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Discovery of Superior Enzymes by Directed
directed evolution ´ enzymes ´ gene technology ´ molecular evolution ´ mutagenesis
1. IntroductionNatural selection has created optimal catalysts that exhibit their
identify individuals showing the desired properties, either by
convincing performance even with a number of sometimes
selection or by screening (Figure 1). After each round, the genes
counteracting constraints. Optimal performance of enzyme
of improved variants are deciphered and subsequently serve as
catalysis does not refer necessarily to maximum reaction rate.
parents for another round of optimization. This review covers the
Rather, it may involve a compromise between specificity, rate,
most important aspects of directed evolution and summarizes
stability, and other chemical constraints; in some cases, it may
key solutions to problems of optimizing and understanding
involve ªintelligentº control of rate and specificity. Because
enzymes are capable of catalyzing reactions under mildconditions and with high substrate specificity that often is
accompanied by high regio- and enantioselectivity, it is notsurprising that a continually increasing number of industrial and
The route of evolutionary optimization of a certain enzyme can
academic reports concern the use of enzyme catalysts in
be described as an adaptive walk in a ªfitness landscapeº that
chemical synthesis as well as in biochemical and biomedical
consists of peaks (sequences with high fitness) which are
connected by ridges and separated by saddles, valleys (sequen-
However, the demands of modern synthesis and their
ces with low fitness), or planes. The fitness landscape is
commercial application were obviously not targeted during
associated to a ªsequence spaceºÐa network-like arrangement
the natural evolution of enzymes. Considering a specific, non-
of all possible amino acid sequences of a given length.[13, 14]
natural application, any property (or combination of properties)
Under the influence of mutation and selection, the enzyme may
of an enzyme may therefore need to be improved. Of course,
walk along the ridges toward peaks, that is, sequences of higher
scientists desired to mimick nature's powerful concepts for
fitness. However, exhaustive searching of all possible protein
tailoring specific enzymatic properties: Following pioneering
sequences for the individual variant with maximum fitness
experiments for evolving molecules in the test tube,[3±6] evolu-
seems like a daunting undertaking because the sequence space
tionary engineering of biomolecules was successfully realized
even for a protein of moderate sequence length of 100 amino
with first selections of functional nucleic acids (ribozymes) by
acids is extraordinarily large (about 10130 sequences). Taking
using the SELEX (systematic evolution of ligands by exponential
again nature as an example, optimal solutions can be found by
enrichment with integrated optimization by non-linear analysis)
exploring only small fractions of the sequence space: A series of
procedure,[7, 8] and with the development of high-affinity ligands
experimental strategies have been developed for generating
(aptamers) by using similar techniques. Meanwhile, evolutionary
mutant libraries in the laboratory which differ in diversity, that is,
engineering, also termed ªdirected evolutionº, has emerged as a
in the extent of covered sequence space, and in approaching
key technology for biomolecular engineering and generated
intelligent solutions for dealing with complexity.
impressive results in the functional adaptation of enzymes toartificial environments.[9±11] Certainly, evolution in the laboratorydoes not come to a halt at the optimization of single genes and
proteins. Recent results excitingly demonstrate the success of
Random mutagenesis at the nucleotide level is a widespread
ªmolecular breedingº of metabolic pathways, and even of
strategy which targets whole genes. This may be achieved by
complete genomes, and the end is not in sight yet.
passing cloned genes through mutator strains,[16, 17] by treating
Directed evolution in the laboratory is highly attractive
because its principles are simple and do not require detailedknowledge of structure, function, or mechanism. Essentially like
natural evolution, directed evolution comprises the iterative
Max Planck Institute for Biophysical Chemistry
implementation of (1) the generation of a ªlibraryº of mutated
genes, (2) its functional expression, and (3) a sensitive assay to
CHEMBIOCHEM 2001, 2, 865 ± 871 WILEY-VCH-Verlag GmbH, D-69451 Weinheim, 2001 1439-4227/01/02/12 $ 17.50+.50/0
extremely rare. Codon-level random mutagenesis ofcomplete genes therefore would be desirable, but hasnot been realized yet.
