There are numerous methods used in in
enzyme modification that can be mainly classified into three different types.
These types are: 1) attaching of the enzyme molecules to some water soluble
polymers 2) polyfunctional substitutions with certain agents used to produce
interior intermolecular linkages and 3) substitutions of the amino-acid groups
on the enzyme surface (Shanmugan and Sathishkumar, 2009).
The methods mentioned above are used
for identifying specific residues at the active site involved in substrate
binding or chemical catalysis; however it has been used for tailoring the
specificities of enzymes (Qi et al.,
2001; Davis, 2003; Svendsen, 2016).
There are many ways that can be used
to achieve enzyme stabilization against thermal inactivation. One of these ways
is cross-linking to a water insoluble carrier with a bi-functional reagent or
covalent coupling to natural and entrapment in gels and synthetic polymers (Najafi
et al., 2005; Shelley, 2011).
Various purification procedures have been
used to isolate proteins and some enzymes have been purified by using more than
one approach. Even though the process of purifying enzymes could be complex at
first sight, however it gets easier through the sequential application of a few
simple methods (Gupta et al., 2016).
Purification of Enzymes
Protein purification is a series of
processes that are mainly aiming to isolate one or a few protein from a complex
mixture which is usually cells, tissues or whole organisms. Protein
purification is essential for the characterization of the structure, function
and interactions of the protein of interest (Iqbal et al., 2016).
Protein and non-protein parts of the
mixture are separated in the purification process, and finally separate the
desired protein from all others is typically the most laborious aspect of
protein purification. Differences in protein size, binding affinity, physio-chemical
properties, and biological activity are exploited in the separation steps (Kennedy,
1990; Iqbal et al., 2016).
Analytical and preparative methods
are mainly the methods used in protein purification. However, the distinction
is not exact, but amount of protein that can practically be purified with that
method is the final deciding factor (Iqbal et al., 2016).
The main goal of analytical methods is
to identify and detect a protein in a mixture, while preparative methods target
producing large quantities of the protein for other purposes, such as industrial
use or structural biology. Bottom line is, the preparative methods can be used
in analytical applications, but not the other way around (Regnier, 1983).
Techniques of Purification
Size exclusion chromatography
Chromatography separates protein in
solution or denaturing conditions through the use of porous gels. Such a
technique is known as “size exclusion chromatography”. The technique
is based on the fact that smaller molecules have to traverse a large volume in
a porous matrix. Therefore, proteins in a certain range in size will require a
variable volume of eluent (solvent) before being collected at the other end of
the column of gel (Kennedy, 1990).
Ion exchange chromatography
Ion exchange chromatography is used
to separate compounds according to the nature and degree of their ionic charge.
The column to be used is chosen based on type and strength of charge. Anion
exchange resins have a negative charge and are used to retain and separate
positively charged compounds, while cation exchange resins have a positively
charged compounds, while cation exchange resins have a positive and are used for
the separation of negatively charged molecules (Kennedy, 1990).
Immobilization of enzymes
While free enzymes are unstable and
cannot be used to meet the economical requirements for an industrial purpose, immobilized
enzymes are used in industrial bioprocesses especially in food, nutritional,
and technology of pharmaceuticals (Sheldon, 2007).
Immobilized enzyme is used in many
ways because of several factors. First, enzyme could be handled easily, second the
ability to reuse costly enzymes, with longer half-lives and less degradation (Shi
et al., 2011), third it helps
preventing the contamination of the substrate with enzyme?protein or other
compounds which decreases purification costs, forth its facile separation from
the product (Spahn and Minteer, 2008).
Among the supports used for enzymes
immobilization are hydrogels and inorganic beads, synthetic organic polymers, smart
polymers and biopolymers (Sheldon, 2007; Salemi, 2010).
Enzyme immobilization uses water
insoluble polysaccharides including agarose, cellulose, chitosan and starch.
Also some proteins including albumin and gelatin have been reported as beads
for the immobilization of enzyme (Krajewska, 2004; Spahn and Minteer, 2008).
Also, some biomaterial such as egg
shell membrane, has been found to be an effective and stable enzyme
immobilization substrate (Choi and Yiu, 2004; Wu et al., 2004). Enzyme
immobilization has been implemented on a larger scale, in the food industry and
in the manufacture of fine chemicals and pharmaceuticals (Krajewska, 2004).
During the process of immobilization,
retention of the activity and the stability must be taken in consideration. It
has been reported that some enzyme activity is lost during immobilization. The
immobilization procedure should be chosen carefully because of the interaction
between enzyme, matrix as well as protein modification (Chen et al., 2014).
Thermostability and pH stability
indicate the capability of the conjugate of enzyme support to resist higher
temperatures or pH at alkaline or acidic sides before occurring denaturation (Shelley,
Storage stability is the ability of
the enzyme to keep its activity under some certain condition of storage.
However, the operational stability does only represent the enzyme function but
it represents the durability of the carrier and concentrations of the inhibitor
in the solution under assay (Raafat et
The general methods used for the
immobilization of enzymes
are various methods used for the immobilization of enzymes. These methods could
be classified mainly into the five groups as shown in Fig. 2: (1) Covalent
binding of the enzyme to a reactive insoluble carrier. (2) Adsorption of enzyme
onto support. (3) Cross-linking of the enzyme protein with glutaraldehyde as a
bifunctional reagents. (4) Entrapment. (5) Encapsulation