Environmental Engineering Reference
In-Depth Information
the amino acid substitution(s) have had the desired effect. This process is typically iterative,
with multiple rounds of mutation and evaluation [32].
Alternatively, a 3D protein structure can be compared with those of related proteins that
vary in the parameter of interest, either by models listed above or by a number of other
homology modeling programs and online servers (see listing of those currently available at
http://ncisgi.ncifcrf.gov/~ravichas/HomMod/). Comparisons among related proteins with
variation in thermostability, for example, have revealed that higher stability generally
correlates with greater proline, arginine, and tyrosine content; lower asparagine, glutamate,
cysteine, and serine content; increased numbers of salt bridges and stabilizing hydrogen
bonds; and a larger fraction of residues in alpha helices [33]. Unfortunately, however, the
effect of a particular amino acid substitution on a protein's thermostability has not proven to
be highly predictable with current understanding [34], typical of the situation with other
parameters of interest as well. This limits speed and throughput, which then limits the
sequence space amenable to testing. Nevertheless, the understanding of factors influencing
protein folding is increasing rapidly, supported in great part by funding for basic research, and
is expected to continually enhance efforts directed toward enzyme rational design [32].
Rational approaches have also entered an era of de novo design in both enzyme active
sites and catalytic antibodies. In one method, the high-energy state of a reaction is modeled
with a protein or antibody side chain geometrically oriented for catalysis. Then, a library of
rotamers, or low-energy side chain conformations, is generated and the novel active sites are
tested for optimal fit with a carrier or scaffold protein. This approach has been demonstrated
successfully in the design of a novel active site for ester hydrolysis within the otherwise inert
protein, thioredoxin [35]. This promises interesting future results for activities of industrial
interest [36].
Rational design is clearly a time- and information-intensive process, and our
understanding of enzymes is far behind that of small molecules. Still, it has succeeded in the
reconfiguration of substrate specificities in oxidoreductases, hydrolases, transferases, and
DNases; the alteration of cofactor requirements; the inversion of reaction stereochemistry;
and the enhancement of enzyme stabilities under various industrial conditions, as well as the
introduction of novel catalytic activities into existing templates [32]. In addition, sufficient
structural information is accumulating for industrially important enzymes, including lipases
and cellulases as well as hydrogenases, that rational design must be considered as one of the
promising molecular techniques available for bioenergy development.
2.5.5. Analytical mutagenesis
. Mutagenesis is also frequently employed to lend insight
into the functions of metabolic pathways, specific genes, or specific regions within a gene,
rather than to create genes with improved function. For these purposes, mutagenic methods
are employed that frequently eliminate the function of a gene altogether. Transposon
insertional mutagenesis is quite popular for this purpose, acting by the random insertion of
large segments of DNA (the transposons) into a genome. Mutants lacking a function of
interest are then assumed to have received an insertion within a gene essential to that
function, and transposon sequences can then be used to locate the insertion. Sequences
flanking the insertion are then investigated, ideally with the help of annotated genomic
databases, to reveal the likely function of the interrupted gene [21].
If the identity of a gene is known, but the regions of amino acids that are most essential to
the activity of the encoded protein are of interest, a technique known as linker-scanning
