155
Generally, the primary structure, i.e., the amino acid sequence, determines the
structure and function of an enzyme molecule or of any protein molecule. It is
important to understand about the critical regions of the molecule for the various
activities of an enzyme and thereby enable to predict the effect of change in specific
amino acids in these areas on different functions. Still the current information about
the relationships between amino acid sequence and the structure and properties of
enzymes obtained from the available database is only partially operative. Enzyme
engineering provides an explanation of the alterations in the structure and function
on the basis of the modifications in amino acid sequence, but it does not provide a
dependable prediction of the influences of specific amino acid changes on the structure and function of enzymes. It might be expected that as more improved softwares
and elaborate databases become available, the structural and functional modifications in enzymes produced by the specified alterations in their amino acid sequences
can be predicted with far greater confidence. Thereby the capability of enzyme
engineering will be enormously enhanced and may have an amazing influence on
enzyme technology.
6.3.2 Steps in Engineering
Protein engineering generally involves three steps: choosing a change in the protein
(engineering strategies, such as rational design or randomization), accomplishing
those modifications (mutagenesis), and evaluating the protein variants for enhanced
properties (screening or selection) (Kazlauskas and Bornscheuer 2009). Selection
of different strategies can lead to a variety of advantages or disadvantages in each of
these steps. The selection of the outstanding method is driven by limitations such as
structural information, various tools and techniques for mutagenesis, and methods
for screening or selection. Many different strategies have been used by protein engineers, and most of those strategies will ultimately yield an improved protein.
Increasing the thermostability of an enzyme is one of the common protein engineering goals. Structure-based approaches, like crystallographic and nuclear magnetic
resonance (NMR) studies, presume that a more rigid enzyme will be more stable at
high temperatures. The structure of an enzyme derived by X-ray crystallography
can be employed to design specific stabilizing interactions such as disulphide bonds
or salt bridges, stabilize the loop regions by removing glycine or introducing proline, or find the most flexible region of the target proteins from the experimental
factors and focus mutagenesis at those regions. An evolution- and bioinformaticsbased approach has the underlying belief that the stability of protein is contributed
by conserved amino acids. The engineering strategy is to analyse similar sequences
and engineer the target protein to simulate the consensus sequence.
Protein engineering generally utilizes two different approaches: rational design
and directed evolution. To create appropriate modifications via site-directed mutagenesis, in rational design, the structure, function, and catalytic mechanism of the
protein must be well understood. However, such acquaintance is absent for most
proteins of interest. According to Bloom and Arnold (2009), even though
6 Enzyme Engineering
Generally, the primary structure, i.e., the amino acid sequence, determines the
structure and function of an enzyme molecule or of any protein molecule. It is
important to understand about the critical regions of the molecule for the various
activities of an enzyme and thereby enable to predict the effect of change in specific
amino acids in these areas on different functions. Still the current information about
the relationships between amino acid sequence and the structure and properties of
enzymes obtained from the available database is only partially operative. Enzyme
engineering provides an explanation of the alterations in the structure and function
on the basis of the modifications in amino acid sequence, but it does not provide a
dependable prediction of the influences of specific amino acid changes on the structure and function of enzymes. It might be expected that as more improved softwares
and elaborate databases become available, the structural and functional modifications in enzymes produced by the specified alterations in their amino acid sequences
can be predicted with far greater confidence. Thereby the capability of enzyme
engineering will be enormously enhanced and may have an amazing influence on
enzyme technology.
6.3.2 Steps in Engineering
Protein engineering generally involves three steps: choosing a change in the protein
(engineering strategies, such as rational design or randomization), accomplishing
those modifications (mutagenesis), and evaluating the protein variants for enhanced
properties (screening or selection) (Kazlauskas and Bornscheuer 2009). Selection
of different strategies can lead to a variety of advantages or disadvantages in each of
these steps. The selection of the outstanding method is driven by limitations such as
structural information, various tools and techniques for mutagenesis, and methods
for screening or selection. Many different strategies have been used by protein engineers, and most of those strategies will ultimately yield an improved protein.
Increasing the thermostability of an enzyme is one of the common protein engineering goals. Structure-based approaches, like crystallographic and nuclear magnetic
resonance (NMR) studies, presume that a more rigid enzyme will be more stable at
high temperatures. The structure of an enzyme derived by X-ray crystallography
can be employed to design specific stabilizing interactions such as disulphide bonds
or salt bridges, stabilize the loop regions by removing glycine or introducing proline, or find the most flexible region of the target proteins from the experimental
factors and focus mutagenesis at those regions. An evolution- and bioinformaticsbased approach has the underlying belief that the stability of protein is contributed
by conserved amino acids. The engineering strategy is to analyse similar sequences
and engineer the target protein to simulate the consensus sequence.
Protein engineering generally utilizes two different approaches: rational design
and directed evolution. To create appropriate modifications via site-directed mutagenesis, in rational design, the structure, function, and catalytic mechanism of the
protein must be well understood. However, such acquaintance is absent for most
proteins of interest. According to Bloom and Arnold (2009), even though
6 Enzyme Engineering