137
The efficient application of biocatalysts requires the availability
of suitable enzymes with high activity and stability under
process conditions, desired substrate selectivity and high
enantioselectivity. However, wild-type enzymes often need to be
optimized to fulfill these requirements. Two rather contradictory
tools can be used on a molecular level to create tailor-made
biocatalysts: directed evolution and rational protein design.
Addresses
*Institute for Chemistry & Biochemistry, Department of Technical
Chemistry & Biotechnology, Greifswald University, Soldmannstrasse 16,
D-17487 Greifswald, Germany;
e-mail: bornsche@mail.uni-greifswald.de
†MPB Cologne GmbH, Neurather Ring 1, D-51063 Köln, Germany;
e-mail: m.pohl@mpb-cologne.com
Current Opinion in Chemical Biology 2001, 5:137–143
1367-5931/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
E enantioselectivity
ee enantiomeric excess
epPCR error-prone PCR
IGPS indole-3-glycerol phosphate synthase
PCR polymerase chain reaction
PRAI phosphoribosylanthranilate isomerase
SDM site-directed mutagenesis
Introduction
The application of enzymes — especially in organic synthesis
— is now well documented in the literature [1•,2–4].
In the past decade, a considerable number of processes
have been commercialized in industry [5•]. Characteristic
features of many biocatalysts, such as high chemo-, regioand
stereoselectivity at ambient temperatures, often
makes them superior to chemical catalysts. In addition,
recent progress in genetic-manipulation techniques
enables the large-scale supply of many enzymes at reasonable
prices. However, identification of new biocatalysts
(for example, by screening of soil samples or strain collections
by enrichment cultures) does not always yield
suitable enzymes for a given synthetic problem. To overcome
this limitation, tailor-made biocatalysts can be
created from wild-type enzymes by protein engineering
using computer-aided molecular modeling and site-directed
mutagenesis, or by directed (molecular) evolution
techniques (Figure 1; see also Update).
Rational design usually requires both the availability of the
structure of the enzyme and knowledge about the relationships
between sequence, structure and mechanism/function,
and is therefore very information-intensive. On the other
hand, rapid progress in solving protein structures by NMR
spectroscopy instead of by X-ray diffraction of crystals and
the enormously increasing number of sequences stored in
public data bases have significantly eased access to data and
structures. Using molecular modeling, it has been possible to
predict how to increase the selectivity, activity and the stability
of enzymes, even if there are no structural data
available and the structure of a homologous enzyme is used
as a model. For details concerning the potential of this
method, readers are referred to a recent review [6•].
Depending on the purpose of the mutagenesis, amino acid
substitutions are often selected by sequence comparison
with homologous sequences. The results have to be carefully
interpreted, however, because minor sequence changes by
a single point-mutation may cause significant structural disturbance.
Thus, comparison of the three-dimensional
structures of mutant and wild-type enzymes are necessary to
ensure that a single mutation is really site-directed.
In sharp contrast, directed evolution (also called evolutive
biotechnology or molecular evolution [7–12]; see also
Update) involves either a random mutagenesis of the gene
encoding the catalyst (e.g. by error-prone PCR) or recombination
of gene fragments (e.g. derived from DNase
degradation, the staggered extension process or random
priming recombination). Libraries thus created are then
usually assayed using high-throughput technologies to
identify improved variants.
For both approaches to protein engineering, the gene(s)
encoding the enzyme(s) of interest, a suitable (usually
microbial) expression system, and a sensitive detection
system are prerequisites.
In this review, we present successful examples of the creation
of suitable biocatalysts, concentrating on those
published since 1999. Methods for the generation of
mutant libraries as well as principles of screening or selection
are out of the scope of this article and readers are
referred to the references given above.