Much effort, however, has been devoted to targeting
single amino acids or selected regions of a proteinwhich have been identified in previous experiments tobe important for a certain function. By focusing only onthe positions of interest and/or their close environment,also the size of a mutant library can be drasticallyreduced (so-called ªdoped librariesº). Methods forrandomizing small gene fragments are among theearliest techniques applied to in vitro evolution.
Typically, they employ the substitution of wild-typegene fragments by synthetic oligonucleotides whichcontain random positions or stretches (random cas-settes),[5, 24] or semi-random ranges (spiked oligonu-cleotides). Randomization of defined positions orregions is achieved with automatic solid-phase DNAsynthesis by programming the desired InternationalUnion of Biochemistry (IUB) mix codes. The introduc-tion of stop codons can be excluded by allowing only Gand C (mix code: S) at the third position of each codon.
Complete permutation of a single amino acid position(saturation mutagenesis) may thus enable the findingof nonconservative replacements which are inaccessi-ble by random point mutagenesis. Meanwhile,automatic solid-phase DNA synthesis also allows forthe selective introduction of codon mixtures by usingtrinucleotide b-cyanoethyl phosphoramidites, evenby combining conventional dimethoxytrityl (DMT)protection with 9-fluorenylmethoxycarbonyl (Fmoc)chemistry.
Recombination of DNA represents an alternative or
Figure 1. A) Schematic representation of directed enzyme evolution by in vivo selection.
additional approach for generating genetic diversity
Target genes are mutated and inserted into a plasmid vector to yield a mutant gene library.
that is based on the mixing and concatenation of
After transformation of a suitable host (which is, for example, auxotrophic with respect to the
genetic material from a number of parent sequences.
target function), selective conditions are applied to the culture. Those cells which express
active target gene variants that perform their activity under the applied constraints can
As compared to random mutagenesis, recombination
survive, whereas other cells harboring inactive gene products die out. B) Schematic
may be advantageous in concentrating beneficial
representation of directed evolution by screening. Bacteria harboring mutant target genes
mutations which have arisen independently and may
are plated and subsequently individualized, for example, in a microwell array. The target
be additive, and likewise, in concentrating deleterious
reaction is started with the addition of such substrates that facilitate the detection of
successfully performed reactions, for example, chromogenic substrates. Desired variants are
mutations which subsequently might be more effi-
ciently purged from the population by selection.[29, 30]DNA shuffling was the first technique introduced forrandom in vitro recombination of gene variants created
single-stranded DNA with various chemical mutagens,[18±20] or by
by random mutagenesis. This method employs the PCR
error-prone PCR.[21, 22] Due to its simplicity and versatility, random
reassembly of whole genes from a pool of short overlapping
PCR mutagenesis emerged as the most common technique
DNA sequences (typical length: 100 ± 300 bp) which are gen-
which can result in mutation frequencies as high as 2% per
erated by random enzymatic fragmentation of different parental
nucleotide position. With alterations of some PCR conditions, the
genes. Alternative protocols include StEP (staggered extension
mutation rate may also be adjusted to lower values. However,
process), and random-priming recombination. In vitro
the number of amino acid substitutions accessible by using
recombination by StEP is forced in a PCR-like reaction with very
error-prone PCR is limited because this reaction biases the
short annealing and extension steps that promote the formation
distribution of mutation type in favor of transitions (A > G, T>
of premature extension products. In following cycles, the
C) and because multiple substitutions within a single codon are
truncated strands may anneal randomly to a parent strand, thus
combining the information of different parent strands. As an
Irrespective of whether a protein library is expressed in a
alternative to DNA shuffling, random-priming recombination
recombinant host or displayed on bacteriophage, the available
produces random fragments for reassembly by annealing of
protein diversity is limited by the transformation or transfection
short, random primers to a certain template gene and extension
efficiencies of bacterial, or eukaryotic cells. Furthermore, the
expression of nonhomologous or even toxic proteins may
There is no reason that the concept of in vitro recombination
severely interfere with some host environments. Thus, selection
should be limited to pools of gene variants generated by
may enrich only those cells which survive by reducing or
random mutagenesis. In an extension of the idea, naturally
circumventing the expression of the specific protein, or by
occuring genes showing high similarity in sequence and
preventing the correct protein folding. Cell-free transcription ±
function can serve as an enormous pool of ªinformationº for
translation systems that were recently developed may provide
the creation of new, chimeric enzymes. Recombination of
the basis for protein evolution in the absence of cells: They
homologous parent genes, also called ªfamily shufflingº,
establish a physical genotype ± phenotype linkage in vitro either
could access yet unexplored regions of the sequence space
by stabilizing the mutual attachment of correctly folded
because it combines genetic variability that has already been
complete protein and its encoding mRNA to the ribosome
selected in nature to be functional.
