Industrial Biotransformations

4.4 Improvements to Enzymes by Molecular Engineering Techniques
later in 1962, together with Perutz, for their studies on the structures of globular proteins.
Since that time the number of novel protein and peptide structures solved by X-ray diffraction
and, more recently, by NMR spectroscopy has reached 30 800 (May 2005); as a consequence
thereof, our knowledge of enzyme architecture and functionality has been improved considerably.
From 1971 onwards, these biological macromolecular structures have been deposited
in the Protein Data Bank (PDB) at Brookhaven National Laboratories [25]. All deposited structures
are available via the internet (http://www.rcsb.org/pdb/).
In addition, methods in the field of recombinant DNA are becoming more and more
“state-of-the-art” technology and widely used in the natural sciences (Table 4.1). In particular,
the polymerase chain reaction (PCR) developed by Mullis (Nobel Prize in 1993) simplifies
the amplification of enzyme coding genes significantly and also the introduction
of site directed mutations [8, 26].
Using the novel techniques of recombinant DNA and the structural knowledge of the
phage T4 lysozyme, in 1988 Matsumura and coworkers published their ground breaking
results on enzyme stabilization by molecular engineering. In site-directed mutagenesis
experiments, two, four or six amino acid residues, spatially close to each other on the
surface of the natural enzyme, were changed into cysteine residues. Consequently, the
variant enzymes contained one, two or three disulfide bonds, hampering the thermal
unfolding of the native enzyme structure. The triple-disulfide variant unfolds at a temperature
23.4 °C higher than the wild-type lysozyme [27–29].
Many examples using the rational design approach to increase enzyme stability (temperature,
pH, organic solvents) or specific activity followed [30]. Successful examples of
rational enzyme design to improve or invert enantioselectivity are relatively rare. However,
Pleiss and coworkers have reported an improvement in the enantioselective hydrolysis
of linalyl acetate by B. subtilis p-nitrobenzyl esterase variants, as predicted by computer
simulations. Furthermore, an inverted enantiopreference using 2-phenyl-3-butin-2yl-acetate
as the model substrate was achieved [31]. Other successful examples of
improved enantioselectivities by rational protein engineering have been presented by the
group working with Hult (Department of Biotechnology, Royal Institute of Technology,
Stockholm, Sweden) on Candida antarctica lipase B (CALB) [32–35] and the group with
Raushel (Department of Chemistry, Texas A&M University, USA) on phosphotriesterase
from Pseudomonas diminuta [36, 37]. Here at least two variants were created showing a
million-fold difference in enantioselectivity towards the substrate ethyl phenyl p-nitrophenyl
phosphate [37].
Nevertheless, there are still fundamental problems when applying rational enzyme
design: (a) the three-dimensional structure of the enzyme and (b) ideas with respect to
the molecular functions of certain amino acid side chains must be available when
rational design has to be applied. An alternative might be the development of a reliable
structural model based on related enzymes. (c) In general it is not possible to predict
exactly the final structure of a variant enzyme using computer simulations; however, the
methods are continuously improving, and include theoretical methods using combined
quantum mechanical and molecular mechanical calculations (QM/MM) [38]; however, in
all simulations the effect of the enzyme dynamics are neglected. (d) Furthermore, solid
state structures derived from crystallography could be different from protein structures
in solution.
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