Directed evolution
Mutants of an esterase from Pseudomonas fluorescens produced
by directed evolution using the mutator strain
Epicurian coli XL1-Red were assayed for altered substrate
specificity using a combination of screening and selection
([13•]; see also Update). Key to the identification of
improved variants acting on a sterically hindered 3-hydroxy
ester was an agar-plate assay system based on pH indicators
that give a change in color upon hydrolysis of the ethyl
ester. Parallel assaying of replica-plated colonies on agar
plates supplemented with the glycerol derivative of the 3hydroxy
ester was used to refine the identification, because
only E. coli colonies producing active esterases had access
Improved biocatalysts by directed evolution and rational
protein design
Uwe T Bornscheuer* and Martina Pohl†
138 Biocatalysis and biotransformation
Figure 1
Rational Protein Design Directed Evolution
Protein structure
1. Planning of mutants
2. Site-directed
mutagenesis
Vectors containing
mutated genes
1. Protein expression
2. Purification
In vitro-recombination
('DNA-shuffling')
Random mutagenesis
Library of mutated genes
Protein expression
in microtiter plates
Selection parameters:
Transformation in
E. coli
Transformation in E. coli
Mutant 1 Mutant 2
Mutant library
> 10,000 clones
Analytics
Mutant
enzymes
Product analysis
achiral, chiral
Protein characterization
- Substrate range
- Kinetics
- Stability
- Enantioselectivity
- Sequencing of genes
Instable Low enantioselectivity
GenesNegative
mutations
Low activity
- Substrate range
- Stability in organic solvents
- Stability towards reaction
conditions
- thermostability
Improved mutant enzymes
Current Opinion in Chemical Biology
Comparison of rational protein design and directed evolution.
During rational protein design, mutants are planned on the basis of
their protein structure and then prepared by SDM. After
transformation in the host organism (e.g. E. coli), the variant is
expressed, purified and analyzed for desired properties. Directed
evolution starts with the preparation of mutant gene libraries by
random mutagenesis, which are then expressed in the host
organism. Protein libraries are usually screened in microtiter plates
using a range of selection parameters. Protein characterization and
product analysis sort out desired and negative mutations. In vitro
recombination by DNA shuffling, for example, can be used for
further improvements. Both protein engineering approaches can
be repeated or combined until biocatalysts with desired properties
are generated.
to the carbon source glycerol, thus leading to enhanced
growth and, in turn, larger colonies. In another example, a
growth selection based on adipyl-leucine or adipyl-serine
was used to identify acylase mutants acting on adipic acid
instead of glutaric acid sidechains in cephalosporins
(CF Sio, JM van der Laan, AM Riemens, WJ Quax, personal
communication). Random mutagenesis of the
α-subunit, then growth selection of active variants followed
by saturation mutagenesis identified three mutants showing
decreased Vmax/KM values for glutaryl substrates and
higher values towards the adipyl analog.
Another alternative for selection is the use of phage display
[14,15] to select for mutants with catalytic activity.
Anchoring the substrate to the phage in such a way that the
product remains bound too, might enable capturing of
active catalysts [16•,17].
In an excellent contribution, Fersht and co-workers [18••]
evolved phosphoribosylanthranilate isomerase (PRAI)
activity from the α/β-barrel scaffold of indole-3-glycerol
phosphate synthase (IGPS). Their success provides a striking
example for testing the ‘conserved scaffold’ hypothesis
of enzyme evolution. Reetz and co-workers [19,20] considerably
increased the enantioselectivity (E) of a lipase from
Pseudomonas aeruginosa PAO1 towards 2-methyldecanoate
by screening mutant libraries using optically pure (R)- or
(S)-p-nitrophenyl ester. Further improvement of the best
mutant (E ~11, 81% enantiomeric excess [ee]) by saturation
mutagenesis led to E ~26 for a mutant bearing five amino
acid substitutions. The recently solved structure of this
lipase [21] suggests that the increased enantioselectivity is
caused by increasing the flexibility of distinct loops of the
enzyme; however, none of the mutations are located near
the binding pocket.
In a similar approach, the enantioselectivity of an esterase
from P. fluorescens was increased from E = 3.5 to E = 5.2–6.6
in a single round of mutation [22]. Here, resorufin esters of
(R)- or (S)-3-phenylbutyric acid were used for the determination
of E values, enabling measurement of fluorescence
in microtiter plates and thereby avoiding problems with
interfering compounds present in the culture medium.
In both examples, the mutants screened for might be
‘adapted’ to the bulky chromophor ester, rather than the
‘true’ ester used in organic synthesis applications (i.e. an
acetate of a racemic alcohol). To overcome this problem,
Reetz and co-workers developed more sophisticated methods
using isotopically labelled acetates [23•] or capillary
electrophoresis [24••]. The latter method allows accurate ee
determination of amines with a throughput of at least 7000
samples per day.
