity. However,
much higher ionic strength is also avoided since some times, high ionic concentration may be
inhibitory to the activity of the enzyme. All the enzymes are pH sensitive. Every enzyme is
stable in a particular pH range only. Every enzyme shows enzyme activity in a particular pH
range only. However, mostly for isolation, pH of the medium is little different than the pH
at which enzyme activity is measured. The pH of the buffer is maintained according to the
nature of the enzyme. Selection of the pH should be such that isolated enzyme should be in
fully active form. In case of plants, presence of the buffer in the isolating medium is more
important since there is accumulation of acids in plant cell vacuoles and these acids get
released in the medium after rupturing. The released acids will influence (decrease) the pH of
the medium and if buffer has good buffering capacity, it will counteract the change in pH by
the acids. However, sometimes, for isolation of the enzymes which act in acidic pH range like
acid phosphatase, it is preferable to use water instead of buffer since release of the acids will
not decrease the pH of water lesser than the optimum pH of the enzyme. In addition to buffer
or water as the case may be, other components are also used as per need. The other
commonly used components in the isolating medium are described below:
If the enzyme under study requires free sulfhydryl group(s) in its structure for exhibiting
activity or in case of plant tissues especially if that tissue accumulates phenolics, many
workers prefer to use a reducing agent. The commonly used reducing agents are cysteine, 2-
mercaptoethanol (β- mercaptoethanol), sodium metabisulfite, dithiothreitol, dithioerythritol,
reduced glutathione etc. and all are used generally in the concentration range 10 to 50 mM.
In some cases, sodium metabisulfite has been used even up to 100 mM. The reducing agent
may get oxidized by air, therefore, it is always dissolved freshly (except 2-mercaptoethanol
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