(called ribosome display), by generating covalent fusions
Homologous recombination may also be achieved in vivo.
between a peptide or protein and its mRNA, or by distribution
Most common are methods based on the transformation of
of a library-based transcription ± translation system within
Saccharomyces cerevisiae with a linearized plasmid and target
aqueous droplets in a water-in-oil emulsion.
gene variants. Intermolecular homology-dependent recombina-tion may occur, which yields a circular plasmid that can be
detected by using a selection marker.
Another concept for exploiting natural sources is called
Functional protein libraries can be created rather easily by using
modular protein design. This idea emphasizes the predominance
one of the strategies described above. Therefore, the most
of a limited number of elementary secondary structure units
challenging step in directed evolution experiments is to develop
which could be adopted by protein sequences having a low
a screening or selection scheme that is sensitive to the
degree of similarity. The permutation of protein modules
properties of interest. Selection can be used in vivo if a
requires nonhomologous recombination for generating func-
substantial growth advantage is conferred to those clones that
tional diversity rather than sequence diversity.
harbor a protein variant with the desired improvement. Mostoften, this is achieved by genetic complementation of hosts thatare deficient in a certain pathway or activity. In other cases, the
positive feedback coupling between a property of interest and
cell survival may be achieved with alteration of genetic contexts,
for example, employing enzyme-specific control elements liketranscriptional promoters. Selective enrichment of only those
The classic evolution experiments with RNA and DNA were
clones that express the particular enzyme function can be very
successful in vitro because nucleic acids represent both function
efficient. It should be noticed, however, that in vivo selection
(phenotype) and genetic information (genotype). The directed
systems usually represent highly specific solutions and often are
evolution of enzymes, however, differs insomuch as diversity is
difficult to implement because microbial hosts are extremely
created on the DNA level, but selection or screening act on the
flexible in circumventing the applied constraints and in invent-
level of encoded protein. Therefore, functional expression of the
ing solutions that are not necessarily related to the targeted
information-carrying DNA libraries, whether generated by ran-
dom mutagenesis or by recombination, is a necessary prereq-
In vitro enrichment procedures that are detached from cell
uisite for the detection of improved enzyme variants. The most
survival may also be termed selection. Originally, these tech-
common approaches for recombinant protein expression em-
niques have been developed for the ªbiopanningº of phage-
ploy the cellular trancription and translation machineries of well-
displayed peptide libraries by binding to a ligand that is
established organisms such as Escherichia coli, Saccharomyces
immobilized on an appropriate column matrix. Recently, the
cerevisiae, or Bacillus subtilis. These cellular expression systems
approach has also been applied to the selective enrichment of
also guarantee the association of a specific protein variant and
phage-displayed functional enzyme libraries. Therefore, the idea
its encoding gene. This is essential for the identification and
of panning optimal binding partners needs to be extended to a
amplification of desired mutants after selection or screening, as
panning of optimal catalysts by using either transition state
well as for further cycles of evolution. Alternatively, a physical
analogues, immobilized suicide substrates, or reaction
link between genotype and phenotype may be established by
substrates that are covalently linked to the same or another
generating fusions between the protein of interest and a
phage via a second fusion.[45±47] However, the enrichment of
bacteriophage coat protein. Following intracellular assembly,
improved enzymes by biopanning remains challenging because
recombinant phages express the protein variants on their
the assessment of phage-displayed enzymes on the basis of their
surface while enclosing the appertaining genetic information
catalytic activity, that is, on the basis of their kinetic parameters,
4. Successful application of directed evolution
Screening is an important alternative to selection which requires
During the past few years, many enzymes have been improved
that the specific property is directly observable by using physical
by directed evolution (Table 2). Some of these new biocatalysts
or biochemical analysis. As compared with in vivo selection, the
are tuned for use in organic synthesis, and commercial
screening approach enables a better control of the applied
applications of some other enzymes are already in sight.