Digital-image screening was used by Arnold’s group [25•]
to identify P450cam variants showing enhanced activity in
naphthalene hydroxylations, in the absence of the cofactor
NADPH, via a ‘peroxide shunt’ pathway. Co-expression of
P450cam with horseradish peroxidase from E. coli converted
hydroxylation products into fluorescent products
amenable by digital screening. In another example, a triple
mutant of P450 BM-3 obtained by directed evolution was
found to hydroxylate indole, producing indigo and indirubin
[26]. Interestingly, the authors screened for P450
variants with altered chain-length specificity, but by
chance discovered variants hydroxylating indole.
Recently, Arnold and co-workers [27•] also reported the
inversion of enantioselectivity of a hydantoinase, from
D-selectivity (40% ee) to moderate L-preference (20% ee at
30% conversion) by a combination of error-prone PCR
(epPCR) and saturation mutagenesis. Only one amino acid
substitution was sufficient to invert enantioselectivity.
Thus, production of L-methionine from D,L-5-(2-methylthioethyl)hydantoin
in a whole-cell system of recombinant
E. coli, also containing a L-carbamoylase and a racemase, at
high conversion became feasible. The substrate specificity
of a peroxidase from Saccharomyces cerevisiae towards guaiacol
was increased 300-fold by means of DNA shuffling [28•].
It is often assumed that improving a biocatalyst in one
direction affects other desired enzyme characteristics. It has
been demonstrated, however, that it is possible to increase
the thermostability of a cold-adapted protease to 60°C
while maintaining high activity at 10°C [29,30•]. The best
psychrophilic subtilisin S41 variant contained only seven
amino acid substitutions resembling only a tiny fraction of
the usual 30–80% sequence difference found between psychrophilic
enzymes and mesophilic counterparts.
In an excellent contribution, researchers at Maxygen (USA)
and Novo Nordisk (Denmark) simultaneously screened for
four properties — activity at 23°C, thermostability, organicsolvent
tolerance and pH-profile — in a library of
family-shuffled subtilisins, and reported variants with considerably
improved characteristics for all parameters [31••].
Instead of improving one specific biocatalyst, the engineering
of entire metabolic pathways by means of directed evolution
is also feasible. The first example has recently been demonstrated
in which phytoene desaturases and lycopene cyclases
were shuffled in the context of a carotenoid biosynthetic
pathway assembled from different bacterial species [32••].
Molecular breeding based on mixing genes and in vitro evolution
using E. coli as host, resulted in the generation of new
products (e.g. fully conjugated and cyclic carotenoids).
Rational protein design
Rational protein design by site-directed mutagenesis
(SDM) is still a very effective strategy to elaborate
improved enzymes. Useful strategies such as the reinforcement
of a promiscuous reaction, change of enzyme
mechanism, substrate specificity, cofactor specificity enantioselectivity,
and stability, as well as the elucidation of
enzyme mechanisms have been reported. Comprehensive
overviews can be found in a number of reviews [33–35].
Improved biocatalysts by directed evolution and rational protein design Bornscheuer and Pohl 139
Rational protein design has successfully been used for stabilization
of enzymes towards thermoinactivation and
oxidation. Examples of successful strategies to enhance thermostability
are the removal of asparagine residues in
α-amylase [36], the introduction of more rigid structural elements
such as proline into α-amylase [37] and D-xylose
isomerase [38], or disulfide bridges to stabilize hen lysozyme
[39]. The introduction of additional hydrophobic contacts
was shown to stabilize 3-isopropylmalate dehydrogenase [40]
and formate dehydrogenase from Pseudomonas sp. [41].
Factors influencing thermostability have recently been elucidated
by a structural comparison of various enzymes from
mesophilic and thermophilic organisms [42].
To increase stability towards oxidation, removal of cysteine
and methionine residues exhibited positive effects in the
case of formate dehydrogenase [43], (R)-3-hydroxybutyrate
dehydrogenase [44], and D-amino acid oxidase [45].
In contrast to directed evolution, successful examples of
improved and inverted enantioselectivity of enzymes by
SDM are rare. The effects of certain mutations are more difficult
to predict compared with those from efforts focusing on
altering other catalytic properties. A very good example is the
inversion of the enantioselectivity of vanillyl-alcohol oxidase
by two point mutations, which switched the enzyme from
(R)- to (S)-specificity (80% ee) with respect to the formation
of 1-(4′-hydroxyphenyl)ethanol by hydroxylation of 4-ethylethanol
[46•]. Another successful example has been reported
by Schmid and co-workers [47,48•], who investigated the
molecular bases of stereoselectivity of two different lipases
by SDM based on molecular modeling studies, and thereby
generated mutants with altered stereoselectivity compared
with that of the wild-type enzyme.
