Protein engineering from a bioindustrial point of view

418 Protein engineering
a/. [9] have successfully applied directed evolution to
improve the whole cell fluorescence by green fluorescent
protein, which is widely used as a reporter for gene
expression and regulation.
An evolutionary approach has been used by You and
Arnold [lo] to give a 471-fold increase in the activity
of subtilisin E in 60% aqueous dimethyl formamide.
Their paper was important in demonstrating the possibility
of engineering enzymes to perform in organic solvents,
very different conditions from their natural environments.
Building upon this work, this group applied the directed
evolution approach to develop an esterase capable of
carrying out a particular deprotection step of an antibiotic
synthesis in aqueous-organic solvents [ll”]. At the time
of writing, two quite different ways of implementing
directed evolution have been published. The Arnold
group [lO,l l**] screens through single and then higher
multiples of variants to cover sequence space systemati-
cally. Stemmer’s group [6-91 relies on examining large
numbers of variants with multiple mutations using high
definition screens or selection to find the best one. The
preferred approach may depend on the nature of the
problem and the type of selection or screens that can be
developed. In fact, the development of high-throughput
screening methods along with screens that accurately
reflect the property that is being engineered for final use,
is a critical area for success when using directed evolution.
Not many publications of screening methods are available,
but a paper describing a simple and effective screening
method for proteases has recently been published [12’].
Sources of material to be engineered
In addition to the substantial effort made by enzyme
suppliers and industrial users to improve existing enzymes,
extremeophiles are being aggressively pursued to provide
new enzymes [13] that are highly thermostable, salt
tolerant, cold active, and so on, depending on the
environment of the native organism. Very interesting
enzymes may be isolated from bacteria on a worm that
resides near thermal vents in the ocean, and are expected
to have a much broader temperature profile of activity
than normal mesophilic or thermophilic enzymes [14].
Such enzymes could function effectively across the variety
of temperature conditions encountered in global laundry
applications.
The de nom design of proteins, although very challenging,
offers the broadest possibilities for new structures. In one
of the first practical applications, a de nova four helix
bundle protein [15] was designed to serve as a source
of scarce amino acids (methionine, threonine, lysine and
leucine) when expressed in the rumen bacteria of dairy
cows [16]. A related concept is to create small protein-like
molecules, either by copying natural motifs [17] or via
templates [l&19], which can serve as ‘molecular scaffolds’
for groups introducing functional properties. For example,
a natural scaffold consisting of the 37 amino acid toxin
from scorpions has been used to design a metal-bindi
site that mimics the one found in carbonic anhydrase [Zr
The successful introduction of the metal-binding si
supports the idea that protein engineering in this mann
may yield functional materials in the future.
Tailoring fundamental properties
Stability
Since the conditions in which industrially importa
proteins are used often differ from their natural e
vironment, stability is a necessary consideration 1
most engineered proteins. The introduction of disulfi
bonds [Zl], chemical cross-links [Z?‘], and salt bridg
[23] has been widely used to increase stability, althou,
not all disulfide bonds increase stability. Recent wo
on barnase by X-ray crystallography [24] and hydrog
exchange [‘25] shows that those disulfide bonds that do n
improve stability disrupt the local structure. The definiti
of an effective salt bridge interaction has perhaps be
sharpened by the recent observation by Tanner et al. [Z!
By comparing a number of glyceraldehyde phosphz
dehydrogenase structures from organisms with differe
heat tolerance they found a strong correlation betwe
increased thermostability and the number of hydrog
bonds involving charged sidechains and neutral partne
On the other hand, for the Arc protein [27], a buried s
bridge between arginine and glutamic acid was found
provide less stability than hydrophobic residues. Althou
not necessarily contradictory to Tanner’s observation
since this is presumably a charge+harge salt bridge, it dc
suggest that the contribution of salt bridges to stabil
remains unresolved.
Activity
Although improving the activity of an industrial enzyr
is often a primary goal, it is also one of the mc
complex. This is partly because in many applicatia
of enzymes the substrate is chemically complex a
heterogeneous. It is reassuring in such a context to fi
the principle of transition state stabilization verified
a relatively simple system. In a study of the hydroly
of acetylcholine by acetycholinesterase [ZS], the cataly
acceleration corresponding to 18 kcal mol-1 of transiti
state stabilization could be accounted for in terms
specific molecular interactions, as identified in the crys
structure of a transition state analog inhibitor with TOQC
californica acetylcholinesterase. A quantitative method
analyze the similarity of substrates and inhibitors, w
respect to their enzyme stabilized transition states,
the purpose of designing more effective transition st;
inhibitors has also recently been published [29].
For more complex reactions, such as the hydrolysis
protein amide bonds by a protease, much less is knov
In a revealing study on the proteolysis of variants o
single chain Monellin (a sweet protein consisting of
amino acids), the extent of proteolysis at a fixed til
correlated with the free energy of protein unfoldi