constraints, and also is more versatile, predominantly in
Enzymes that exhibit increased activity in aqueous-organic
unnatural environments, or with unnatural substrates. The
solvents were among the first products of directed-evolution
number of individual mutants that can be tested in a certain
experiments.[52, 53] These biocatalysts enable the performance of
period of time (throughput), however, may be lowerÐdepend-
reactions at increased substrate solubility and stability, and thus
ing on the enzymatic reaction and the sensitivity of the applied
effect altered reaction equilibria, higher reaction rates, and
higher product yields. The directed evolution of a large number
The comparative assessment of individual mutants usually
of enzymes that exhibit increased performance at elevated
requires that the mutant libraries are diluted and distributed.
temperatures has been driven by a similar motiva-
This can either be achieved by conventional plating of trans-
tion.[26, 32, 54±58, 60±62] Furthermore, increased thermostability may
formants on agar plates or filter membranes, or by distribution of
be beneficial regarding the long-term stability of proteins at
the mutant pool in a microtiter format (usually 96- or 384-well
plates, but also formats with higher sample density, including
Directed evolution has also been employed to improve the
silicon wafers). This time-consuming step is sometimes accel-
expression and folding of recombinant eukaryotic enzymes
erated by using robotic systems. Common assays are based on
which fail to adopt their active conformation in a heterologous
visual or spectroscopic detection, for example formation, alter-
host, or which are expressed in an artificial context, for example,
ation, or destruction of colors or fluorescence characteristics. The
in the form of a fusion protein.[63±66] Likewise, the secretion of
determination of the optical parameters can also be accom-
correctly folded enzymes has been facilitated by using evolu-
plished by using automatic plate-reading systems, which enable
tionary optimization. In most of these cases, the increased
a normalization of measured intensity values to the respective
levels of expression and native folding have been attributed to
cell densities and, furthermore, may be used for monitoring
few point mutations within the structural genes.
reaction kinetics. There is an increasing tendency toward
The narrow range of substrates that are accepted by natural
automation and parallel processing by using decreasing sample
enzymes often retards or prevents their use in new synthetic and
volumes and concentrations, for example by applying the highly
commercial applications. By far most results therefore reflect the
sensitive fluorescence correlation spectroscopy (FCS) technique,
efficient tuning of catalytic efficiency toward nonnatural sub-
which requires concentrations in the femtomolar range.
strates.[34, 42, 43, 65, 68±88] Similarly, the enantioselectivity of specific
Alternative approaches like confocal fluorescence coincidence
bioconversions has been significantly improved by using evolu-
analysis (CFCS) or a fluorescence-activated cell sorter (FACS)
tionary approaches.[89±91] Enzymes with altered substrate speci-
can directly analyze single cells or proteins and, thus, gain
ficity that yield yet inavailable products have also recently been
(ultra)high throughput by avoiding the transfer of individuals.
generated by using the molecular breeding of new biosynthetic
Disregarding whether a specific directed-evolution experi-
pathways. Together with similar attempts at mixing subunits
ment employs a selection or screening approach (for a
of multi-enzyme complexes, this approach opens up the
comparison, see Table 1), it should finally be emphasized that
horizon toward new biologically active compounds.
it is important to choose selective constraints that precisely
The conversion of a specific enzymatic activity into another
reflect the desired property. Otherwise, ªyou get what you screen
has very recently been achieved by using the methods of
directed evolution, and by using a combination of rational and
Table 1. Comparison of strategies for searching improved biocatalysts.