Compared with factors influencing stereospecificity and
enantioselectivity, the prediction of molecular factors effecting
catalytic activity are easier to predict. In the case of
pyruvate decarboxylase from Zymomonas mobilis, mutation of
Trp392, a bulky residue in the substrate-binding channel,
increased the carboligase side-reaction of the enzyme by a
factor of six, without negatively influencing the stability and
the enantioselectivity [49]. Sterical principles were also valid
in the case of PepC, which is an aminopeptidase, because of
four carboxy-terminal residues interacting with the active site.
Removal of the carboxy-terminal tetrapeptide converted the
aminopeptidase into an oligopeptidase ([50]; see also
Update). The substrate range of P450cam was extended from
camphor to polycyclic aromatic hydrocarbons by mutation of
two aromatic residues (Phe87 and Tyr96) in the substrateaccess
channel [51]. On the basis of a homology model,
various mutants of PER-1 β-lactamase have been generated
with improved activity towards different β-lactam-based
antibiotics [52]. By point mutation of Gln27 — a residue
located in a cap closing the back of the active site — the catalytic
properties of an Aspergillus fumigatus phytase were
successfully optimized [53]. The sidechain of this non-conserved
residue is in close contact with the substrate during
catalysis. Thus, mutation of glutamine to leucine or
isoleucine prevents the formation of a hydrogen bond
between the substrate and the sidechain. Alteration of substrate
specificity was achieved by SDM of human leukocyte
5-lipoxygenase, yielding a 15-lipoxygenating biocatalyst [54•].
An example for the generation of a new enzymatic activity by
protein design has been reported by the Christen group [55],
who obtained a dicarboxylic amino acid β-lyase, an enzyme
not found in nature, by SDM of a tyrosine phenol-lyase.
Combination of both methods
An impressive example of the use of directed evolution
and rational protein design was shown by researchers at
Novo Nordisk (Bagsvaerd, Denmark). They targeted a
heme peroxidase from Coprinus cinereus to be used as a dyetransfer
inhibitor in laundry detergent [56••]. Screening for
improved stability was performed by measuring residual
activity after incubation under conditions mimicking those
in a washing machine (e.g. pH 10.5, 50°C, 5–10 mM peroxide).
Mutants were obtained by epPCR (out of 64,000)
as well as by rational design. Surprisingly, for both methods
sequencing of the best variants identified position Glu239
to be crucial for success. Saturation mutagenesis showed
that replacement with glycine — as predicted by computer-modeling
— gave the best performance. Subsequent
in vivo shuffling led to dramatic improvements, yielding a
mutant with 174 times the thermal stability and 100 times
the oxidative stability of the wild-type peroxidase.
Another example of the successful combination of both
mutagenesis strategies is the improvement of the thermal
stability of a 3-isopropylmalate dehydrogenase variant from
Bacillus subtilis, which was obtained by a triple mutation
using SDM followed by epPCR. The latter mutagenesis
identified two further stabilizing point-mutations, which
were then combined using SDM [57].
Conclusions
Recent work in the area of protein engineering, summarised
in this article, shows that rational design and directed evolution
are both applicable to creating desired mutant enzymes,
although the positions of mutations often differ considerably.
For rational protein design, those amino acid residues that
appear ‘logical’ to the researcher examining the three-dimensional
structure are usually modified (i.e. they are close to the
active site, the binding pocket etc.). In sharp contrast,
sequencing of variants obtained by directed evolution followed
by their structural analysis very often reveals that
mutations are far away from the place where the reaction
takes place (as was shown for improved enantioselectivity
[19] and altered substrate specificity [13•] of hydrolases). First
examples from careful analysis of three-dimensional structures
of mutants and wild types help to identify how
‘irrational’ mutations affect the catalytic properties of a biocatalyst
[58••,59••]. Further work will surely help to increase
our understanding of structure/function relationships and will
see more combinations of both protein engineering tools.
140 Biocatalysis and biotransformation
Update
Recent work has shown that the chain-length specificity of
P450 BM-3 can be modulated by rational design [60]. In contrast
to the wild-type, a variant bearing five mutations accepted
a p-nitrophenoxyoctanoic acid (C8-pNCA), whilst the activity
towards C10- and C12-pNCA remained unchanged.
