165
identifying the consensus sequences and amino acid substitutions that generate stable enzymes. These data can be used to design small libraries with a huge percentage
of catalytically active variants, which have been used to discover enzymes with
highly efficient biocatalytic properties (Jochens and Bornscheuer 2010).
Multiple amino acid substitutions are usually required for large changes in
enzyme properties. More and more powerful screening could be considered as the
simplest solution to this problem. High-throughput methods like fluorescenceactivated cell sorting, which are capable of screening tens of millions of variants in
a short time, can be used to monitor the alterations in substrate specificity (Becker
et al. 2008; Fernandez-Alvaro et al. 2011).
As of now, the perfect way to create multiple mutations is to add them simultaneously, but to limit the choices using statistical or bioinformatics methods. Jochens
and Bornscheuer (2010) used this method to enhance the enantioselectivity of a
Pseudomonas fluorescens esterase. Weinreich and co-workers (2006) studied the
effectiveness of cooperative interactions (mutations) in the development of a
β-lactamase with advanced properties. In this study, the reaction rate was enhanced
by mutation A, but this mutation destabilized the β-lactamase. The overall effect
was slightly advantageous, whereas the reaction rate remained unaffected by mutation B but stabilized the β-lactamase; by itself, it had no effect. In combination,
mutations A and B were greatly advantageous because the β-lactamase showed
higher reaction rate and preserved its stability, but addition of mutations stepwise
will most probably miss these kinds of synergism.
The integration of enzymes with nanomaterials and in complex multi-enzyme
assemblies holds potentials for the future (McDonald et al. 2007). Enzyme immobilization strategy was in use since the early days of biocatalysis, but it may be more
efficient when the biocatalyst’s surface orientation is controlled. Future protein engineering has to focus on the challenges that emerge through the interfacing of individual biocatalysts with other proteins in a metabolic pathway or support matrices.
‘Peptidomimetics’ is another essential approach that finds utilization in protein
engineering and is considered as an important method for medical and bioorganic
chemistry. It involves imitating or hindering the activity of enzymes or natural peptides upon design and synthesis of peptide analogues that are metabolically stable.
Peptidomimetics is considered as an important approach for medical and bioorganic
chemistry. A variety of synthesis methods such as the use of a common intermediate, solid-phase synthesis, and combinatorial approaches are used in peptidomimetics (Trabocchi and Guarna 2014). Protein engineering also uses another technique
called ‘flow cytometry’, which is an effective approach for single-cell analysis
(Mattanovich and Borth 2006; Wittrup 2001).
Another valuable protein engineering approach which is utilized in remodelling
enzyme properties and function is the ‘designed divergent evolution’, whose methodology works based on the theories of divergent molecular evolution. According to
the theory of divergent evolution, initially enzymes with more specific functions
have evolved from those enzymes with promiscuous functions. Secondly, this process is driven by a few amino acid substitutions; and finally, the effects of double/
multiple mutations are usually additive. Thus, this approach permits the selection of
6 Enzyme Engineering
identifying the consensus sequences and amino acid substitutions that generate stable enzymes. These data can be used to design small libraries with a huge percentage
of catalytically active variants, which have been used to discover enzymes with
highly efficient biocatalytic properties (Jochens and Bornscheuer 2010).
Multiple amino acid substitutions are usually required for large changes in
enzyme properties. More and more powerful screening could be considered as the
simplest solution to this problem. High-throughput methods like fluorescenceactivated cell sorting, which are capable of screening tens of millions of variants in
a short time, can be used to monitor the alterations in substrate specificity (Becker
et al. 2008; Fernandez-Alvaro et al. 2011).
As of now, the perfect way to create multiple mutations is to add them simultaneously, but to limit the choices using statistical or bioinformatics methods. Jochens
and Bornscheuer (2010) used this method to enhance the enantioselectivity of a
Pseudomonas fluorescens esterase. Weinreich and co-workers (2006) studied the
effectiveness of cooperative interactions (mutations) in the development of a
β-lactamase with advanced properties. In this study, the reaction rate was enhanced
by mutation A, but this mutation destabilized the β-lactamase. The overall effect
was slightly advantageous, whereas the reaction rate remained unaffected by mutation B but stabilized the β-lactamase; by itself, it had no effect. In combination,
mutations A and B were greatly advantageous because the β-lactamase showed
higher reaction rate and preserved its stability, but addition of mutations stepwise
will most probably miss these kinds of synergism.
The integration of enzymes with nanomaterials and in complex multi-enzyme
assemblies holds potentials for the future (McDonald et al. 2007). Enzyme immobilization strategy was in use since the early days of biocatalysis, but it may be more
efficient when the biocatalyst’s surface orientation is controlled. Future protein engineering has to focus on the challenges that emerge through the interfacing of individual biocatalysts with other proteins in a metabolic pathway or support matrices.
‘Peptidomimetics’ is another essential approach that finds utilization in protein
engineering and is considered as an important method for medical and bioorganic
chemistry. It involves imitating or hindering the activity of enzymes or natural peptides upon design and synthesis of peptide analogues that are metabolically stable.
Peptidomimetics is considered as an important approach for medical and bioorganic
chemistry. A variety of synthesis methods such as the use of a common intermediate, solid-phase synthesis, and combinatorial approaches are used in peptidomimetics (Trabocchi and Guarna 2014). Protein engineering also uses another technique
called ‘flow cytometry’, which is an effective approach for single-cell analysis
(Mattanovich and Borth 2006; Wittrup 2001).
Another valuable protein engineering approach which is utilized in remodelling
enzyme properties and function is the ‘designed divergent evolution’, whose methodology works based on the theories of divergent molecular evolution. According to
the theory of divergent evolution, initially enzymes with more specific functions
have evolved from those enzymes with promiscuous functions. Secondly, this process is driven by a few amino acid substitutions; and finally, the effects of double/
multiple mutations are usually additive. Thus, this approach permits the selection of
6 Enzyme Engineering