selection linkage between desired activity and cell survival
false positives (viable but undesired mutants)
screening individualization of mutant clones; in some cases, direct testing of each single clone for the
multiple pipetting/washing/transfer steps
isolation of mutant proteins from competitive
cellular activitiesoften: need for fluorogenic or chromogenic sub-
assays in nonnatural environments (artificial
low throughput (ca. 105 individual mutants in
qualitative and quantitative assay of one or
Table 2. Examples of enzymes that were successfully optimized by using directed evolution.[a]
suppressor tRNAs with diverse ribosomally
(1) increased activity toward b-branched amino
activity toward polychlorinated biphenyls (PCBs) degradation of various PCBs, polychlorinated benzene,
thermostability at 658C without decrease in
b-glucosidase CelB (Pyrococcus furiosus) increased catalytic activity at 208C
retention of function after glutaraldehyde
increased stability toward glutaraldehyde and
inactivation of 20000-fold higher concentration of cefo-
sixfold higher PRAI activity than wild-type enzyme
PRAI activity while retaining ProFARI activity
PRAI activity 3 ± 11 Â 104 lower than wild-type; ProFARI
increase in intrinsic peroxidase activity
activity of monomeric and hexameric enzyme
increased activity toward naphthalene in the
enantioselectivity and increased activity
conversion into L-hydantoinase, 5-fold activity
316-fold decrease in LD100 of transformed E. coli in the
presence of AZT,[b] 11-fold decrease with ddC[b]
hydrolysis of sterically hindered 3-hydroxy esters activity with an increase in enantiomeric excess from
increased thermal and oxidative stability
174-fold thermostability, 100-fold oxidative stability
1000-fold increase in specificity towards p-nitrophenyl
furanoside, 66-fold increase in specific activity
activity toward a range of new substrates
DNA-dependent DNA polymerase activity and resistance 
increased activity toward ABTS[b] and guaiacol
total, 40-fold; ABTS, 5.4-fold; guaiacol, 2.3-fold
(B. subtilis)kanamycin nucleotidyl transferase
(1) 200-fold increase in t1/2 at 60 ± 658C
increased activity in the absence of cofactor
increase in enantiomeric excess from 2% to 81%
lipases (Staphylococcus hyicus, S. aureus) substrate specificity (phospholipids vs. short-
3-fold increase in activity toward long-chain pNB[b] esters 
substrate specificity (activity on phospholipids)
11.6-fold increase in abolute phopholipase activity,
11.5-fold increase in phospholipase/lipase ratio
6-fold increase in yield of D-amino acids
fusion proteinO6-alkylguanine alkyltransferase
reduction of BG inhibitory concentration to 50%
2.7 ± 5.5-fold decrease in mutation frequency
phytoene desaturase, lycopene cyclase new carotenoid pathway
synthesis of 3,4,3',4'-tetradehydrolycopene and torulene
148C increase in Tm without any decrease in activity at
increased activity in aqueous/organic solvents
50 ± 150-fold activity toward different pNB esters in
6-fold activity, while retaining 3.3-fold lower activity at 708C 
altered specificity and increased activity
20 ± 45-fold increase in rate and a greater selectivity
increased activity at decreased temperature
178C increase in Topt, increased activity at all temperatures 
functional complementation of E. coli DNA
retention of activity of active-site mutants
43-fold toward ganciclovir, 20-fold toward aciclovir
[a] See also ref. . [b] Abbreviations: ABTS 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid; AZT 3'-azido-3'-deoxythymidine; BG O6-benzylgua-
nine; ddC dideoxycytidine; IGPS indoleglycerol phosphate synthase; pNB para-nitrobenzoate; PRAI phosphoribosyl anthranilate isomerase; ProFARI
N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase.
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ARTICLE IN PRESS The Journal of Emergency Medicine, Vol. xx, No. x, pp. xxx, 2009Copyright © 2009 Published by Elsevier Inc. doi:10.1016/j.jemermed.2008.06.029 Original Contributions EFFICACY AND COST COMPARISONS OF BRONCHODILATATOR ADMINISTRATION BETWEEN METERED DOSE INHALERS WITH DISPOSABLE SPACERS AND NEBULIZERS FOR ACUTE ASTHMA IN AN INNER-CITY ADULT POPULATION Su
INFORMED CONSENT FOR CHEMICAL EXFOLIATION TREATMENT Please read and initial after each paragraph. I have been given the Skin Care History Questionnaire and have read and answered the I have discussed any further questions and or concerns that I may have with my Skin Care My Skin Care Specialist has answered any questions I have regarding my post care. I acknowledge my obligations to clo