Enzymes - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 356

is enclosed within a porous membrane. The membrane can be polymeric or an enriched inter-
facial phase formed around a micro drop.
Despite the aforementioned advantages, enzyme entrapment may have its inherent prob-
lems, such as enzyme leakage into solution, significant diffusion limitations, reduced enzyme
activity and stability, and lack of control of microenvironmental conditions. Enzyme leakage
can be overcome by reducing the molecular weight cutoff of membranes or the pore size of
solid matrices. Diffusion limitations can be eliminated by reducing the particle size of
matrices and/or capsules. Reduced enzyme activity and stability are due to unfavorable
microenvironmental conditions, which are difficult to control. However, by using different
matrices and chemical ingredients, by changing processing conditions, and by reducing
particle or capsule size, more favorable microenvironmental conditions can be obtained.
Diffusion barrier is usually less significant in microcapsules as compared to gel beads.
8.3.1.2. Surface Immobilization
The two major types of immobilization of enzymes on the surfaces of support materia1s
are adsorption and covalent binding.
Adsorption is the attachment of enzymes on the surfaces of support particles by weak phys-
ical forces, such as van der Waals or dispersion forces. The active site of the adsorbed enzyme
is usually unaffected, and nearly full activity is retained upon adsorption. However, desorp-
tion of enzymes is a common problem especially in the presence of strong hydrodynamic
forces, since binding forces are weak. Adsorption of enzymes may be stabilized by cross-link-
ing with glutaraldehyde. Glutaraldehyde treatment can denature some proteins. Support
materials used for enzyme adsorption can be inorganic materials, such as alumina, silica,
porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such
as cellulose (CMC, DEAE-cellulose), starch, and activated carbon, and ion-exchange resins,
such as Amberlite, Sephadex, and Dowex. The surfaces of the support materials may need
to be pretreated (chemically or physically) for effective immobilization.
Covalent binding is the retention of enzyme on support surfaces by covalent bond forma-
tion. Enzyme molecules bind to support material via certain functional groups, such as
amino, carboxyl, hydroxyl, and sulfhydryl groups. These functional groups must not be in
the active site. One common trick is to block the active site by flooding the enzyme solution
with a competitive inhibitor prior to covalent binding. Functional groups on support material
are usually activated by using chemical reagents, such as cyanogen bromide, carbodiimide,
and glutaraldehyde. Support materials with various functional groups and the chemical
reagents used for the covalent binding of proteins are listed in Table 8.4 .
Binding groups on the protein molecule are usually side groups (R) or the amino or
carboxyl groups of the polypeptide chain. The cross-linking of enzyme molecules with each
other using agents such as glutaraldehyde, bis-diazobenzidine, and 2,2-disulfonic acid is
another method of enzyme immobilization. Cross-linking can be achieved in several different
ways: enzymes can be cross-linked with glutaraldehyde to form an insoluble aggregate,
adsorbed enzymes may be cross-linked, or cross-linkingmay take place following the impreg-
nation of porous support material with enzyme solution. Cross-linking may cause significant
changes in the active site of enzymes, and also severe diffusion limitations may result.
The most suitable support material and immobilization method vary depending on the
enzyme and particular application. Two major criteria used in the selection of support