Enzymes are formed in all living
organisms where they catalyze and regulate essential chemical reactions needed
for the life of organism (Nisha and Divakaran, 2014).
Enzymes are proteins in nature. They
are fragile and large molecules. Hence enzymes are completely different from the
well-known organic and inorganic catalysts. Soluble enzymes are regarded as being
instable and sensitive to process conditions (Biro et al., 2008; Buchholz
et al., 2012).
Enzymes as biocatalysts
Enzymes are biocatalysts which have different
applications in industrial chemistry (Wohlgemuth, 2010). This
application includes purified enzymes, immobilized enzymes or immobilized cells
as catalysts for the process mentioned above (Schmid et al., 2001; Gong et al.,
2012). The development of biocatalysts is completely targeted to the
progress of protein expression, metabolic engineering, large-scale genome
sequencing and detected evolution (Bornscheuer et al., 2012).
Biocatalysts have a critical importance for
processes of industrial, pharmaceutical and biotechnological application (Sanchez
and Demain, 2010). The success of enzyme application for any enzymatic
processes depends on the cost competitiveness as well as the well-established
chemical methods (Tufvesson et al.,
When being compared to chemical
catalysts, it is noted that enzymes are more incline to be consequently and are
used in performing molecular transformations which cannot be achievable by ordinary
chemical catalysis (Liese et al.,
Enzymes which are thermostable at high
temperatures are more desirable in industrial applications. The rate of
reaction typically increases every 10°C increase in temperature thus most
enzymes do not withstand high temperatures over higher than 40°C and they can
be denatured at extreme values of pH (Cornish-Bowden, 2004).
When applied to the industrial
biocatalysts area, enzymes are proven to provide a great success. Various
factors may affect the application of biocatalysts, such factors are enzyme
promiscuity, screening technologies as well as robust computational methods for
improving the properties of enzyme available for the applications (Adrio and
In fact, the biotechnological
processes have many advantages over well-established chemical processes such as
having less catalyst waste, increased catalyst efficiency as well as a lower
energy demand. They might be around 150 biocatalytic processes that are being
applied in industry (Panke and Wubbolts, 2005). However, the new
development in protein engineering made it easier to successfully use
particular enzyme characteristics in industrial purpose (Lutz, 2010).
According to the fact that enzymes
are involved in all aspects of biochemical conversion varying from the simple
enzyme or fermentation conversion leading to the complex techniques in genetic
engineering, it is fair to say that enzymes are considered as a focal point of
biotechnological processes (Ebbs, 2004).
Environmental and genetic manipulations
can be used to increase the enzyme levels. Thousand-fold increases have been observed
for catabolic enzymes, and biosynthetic enzymes have been increased several
hundred-fold (Burns and Dick, 2002).
Many disadvantages have been noted in
the processes of different industries such as the production of pharmaceuticals
and chemicals. These disadvantages may include the need for high temperature, low
catalytic efficiency, low pH and high pressure. Not to mention that using
organic solvents produces pollutants and organic waste. Enzymes such as
biocatalysts are more useful for the applications mentioned above because they
work under mild reaction conditions, have a long half-life and they work on
unnatural substrates (Johnson, 2013).
Furthermore, enzymes can be
chemically-modified or selected genetically for improving some characteristics such
as substrate specificity, stability as well as specific activity. However, some
disadvantages are found in enzymes including the requirement of certain
co-factor by enzymes. There are different ways that can be used in order to
solve such a problem among which using the whole cells as well as recycling of
cofactor (Baici, 2015).
Reports show that enzymes isolated
from microbes are applied in pharmaceuticals as diagnostic reagents, as
reagents for the production of chemicals, food additives, the manufacture of
detergents, the treatment of industrial wastes and bioremediation (Baxter
and Cummings, 2006).
Stability of enzyme
Stability of enzymes is an important
concern especially during thermal processing. Losing enzyme activity at high
temperature ranges is directly related to variations of enzyme conformation (Cui
et al., 2008, Fu et al., 2010). One can estimate this through thermodynamic
parameters and Arrhenius equation (Marangoni, 2003).
In a nutshell, enzyme stability is absolutely
essential in basic and applied enzymology. Enzyme stabilization principles
could only be understood through illustrating how enzymes lose their activity
followed by deriving the structure stability relationships existing in
enzymatic molecules (Plou et al.,
The most important outcome of using
enzymes is to produce useful compounds. Since the fact that enzymes are
unstable and can be quickly inactivated through different mechanisms, they
cannot be the proper catalysts for industrial applications. Having a stable
enzyme in soluble form is inevitable to achieve the storage of purified enzymes
and the purification processes as well (Aehle, 2007).
Different strategies have been used in
order to enhance enzyme stability. The well-known methods for obtaining soluble
stable enzymes are: 1) chemical modifications of enzymes and 2) use of
additives (Taravati et al., 2007, Shelley, 2011).
Additives are soluble compounds that
have a particular effect on the thermostability of the enzyme protein. Remarkable
effect on the enzyme stability is noticed when particular compounds to enzyme
solutions are added. Such additives are polymers, polyhydrilic, sugar, alcohols,
and other organic solvents (Polaina and Maccabe, 2007).
Adding certain types of chemicals could
be used in avoiding such conformational changes of the enzyme. These chemicals include
polyols which is mainly used to promote numerous hydrogen bonds or salt-bridge
formation between amino acid residues. These bonds or bridges make the enzyme
molecule more rigid, hence it becomes more resistant to the thermal unfolding (George
et al., 2001; Costa et al., 2002). However, the choosing
of the appropriate additive depends on the enzyme structure.
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 intended to isolate one or a few protein from a complex mixture,
usually cells, tissues or whole organisms. Protein purification is vital 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 detect and identify a protein in a mixture, whereas preparative methods aim
to produce 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).