These onto support. (2) Entrapment. (3) Cross-linking
These enzyme immobilization methods
can be classified into the five groups as shown in Fig. 2: (1) Adsorption of
enzyme onto support. (2) Entrapment. (3)
Cross-linking of the enzyme protein with glutaraldehyde as a bifunctional
reagents. (4) Covalent binding of the enzyme to a reactive insoluble carrier.
The easiest way for enzyme
immobilization is the physical adsorption of the enzyme protein onto a solid
carrier. This method depends on the physical interaction between the enzyme
protein and the surface of the carrier. This can be brought out by mixing
enzyme with the carrier (Johnson et
al., 1996; Jegannathan et al.,
Adsorption is characterized by the
fact that no reagents but only little activation steps are demanded (Nisha et al., 2012). Therefore, adsorption
is cheap and less distributive for enzyme protein 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, 2014).
Due to the week bonds involved in the
process, adsorption of the protein resulting from changes in temperature, pH,
ionic strength or even the more presence of substrate, is often observed as a
disadvantage (Zhang et al., 2013).
Another disadvantage is non-specific
adsorption of other proteins since the immobilized enzyme is being used (Zucca
and Sanjust, 2014). This may change the properties of the immobilized
enzyme or the substrate and the velocity of enzyme catalysis may be decreased
and this depends on the mobility of substrate and enzyme (Rao et al., 1998; Shelley, 2011).
Confining of a particular enzyme
inside lattices for polymerized gels in considered as other method for enzyme immobilization
(Zhang et al., 2013). This
facilitates the free diffusion of low molecular weight substrate as well as the
products of reaction. The known
polymerization of the hydrophilic matrix in aqueous enzyme solution is followed
by breaking up the polymeric mass to certain particle of the desired size (Lam
and Malikin, 1994).
Calcium alginate hydrogel beads are
commonly used as carriers in the entrapment of biocatalyst (Li and Li, 2010;
Shen et al., 2011). This method
of low cost, high porosity and simplicity of preparation, however this material
has some limitations due to including high bimolecule leakage, biocompatibility
and large pore size (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 suitable for other enzymes such
as dextranase, trypsin and ribonuclease (Marangoni, 2002).
The pore size for synthetic gels of
the polyacrylamide may result in leakage of some entrapped enzyme, 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).
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) ?-carboxyle group of the
chain end and ?- and ?- carboxyl groups of aspartic and glutamic acid, 2) the
imidazole group of histidine, 3) ?-amino
groups of the chain and amino groups of lysine and arginine , 4) the phenol
ring of tyrosine, 5) –SH group of cysteine, 6) –OH groups of serine and
threonine, and 7) the indole group of tryptophan (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).
The disadvantage of this method is
that it often causes the low activity recovery which is resulted from the
destruction of enzyme active conformation during immobilization reaction. Also,
the multipoint attachment to the supports or steric hindrance of enzyme or the
strong strength of covalent binding causes low activity recovery (Zhang et al., 2013).
In this method enzymes are physically
entrapped inside a porous matrix. Bonds involved 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 is adjusted to prevent the loss of enzyme.
size of the matrix can be adjusted with the concentration of the polymer used.
Agar-agar and carrageenan have comparatively large pore sizes. The greatest
disadvantage of this method is that there is a possibility of leakage of low
molecular weight enzymes from the matrix. Examples of commonly used matrixes
for entrapment are: polyacylamide gels, cellulose triacetate, agar, gelatin,
carrageenan and alginate (Dwevedi, 2016).