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Protein engineering via directed evolution or rational design has developed as a very
powerful mechanism to design and modify the properties of enzymes. The use of
computational tools in combination with directed evolution and rational protein
design is becoming more and more important in order to explore enzyme sequence
space and to develop modified or unique enzymes. The advancements in these areas
should support in expanding the application of microbial enzymes in industry.
This engineering technology rapidly found its use for a wide range of proteins,
especially in the field of biocatalysis. The application of directed evolution has been
made more easy and feasible with the development of a diverse set of molecular
biology tools to create well-balanced mutant libraries as well as suitable highthroughput screening methods. Directed evolution has developed as a standard
methodology in protein engineering, which can be used complementarily or in combination with rational protein design to meet the various demands for industrially
applicable biocatalysts.
The technology of enzyme engineering has enormous, virtually limitless, potential. But the realization of this potential is limited by several factors. An awareness
of the three-dimensional structure of an enzyme provides the basis for deciding the
changes to be made in the amino acid sequence of the enzyme. Data on this aspect
of many enzymes are not available at present; this limits the application of enzyme
engineering to such enzymes. At present, the knowledge of the exact interactions,
viz., hydrogen bonding, electrostatic interactions, and hydrophobic interactions,
between the active site of an enzyme and the substrate of this enzyme is, at best,
incomplete. This knowledge is essential for the prediction of the effect of a change
in a specific amino acid of the active site of an enzyme. Various factors that determine the stability of proteins are not well known. But salt bridges and other electrostatic interactions are known to confer thermostability. Therefore, many enzyme
engineering experiments are designed to generate these data, which are then used to
plan further experiments.
As the basis of protein engineering is established well, eventually protein engineering will shift towards rational design. Identification of the best protein engineering strategy will help in the advancement of protein engineering more quickly.
If we compare different strategies, solutions can be attained with less effort. It is
unlikely that one strategy will prevail, as each problem differs in its objectives,
amount of information available, and specificities of the protein. Comparison of
different strategies will also help in establishing fundamentals of protein engineering and enhancing our knowledge of enzyme mechanism. This knowledge will
make rational design more reliable and further speed up the path to solutions.
Regardless of the advances in the field of enzyme engineering, major challenges
still prevail to harnessing the benefits of biocatalysis completely. Even though the
field of enzyme engineering has advanced much rapid than a decade ago, mutating
30–40 amino acids and screening tens of thousands of samples still need an enormous research team. Most of the engineering strategies yield improved variants and
help to find them quicker, but decision on the selection of better strategies still
remains unclear. Direct comparison of different strategies for similar problems and
examination of assumptions behind different strategies will help in identifying the
most powerful or productive ones.
S.M. Basheer and S. Chellappan
Protein engineering via directed evolution or rational design has developed as a very
powerful mechanism to design and modify the properties of enzymes. The use of
computational tools in combination with directed evolution and rational protein
design is becoming more and more important in order to explore enzyme sequence
space and to develop modified or unique enzymes. The advancements in these areas
should support in expanding the application of microbial enzymes in industry.
This engineering technology rapidly found its use for a wide range of proteins,
especially in the field of biocatalysis. The application of directed evolution has been
made more easy and feasible with the development of a diverse set of molecular
biology tools to create well-balanced mutant libraries as well as suitable highthroughput screening methods. Directed evolution has developed as a standard
methodology in protein engineering, which can be used complementarily or in combination with rational protein design to meet the various demands for industrially
applicable biocatalysts.
The technology of enzyme engineering has enormous, virtually limitless, potential. But the realization of this potential is limited by several factors. An awareness
of the three-dimensional structure of an enzyme provides the basis for deciding the
changes to be made in the amino acid sequence of the enzyme. Data on this aspect
of many enzymes are not available at present; this limits the application of enzyme
engineering to such enzymes. At present, the knowledge of the exact interactions,
viz., hydrogen bonding, electrostatic interactions, and hydrophobic interactions,
between the active site of an enzyme and the substrate of this enzyme is, at best,
incomplete. This knowledge is essential for the prediction of the effect of a change
in a specific amino acid of the active site of an enzyme. Various factors that determine the stability of proteins are not well known. But salt bridges and other electrostatic interactions are known to confer thermostability. Therefore, many enzyme
engineering experiments are designed to generate these data, which are then used to
plan further experiments.
As the basis of protein engineering is established well, eventually protein engineering will shift towards rational design. Identification of the best protein engineering strategy will help in the advancement of protein engineering more quickly.
If we compare different strategies, solutions can be attained with less effort. It is
unlikely that one strategy will prevail, as each problem differs in its objectives,
amount of information available, and specificities of the protein. Comparison of
different strategies will also help in establishing fundamentals of protein engineering and enhancing our knowledge of enzyme mechanism. This knowledge will
make rational design more reliable and further speed up the path to solutions.
Regardless of the advances in the field of enzyme engineering, major challenges
still prevail to harnessing the benefits of biocatalysis completely. Even though the
field of enzyme engineering has advanced much rapid than a decade ago, mutating
30–40 amino acids and screening tens of thousands of samples still need an enormous research team. Most of the engineering strategies yield improved variants and
help to find them quicker, but decision on the selection of better strategies still
remains unclear. Direct comparison of different strategies for similar problems and
examination of assumptions behind different strategies will help in identifying the
most powerful or productive ones.
S.M. Basheer and S. Chellappan