163
Even though a large database is available for sequence–structure correlations,
which grows expeditiously along with advanced softwares, currently it is inadequate to predict three-dimensional changes arising as a result of various substitutions. The major hurdle in engineering is in evaluating the overall effects on the new
structure. Hence, protein engineering is currently considered a random process
which may be utilized with only little realistic possibility for immediate success.
Probably quite small alteration in sequence may bring about considerable conformational changes and even affect the rate-determining step in the enzymatic catalysis. Nevertheless, it is reasonable to expect that, if an adequately accurate database
plus appropriate software are provided, the relative possibility of success will boost
in the future, and the products of protein engineering will provide a major impact on
enzyme technology.
Considerable effort has been spent on engineering more thermophilic enzymes.
The enzymes from thermophiles are usually only 20–30 kJ more stable than their
mesophilic counterparts. The thermophilic property can be attained by the inclusion
of few extra hydrogen bonds, an internal salt link, or extra internal hydrophobic
residues, giving a slightly more hydrophobic core. All of these modifications are
less enough to be accomplished by protein engineering. The secondary structure of
the enzyme must be conserved, in order to establish a more predictable outcome.
Wherever allowable, small increases in the interior hydrophobicity may also
increase the thermostability by substituting interior glycine or serine residues with
alanine. It should be understood that creating an enzyme more thermostable reduces
its overall flexibility and, hence, it is possible that the artificial enzyme thus produced will have decreased catalytic potential.
In the food and feed industry, the integration of enzymes has been a strong
approach, but it is clear that dedicated research efforts should be constantly provided to make this application of biological agents more effective and/or diversified.
In spite of the success obtained in the field of enzyme engineering, there is a deficiency of a set of universally applicable rules, including both technical and economic requirements. However, it can be anticipated that attempts will be made
towards developing novel immobilized enzymes with relevant physical, chemical,
and geometric features that can be used in various reactor configurations and that
respond to the economic requirements for large-scale utilization.
According to Behe and Snoke (2004), when the desired modification involves a
concurrent alteration in several amino acids, it is not expected to be achieved by the
random approach. In several cases, the better option is a combination of approaches
to develop the necessary structure or function and its improvement by random techniques. Park et al. (2006) modified a metallohydrolase through designed deletion
and insertion of various structural loops in the active site to form a novel enzyme
with a diverse catalytic function and then applied random techniques to enhance the
designed activity. Bloom et al. (2006) established that the stability of the protein
scaffold enhances its evolvability. Specifically, when the stability of a protein scaffold is increased, it may be desirable to make mutations that create a truly new
property. This approach can be very hopeful in finding new enzymatic solutions that
otherwise can be missed in the functional screening.
6 Enzyme Engineering
Even though a large database is available for sequence–structure correlations,
which grows expeditiously along with advanced softwares, currently it is inadequate to predict three-dimensional changes arising as a result of various substitutions. The major hurdle in engineering is in evaluating the overall effects on the new
structure. Hence, protein engineering is currently considered a random process
which may be utilized with only little realistic possibility for immediate success.
Probably quite small alteration in sequence may bring about considerable conformational changes and even affect the rate-determining step in the enzymatic catalysis. Nevertheless, it is reasonable to expect that, if an adequately accurate database
plus appropriate software are provided, the relative possibility of success will boost
in the future, and the products of protein engineering will provide a major impact on
enzyme technology.
Considerable effort has been spent on engineering more thermophilic enzymes.
The enzymes from thermophiles are usually only 20–30 kJ more stable than their
mesophilic counterparts. The thermophilic property can be attained by the inclusion
of few extra hydrogen bonds, an internal salt link, or extra internal hydrophobic
residues, giving a slightly more hydrophobic core. All of these modifications are
less enough to be accomplished by protein engineering. The secondary structure of
the enzyme must be conserved, in order to establish a more predictable outcome.
Wherever allowable, small increases in the interior hydrophobicity may also
increase the thermostability by substituting interior glycine or serine residues with
alanine. It should be understood that creating an enzyme more thermostable reduces
its overall flexibility and, hence, it is possible that the artificial enzyme thus produced will have decreased catalytic potential.
In the food and feed industry, the integration of enzymes has been a strong
approach, but it is clear that dedicated research efforts should be constantly provided to make this application of biological agents more effective and/or diversified.
In spite of the success obtained in the field of enzyme engineering, there is a deficiency of a set of universally applicable rules, including both technical and economic requirements. However, it can be anticipated that attempts will be made
towards developing novel immobilized enzymes with relevant physical, chemical,
and geometric features that can be used in various reactor configurations and that
respond to the economic requirements for large-scale utilization.
According to Behe and Snoke (2004), when the desired modification involves a
concurrent alteration in several amino acids, it is not expected to be achieved by the
random approach. In several cases, the better option is a combination of approaches
to develop the necessary structure or function and its improvement by random techniques. Park et al. (2006) modified a metallohydrolase through designed deletion
and insertion of various structural loops in the active site to form a novel enzyme
with a diverse catalytic function and then applied random techniques to enhance the
designed activity. Bloom et al. (2006) established that the stability of the protein
scaffold enhances its evolvability. Specifically, when the stability of a protein scaffold is increased, it may be desirable to make mutations that create a truly new
property. This approach can be very hopeful in finding new enzymatic solutions that
otherwise can be missed in the functional screening.
6 Enzyme Engineering