Phospholipase activity was introduced into a
Staphylococcus aureus lipase by directed evolution using
epPCR and gene shuffling [61]. The best variant contained
six mutations and displayed a 11.6-fold increase in
phospholipase activity and a 11.5-fold increased phospholipase
: lipase ratio compared to the wild type. A very
brief opinion on the same subject as this review compares
mutational and selective mechanisms and basic requirements
of rational design and directed evolution [62].
Also, two reviews summarizing recent achievements and
future prospects in the field of directed evolution were
recently published [63,64].
Acknowledgements
UT Bornscheuer thanks the German research foundation (DFG, Bonn,
Germany) for financial support. M Pohl is grateful to the DFG for funding in
frame of Sonderforschungsbereich 380 and to the BMBF (Bonn, Germany).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
••of outstanding interest
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• Regio- and Stereoselective Biotransformations. Weinheim: WileyVCH;
1999.
This monograph provides a comprehensive and up-to-date summary of stereoselective
and regioselective biotransformations using different hydrolases.
2. Faber K: Biotransformations in Organic Chemistry. Berlin: Springer; 2000.
3. Patel RN: Stereoselective Biocatalysis. New York: Marcel Dekker; 2000.
4. Bornscheuer UT: Enzymes in Lipid Modification. Weinheim:
Wiley-VCH; 2000.
5. Liese A, Seelbach K, Wandrey C: Industrial Biotransformations.
• Weinheim: Wiley-VCH; 2000.
This book lists a wide variety of industrialized processes with a plethora of
detailed information. The division based on enzyme nomenclature provides
rapid access to the given data.
6. Kazlauskas RJ: Molecular modeling and biocatalysis: explanations,
• predictions, limitations, and opportunities. Curr Opin Chem Biol
2000, 4:81-88.
A good overview about successful and less successful applications of molecular
modeling as a tool to analyze and predict the function of certain amino
acid sidechains in enzymes.
7. Arnold FH, Volkov AA: Directed evolution of biocatalysts. Curr Opin
Chem Biol 1999, 3:54-59.
8. Bornscheuer UT: Directed evolution of enzymes. Angew Chem Int
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9. Reetz MT, Jaeger K-E: Superior biocatalysts by directed evolution.
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10. Petrounia IP, Arnold FH: Designed evolution of enzymatic
properties. Curr Opin Biotechnol 2000, 11:325-330.
11. Tobin MB, Gustafsson C, Huisman GW: Directed evolution: the
‘rational’ basis for ‘irrational’ design. Curr Opin Struct Biol 2000,
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12. Sutherland JD: Evolutionary optimisation of enzymes. Curr Opin
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13. Bornscheuer UT, Altenbuchner J, Meyer HH: Directed evolution of
• an esterase: screening of enzyme libraries based on
pH-indicators and a growth assay. Bioorg Med Chem 1999,
7:2169-2173.
Although this assay is very useful to screen large libraries in an agar-plate
format, it is rather restricted to the identification of a first active enzyme variant.
Identification of more active mutants in subsequent generations is therefore
difficult to perform.
14. Matthews DJ, Wells JA: Substrate phage: selection of protease
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15. Verhaert RMD, Duin JV, Quax WJ: Processing and functional display
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A selection of catalysts was achieved by binding the reaction product to the
surface of a phage to which the enzyme is fused. Access using specific antiproduct
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17. Pedersen H, Hölder S, Sutherlin DP, Schwitter U, King DS,
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18. Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Directed
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Careful design of the basic PRAI loop system into IGPS created the scaffold
for directed evolution by DNA-shuffling and StEP (staggered extention
process). Active variants were identified by an agar-plate assay using an
E. coli strain that does not grow in the absence of tryptophan, which in turn
is the product of PRAI activity. The mutant obtained had 28% identity to
PRAI and 90% identity to IGPS.
19. Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW,
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20. Reetz MT: Evolution in the test tube as a means to create
enantioselective enzymes for use in organic synthesis. Sci Prog
2000, 83:157-172.
21. Nardini M, Lang DA, Liebeton K, Jäger KE, Dijkstra BW: Crystal
structure of Pseudomonas aeruginosa lipase in the open
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J Biol Chem 2000, 275:31219-31225.
22. Henke E, Bornscheuer UT: Directed evolution of an esterase from
Pseudomonas fluorescens. Random mutagenesis by error-prone
PCR or a mutator strain and identification of mutants showing
enhanced enantioselectivity by a resorufin-based fluorescence
assay. Biol Chem 1999, 380:1029-1033.
