370
Other Applications
Protein Engineering and Immobilization
Biomolecules that are physically localized in a certain defined region of space with
retention of their catalytic activities, and which can be used repeatedly and continuously are defined as immobilization (Brena and Batista-Viera 2006). In industrial
field, enzyme immobilization is a progressing field due to its multiple functions
including reuse of enzymes for the same reaction, longer half lives, low degradation
and prevention of substrate contamination with enzymes or other compounds
(Abdel-majeed et al. 2012). However, recent developments in protein engineering
have revolutionized the development of commercially available enzymes into better
industrial catalysts than provided by immobilization. Protein engineering and
immobilization techniques are sequential and compatible strategies for the enhancement of enzyme properties. In many industrial processes, enzymes are immobilized
to increase its stability and immobilized enzymes can be easily separated from reaction mixture and used again, thus, leading to economy of the process.
Immobilization have three most common methods viz; adsorption, entrapment,
and crosslinking or covalently binding to a support. Proteins are immobilized either
by physical adsorption to the surface of the nanoparticle or by covalent bonding to
previously functionalized nanoparticles. Physical adsorption and covalent binding
both decrease or avoid enzyme leaching, but binding to a planar surface can result
in decreased stability or even protein denaturation (Brode et al. 1996). Crosslinking
of enzymes typically enhances their stability at the expense of reduced activity.
Microencapsulation into micelles or micellar polymers offers the highest potential
to significantly improve enzyme lifetime and stop enzyme leaching, although problems of mass transfer may occur. Covalent binding of an enzyme to a carrier has the
significance that the enzyme is tightly fixed. This is due to the fact that the multiple
covalent bonds formation between the enzyme and the carrier decreases conformational flexibility and thermal vibrations, thus preventing protein unfolding and
denaturation (Singh et al. 2010; Hanefeld et al. 2009). Multipoint and multi subunit
covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in
improved enzyme stability. However, there are few drawbacksrelated to enzyme
immobilization including enzyme leakage owing to formation of weak bonds
between enzyme and the carrier due to changes in temperatures, pH, ionic strength
or even the mere presence of substrate (Dariush 2003). Another problem is restricted
diffusion of high molecular weight substrates such as ribonuclease, trypsin and dextranase into entrapped enzymes Sankaran et al. 1989. Furthermore, selection of conditions for immobilization by covalent binding is more difficult than in other carrier
binding methods.
Protein engineering however, can counteract these drawbacks by altering amino
acid composition of enzymes that contain sequences of several hundred amino acids
folded in a unique three-dimensional structure. Protein engineering is a faster, more
F. Jhan et al.
Other Applications
Protein Engineering and Immobilization
Biomolecules that are physically localized in a certain defined region of space with
retention of their catalytic activities, and which can be used repeatedly and continuously are defined as immobilization (Brena and Batista-Viera 2006). In industrial
field, enzyme immobilization is a progressing field due to its multiple functions
including reuse of enzymes for the same reaction, longer half lives, low degradation
and prevention of substrate contamination with enzymes or other compounds
(Abdel-majeed et al. 2012). However, recent developments in protein engineering
have revolutionized the development of commercially available enzymes into better
industrial catalysts than provided by immobilization. Protein engineering and
immobilization techniques are sequential and compatible strategies for the enhancement of enzyme properties. In many industrial processes, enzymes are immobilized
to increase its stability and immobilized enzymes can be easily separated from reaction mixture and used again, thus, leading to economy of the process.
Immobilization have three most common methods viz; adsorption, entrapment,
and crosslinking or covalently binding to a support. Proteins are immobilized either
by physical adsorption to the surface of the nanoparticle or by covalent bonding to
previously functionalized nanoparticles. Physical adsorption and covalent binding
both decrease or avoid enzyme leaching, but binding to a planar surface can result
in decreased stability or even protein denaturation (Brode et al. 1996). Crosslinking
of enzymes typically enhances their stability at the expense of reduced activity.
Microencapsulation into micelles or micellar polymers offers the highest potential
to significantly improve enzyme lifetime and stop enzyme leaching, although problems of mass transfer may occur. Covalent binding of an enzyme to a carrier has the
significance that the enzyme is tightly fixed. This is due to the fact that the multiple
covalent bonds formation between the enzyme and the carrier decreases conformational flexibility and thermal vibrations, thus preventing protein unfolding and
denaturation (Singh et al. 2010; Hanefeld et al. 2009). Multipoint and multi subunit
covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in
improved enzyme stability. However, there are few drawbacksrelated to enzyme
immobilization including enzyme leakage owing to formation of weak bonds
between enzyme and the carrier due to changes in temperatures, pH, ionic strength
or even the mere presence of substrate (Dariush 2003). Another problem is restricted
diffusion of high molecular weight substrates such as ribonuclease, trypsin and dextranase into entrapped enzymes Sankaran et al. 1989. Furthermore, selection of conditions for immobilization by covalent binding is more difficult than in other carrier
binding methods.
Protein engineering however, can counteract these drawbacks by altering amino
acid composition of enzymes that contain sequences of several hundred amino acids
folded in a unique three-dimensional structure. Protein engineering is a faster, more
F. Jhan et al.