Enzymes noted that enzymes are more incline to

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).

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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
demain, 2014).

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 have
a long half-life, work under slight reaction conditions 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

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).

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
al., 2012).

The general methods used for the
immobilization of enzymes

There 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. 



The easiest way for enzyme
immobilization is the physical adsorption of the enzyme protein onto a solid
carrier. Such a method depends on the physical interaction between the surface
of the carrier and the enzyme protein. This can be done through mixing enzyme
with the carrier (Johnson et al.,
1996; Jegannathan et al., 2008).

Adsorption is characterized by the
fact that it does not demand reagents but only little activation steps (Nisha
et al., 2012). Hence, adsorption
is less distributive and cheap for enzyme protein when compared to chemical
methods of attachment. The binding occurs by hydrogen bonds, salt linkage as
well as Vander Waals forces (Brady and Jordan, 2009; Zucca and Sanjust,

Because of the week bonds involved in
the process, adsorption of the protein resulting from changes in pH, temperature,
ionic strength or even the more presence of substrate is often considered as a
disadvantage (Zhang et al., 2013).

One more disadvantage is non-specific
adsorption of other proteins since the immobilized enzyme is being used (Zucca
and Sanjust, 2014). This may result in changing the properties of the substrate
or the immobilized enzyme and the velocity of enzyme catalysis may be decreased
depending on the mobility of enzyme and substrate (Rao et al., 1998; Shelley, 2011).


Another method for enzyme immobilization
is encapsulation; which means the confinement of a particular enzyme inside
lattices for polymerized gels (Zhang et
al., 2013). This accelerates the free diffusion of substrate with low molecular
weight substrate as well as the products of reaction.  The known polymerization of the hydrophilic
matrix in aqueous enzyme solution is then followed by the breakage of the
polymeric mass into certain particle of the desired size (Lam and Malikin,

The entrapment of biocatalyst usually
uses calcium alginate hydrogel beads as carriers (Li and Li, 2010;
Shen et al., 2011). This method has
many advantages such as its simplicity of preparation, high porosity and low
cost, however this material still has some limitations since it has large pore
size, high bimolecule leakage and biocompatibility (Li and Li, 2010; Zucca
and Sanjust, 2014).

Since no bond formation in occlusion
process between the polymer matrix and the enzyme it is applicable method that
in theory, involves no disruption of the protein molecules (Sassolas et al., 2012). Other disadvantage
for this method is that only substrate of low molecular weight can diffuse
quickly to the enzyme protein. This method is absolutely suitable for other
enzymes such as ribonuclease, dextranase and trypsin (Marangoni, 2002).

A leakage in some entrapped enzyme might
occur due to the pore size for synthetic gels of the polyacrylamide, sometimes
after prolonged washing (Grosova et
al., 2008; Zucca and Sanjust, 2014).


Enzyme immobilization has been
carried out by intermolecular cross-linking of the protein, either to bind to
other protein molecules or to bifunctional reagents on an insoluble support
matrix (Sheldon, 2007; Tran and Belkus, 2011).

Cross-linking is used in conjugation
with one of other methods (Lam and Malikin, 1994; Zucca and Sanjust, 2014).
The covalent cross-linking with polymers, such as glutaraldehyde have been used
to increase the encapsulation efficiency and control release of enzyme from the
gel matrix (Li and Li, 2010; Zucca and Sanjust, 2014).

Using cross-linking method for enzyme
immobilization is relatively cheap. Many aldehydes and other cross-linking
agent are used for this purpose (Kurby, 1990; Zucca and Sanjust, 2014).

Covalent binding:

The formation of covalent bonds
between the enzyme and the support matrix is used as immobilization method. The
choice is limited by the fact do not cause loss of enzymatic activity and the
active site of the enzyme must be unaffected by the used reagent (Copeland,
2004; Zhang et al., 2013).

The suitable functional groups of
proteins suitable for covalent binding include : 1) the indole group of
tryptophan 2) the imidazole group of histidine, 3)  ?-amino groups of the chain and amino groups
of lysine and arginine , 4) –SH group of cysteine, 5) the phenol ring of
tyrosine, 6) –OH groups of serine and threonine, and 7) ?-carboxyle group of
the chain end and ?- and ?- carboxyl groups of aspartic and glutamic acid (Marangoni,
2002;  Singh,2009).

Aminoethyl cellulose has been
attached to the carboxylic acid residues of enzyme protein in the presence of
carbodiimide. It has been reported that SH residues of enzyme protein have been
linked to the thiol groups present in the cross-linked copolymer of acrylamide
and non-acrylol cysteine (Copland, 2000; Nisha et al., 2012).

This method has one disadvantage which
is that it often causes the low activity recovery which is resulted from the
destruction of enzyme active conformation during immobilization reaction. The
multipoint attachment of the enzyme to the supports or steric hindrance of
enzyme or the strong strength of covalent binding causes low activity recovery (Zhang
et al., 2013).


The last method used for the immobilization
of enzymes is entrapment. In this method enzymes are physically entrapped
inside a porous matrix. Bonds used in stabilizing the enzyme to the matrix may
be covalent or non-covalent. The matrix used will be a water-soluble polymer.
The form and nature of matrix varies with different enzymes. Pore size of
matrix could be adjusted to prevent the loss of enzyme.

Pore size of the matrix can be
adjusted with the concentration of the polymer used. Agar-agar and carrageenan
have comparatively large pore sizes. However, the main disadvantage of this
method is that there is a possibility of leakage of low molecular weight
enzymes from the matrix. Such matrixes used for entrapment are: carrageenan, polyacylamide
gels, agar, cellulose triacetate, gelatin, and alginate (Dwevedi, 2016).  


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