cannot reach the active sites of the entrapped or encapsulated enzymes because of
small pores in the polymer network. Low loading capacity and possible deactivation
during immobilization represent another drawback of these techniques (Hwang and
Gu 2013; Ahmad and Sardar 2015).
Carrier-free immobilized enzymes do not require a support and are prepared by
direct cross-linking of different enzyme preparations. Several modifications such as
cross-linked dissolved enzymes (CLEs), cross-linked enzyme aggregates (CLEAs),
cross-linked enzyme crystals (CLECs), and cross-linked spray-dried enzymes
(CLSDs) have been developed to date (Cao et al. 2003). The most common material
is CLEAs. Preparation of CLEAs is relatively simple and consists of two steps.
Soluble enzymes are precipitated into aggregates by water-miscible organic solvents, salts, or nonionic polymers. The process does not require highly pure
enzymes. Then the aggregates are cross-linked by bi- or multifunctional reagents.
Glutaraldehyde is a typically used agent because of high reactivity and low cost.
CLEAs are easy to recover and reuse and have higher operational stability. By
precise control of the process, CLEAs can be prepared as uniformly sized crosslinked aggregates (Nguyen and Yang 2014). Production of CLEAs is a relatively
simple and cheap process. Carrier-free immobilization has several drawbacks
though. Enzymes frequently undergo conformational changes that can cause the
enzyme activity loss. Production of large and stable enzyme aggregates without
denaturation can be problematic since the process of cross-linking is difficult to
control and can result in mass transfer limitations of large substrates (Sheldon and
van Pelt 2013).
Many variations based on combinations of these basic immobilization techniques
have been developed. Further physicochemical factors, advantages and drawbacks
of each technique should be considered when planning immobilization of enzymes;
they are reviewed by Hanefeld et al. (2009), Hwang and Gu (2013), and Zucca and
Sanjust (2014), respectively.
16.6 Nanomaterials
A broad variety of carrier materials are available for enzyme immobilization
(Fig. 16.1) (Datta et al. 2013). Nanoparticles made of silica, magnetite, and titanium
are the most common materials employed for biocatalysis. More recently, novel
nanomaterials such as nanotubes, nanofibres, nanogels, and graphene oxide
nanosheets have been tested. Nanomaterials exhibit unique physicochemical properties. Especially large specific surface area and effective enzyme loading are the key
factors that determine the biocatalyst efficiency. High mechanical strength and
minimal diffusional problems represent further advantages offered by nanomaterials
(Cipolatti et al. 2014). The production of nanomaterials is usually a cheap process.
Several reviews on the immobilization of various enzymes onto different types of
nanomaterials have been published earlier (Gupta et al. 2011; Cipolatti et al. 2014;
350
M. Čvančarová et al.
small pores in the polymer network. Low loading capacity and possible deactivation
during immobilization represent another drawback of these techniques (Hwang and
Gu 2013; Ahmad and Sardar 2015).
Carrier-free immobilized enzymes do not require a support and are prepared by
direct cross-linking of different enzyme preparations. Several modifications such as
cross-linked dissolved enzymes (CLEs), cross-linked enzyme aggregates (CLEAs),
cross-linked enzyme crystals (CLECs), and cross-linked spray-dried enzymes
(CLSDs) have been developed to date (Cao et al. 2003). The most common material
is CLEAs. Preparation of CLEAs is relatively simple and consists of two steps.
Soluble enzymes are precipitated into aggregates by water-miscible organic solvents, salts, or nonionic polymers. The process does not require highly pure
enzymes. Then the aggregates are cross-linked by bi- or multifunctional reagents.
Glutaraldehyde is a typically used agent because of high reactivity and low cost.
CLEAs are easy to recover and reuse and have higher operational stability. By
precise control of the process, CLEAs can be prepared as uniformly sized crosslinked aggregates (Nguyen and Yang 2014). Production of CLEAs is a relatively
simple and cheap process. Carrier-free immobilization has several drawbacks
though. Enzymes frequently undergo conformational changes that can cause the
enzyme activity loss. Production of large and stable enzyme aggregates without
denaturation can be problematic since the process of cross-linking is difficult to
control and can result in mass transfer limitations of large substrates (Sheldon and
van Pelt 2013).
Many variations based on combinations of these basic immobilization techniques
have been developed. Further physicochemical factors, advantages and drawbacks
of each technique should be considered when planning immobilization of enzymes;
they are reviewed by Hanefeld et al. (2009), Hwang and Gu (2013), and Zucca and
Sanjust (2014), respectively.
16.6 Nanomaterials
A broad variety of carrier materials are available for enzyme immobilization
(Fig. 16.1) (Datta et al. 2013). Nanoparticles made of silica, magnetite, and titanium
are the most common materials employed for biocatalysis. More recently, novel
nanomaterials such as nanotubes, nanofibres, nanogels, and graphene oxide
nanosheets have been tested. Nanomaterials exhibit unique physicochemical properties. Especially large specific surface area and effective enzyme loading are the key
factors that determine the biocatalyst efficiency. High mechanical strength and
minimal diffusional problems represent further advantages offered by nanomaterials
(Cipolatti et al. 2014). The production of nanomaterials is usually a cheap process.
Several reviews on the immobilization of various enzymes onto different types of
nanomaterials have been published earlier (Gupta et al. 2011; Cipolatti et al. 2014;
350
M. Čvančarová et al.