23. Reetz MT, Becker MH, Klein HW, Stöckigt D: A method for high
• throughput screening of enantioselective catalysts. Angew Chem
Int Ed Engl 1999, 38:1758-1761.
Screening for enzymes showing enhanced enantioselectivity was achieved by
using isotopically labelled substrates in combination with high-throughput mass
spectrometry at a capacity of about 1000 samples per day. Thus, mutants can
be assayed with the ‘true’ substrate bearing no bulky chromophoric groups.
24. Reetz MT, Kühling KM, Deege A, Hinrichs H, Belder D: Super-high
•• throughput screening of enantioselective catalysts by using
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39:3891-3893
Chiral-modified electrolytes used for capillary electrophoresis allowed efficient
separation of a range of FITC-labelled amines in a specially designed
capillary electrophoresis system amenable to direct microtiter plate sampling.
The authors claim that shorter analysis times enable super-highthroughput
of up to 30,000 samples per day. This elegant method should be
easily applicable to the separation of other chiral molecules, thus enabling
the efficient screening of huge mutant libraries.
25. Joo H, Lin Z, Arnold FH: Laboratory evolution of peroxide-mediated
• cytochrome P450 hydroxylation. Nature 1999, 399:670-673.
Directed evolution of a cofactor-free monooxygenase in combination with a
well-designed high-throughput screening eased access to improved variants.
26. Li Q-S, Schwaneberg U, Fischer P, Schmid RD: Directed evolution
of the fatty acid hydroxylase P450 BM-3 into an indolehydroxylating
catalyst. Chem Eur J 2000, 6:1531-1536.
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• directed evolution of hydantoinase for improved production of
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This example of inverting enantioselectivity of an enzyme is especially interesting,
because it addresses a problem with high impact on an industrial application.
28. Iffland A, Tafelmeyer P, Saudan C, Johnson K: Directed molecular
• evolution of cytochrome c peroxidase. Biochemistry 2000,
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Interestingly, sequencing revealed that in the superfamily of peroxidase the
fully conversed residue Arg48 was changed to histidine. The authors believe
that this mutation plays a key role in the increase in activity toward the phenolic
substrate guaiacol.
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by saturation mutagenesis: rapid improvement of protein
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• Directed evolution study of temperature adaptation in a
psychrophilic enzyme. J Mol Biol 2000, 297:1015-1026.
Directed evolution gave mutants showing stability and high activity over a
very broad temperature range.
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DNA family shuffling of 26 protease genes yielded chimeras possessing
positive combinations of properties of individual parents.
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An excellent contribution exemplifying that directed evolution can be applied
for molecular breeding of entire pathways.
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substitution in an alkaline, liquefying αα-amylase from Bacillus sp.
strain KSM-1378. Biosci Biotechnol Biochem 1999, 63:1535-1540.
39. Ueda T, Masumoto K, Ishibashi R, So T, Imoto T: Remarkable
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of the thermal stability of a partially stabilized Bacillus subtilis
3-isopropylmalate dehydrogenase variant by random and sitedirected
mutagenesis. Eur J Biochem 1999, 260:499-504.
41. Rojkova AM, Galkin AG, Kulakova LB, Serov AE, Savitsky PA,
Fedorchuk VV, Tishkov VI: Bacterial formate dehydrogenase.
Increasing the enzyme thermal stability by hydrophobization of
αα-helices. FEBS Lett 1999, 445:183-188.
42. Kumar S, Tsai CJ, Nussinov R: Factors enhancing protein
thermostability. Protein Eng 2000, 13:179-191.
43. Slusarczyk H, Felber S, Kula MR, Pohl M: Stabilization of NADdependent
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A very nice study on alteration of enantioselectivity based on structural comparison
of two members of structurally related FAD-dependent oxidoreductases, of
which one is (R)-specific and the other (S)-specific. Site-directed mutagenesis
introduced (S)-selectivity in the (R)-selective wild-type enzyme. Structural analysis
of the mutant enzyme revealed that the mutations are really site-directed.
47. Scheib H, Pleiss J, Stadler P, Kovac A, Potthoff AP, Haalck L,
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48. Scheib H, Pleiss J, Kovac A, Paltauf F, Schmid RD: Stereoselectivity
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to a complex problem. Protein Sci 1999, 8:215-221.
Triglyceride derivatives bearing non-carboxylic ester groups in sn2-position
(amide-, ether-, alkyl- groups) lead to lowered and even inverted stereoselectivity
in hydrolysis using lipase mutants.
49. Pohl M: Optimierung von Biokatalysatoren für technische
Prozesse. Chem Ing Technik 2000, 72:883-885. [Title translation:
Optimization of biocatalysts for technical processes.]
50. Mata L, Gripon JC, Mistou MY: Deletion of the four C-terminal
residues of PepC converts an aminopeptidase into an
oligopeptidase. Protein Eng 1999, 12:681-686.
51. Harford-Cross CF, Carmichael AB, Allan FK, England PA, Rouch DA,
Wong LL: Protein engineering of cytochrome p450(cam)
(CYP101) for the oxidation of polycyclic aromatic hydrocarbons.
Protein Eng 2000, 13:121-128.
52. Bouthors AT, Delettre J, Mugnier P, Jarlier V, Sougakoff W: Sitedirected
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242 in PER-1 ββ-lactamase hydrolysing expanded-spectrum
cephalosporins. Protein Eng 1999, 12:313-318.
53. Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L,
van Loon AP, Pasamontes L: Optimization of the catalytic
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54. Schwarz K, Walther M, Anton M, Gerth C, Feussner I, Kühn H:
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site-directed mutagenesis. J Biol Chem 2001, 276:773-779.
Introduction of space-filling amino acid residues in the substrate-binding
pocket of human 5-lipoxygenase altered substrate specificity. Single mutants
gave no effect, but a quadruple mutant was found to be sufficient.
55. Mouratou B, Kasper P, Gehring H, Christen P: Conversion of
tyrosine phenol-lyase to dicarboxylic amino acid ββ-lyase, an
enzyme not found in nature. J Biol Chem 1999, 274:1320-1325.
56. Cherry JR, Lamsa MH, Schneider P, Vind J, Svendsen A, Jones A,
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An impressive example of the combination of site-directed mutagenesis,
epPCR and DNA shuffling.
57. Akanuma S, Yamagishi A, Tanaka N, Oshima T: Further improvement
of the thermal stability of a partially stabilized Bacillus subtilis
3-isopropylmalate dehydrogenase variant by random and sitedirected
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58. Spiller B, Gershenson A, Arnold FH, Stevens RC: A structural view
•• of evolutionary divergence. Proc Natl Acad Sci USA 2000,
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Comparison of structures of different mutants of a p-nitrobenzyl esterase
(one evolved for increased activity in aqueous-organic solvents, the other for
increased thermostability) were compared with the wild-type structure.
Careful analysis of structural changes identified networks of mutations that
collectively remodel the active site in a way hardly predictable by rational
design. Only a few of the common motifs that are believed to be involved in
making an enzyme thermophilic were found.
59. Oue S, Okamoto A, Yano T, Kagamiyama H: Redesigning the
•• substrate specificity of an enzyme by cumulative effects of the
mutations of non-active site residues. J Biol Chem 1999,
274:2344-2349.
Directed evolution created an aspartate aminotransferase showing 2.1 × 106fold
increased activity towards the non-native substrate, valine. Interestingly,
only one of 17 mutations appears to contact the substrate, none interacted with
142 Biocatalysis and biotransformation
the cofactor. Structural analysis revealed that the active site is remodelled, the
subunit interface is altered and the domain enclosing the substrate is shifted.
60. Li Q-S, Schwaneberg U, Fischer M, Schmitt J, Pleiss J, Lutz-Wahl S,
Schmid RD: Rational evolution of a medium chain-specific cytochrome
P450 BM-3 variant. Biochim Biophys Acta 2001, 1545:114-121.
61. van Kampen MD, Egmond MR: Directed evolution: from a
staphylococcal lipase to a phospholipase. Eur J Lipid Sci Technol
2000, 102:717-726.
62. Chen R: Enzyme engineering: rational redesign versus directed
evolution. Trends Biotechnol 2001, 19:13-14.
63. Reetz MT: Combinatorial and evolution-based methods in the
creation of enantioselective catalysts. Angew Chem Int Ed Engl
2001, 40:284-310.
64. Arnold FH: Combinatorial and computational challenges for
biocatalyst design. Nature 2001, 409:253-257.
Improved biocatalysts by directed evolution and rational protein design Bornscheuer and Pohl 143