Strategies for the discovery and engineering of enzymes forbiocatalysisTimo Davids, Marlen Schmidt, Dominique Bottcher andUwe T Bornscheuer
Available online at www.sciencedirect.com
Protein engineering is the most important method to overcome
the limitations of natural enzymes as biocatalysts. The past few
years have seen a tremendous increase in novel concepts to
facilitate the design of mutant libraries for focused directed
evolution mostly guided by advanced bioinformatic tools. In
addition, advanced high-throughput methods were developed
using, for example, FACS analysis or microfluidic systems.
These achievements significantly facilitate the tailor-made
design of enzymes to make them suitable for industrial
applications.
Address
Institute of Biochemistry, Department of Biotechnology and Enzyme
Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17487
Greifswald, Germany
Corresponding author: Bornscheuer, Uwe T (uwe.bornscheuer@uni-
greifswald.de)
Current Opinion in Chemical Biology 2013, 17:215–220
This review comes from a themed issue on Biocatalysis and
Biotransformation
Edited by Nicholas J Turner and Matthew D Truppo
For a complete overview see the Issue and the Editorial
Available online 21st March 2013
1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.cbpa.2013.02.022
IntroductionEnzymes have found numerous applications as biocata-
lysts for synthetic organic chemistry due to their high
stereoselectivity required for the creation of chiral com-
pounds, but also for targets where mild reaction con-
ditions are important, such as in the conversion of
labile compounds, in protection/deprotection steps or
where chemoselectivity and regioselectivity are needed.
Furthermore, engineered enzymes became useful for the
pretreatment of renewable resources as starting materials
such as in the degradation of lignocellulose to make
biofuels of the 2nd or 3rd generation. As recently pointed
out in our review [1��] ‘‘in the past, an enzyme-based processwas designed around the limitations of the enzyme; today, theenzyme is engineered to fit the process specifications’’, biocata-
lysis became a mature technology through several waves
of innovations. Initially only wild-type enzymes could be
used, but the advent of recombinant DNA technology
enabled the cloning and functional expression of the
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enzyme of interest. First protein engineering methods
using rational protein design or directed evolution (by
error-prone PCR or gene shuffling in combination with
high-throughput screening and iterative cycles of im-
provement) allowed alteration of the properties of the
enzyme to meet limitations in their application to a
reasonable extent. Novel tools of protein engineering
open up new avenues to achieve the goal of industrially
useful biocatalysts significantly faster and even allow to
create enzyme variants where the wild-type had no
activity at all as demonstrated for a transaminase to make
the drug sitagliptin [2]. Numerous examples of successful
protein engineering can be found in books [3–5] and
recent reviews [1��,6–8,9��,10,11�]. Further examples
for the application of various enzymes in biocatalysis
can be found elsewhere in this issue. This contribution
covers recent developments published mostly in 2010–2012 focusing on novel concepts for enzyme discovery, for
the creation of mutant libraries and identification of hits,
and bioinformatic tools to guide protein engineering
strategies (Figure 1). The de novo computational design
of enzymes is outside of the scope of this review and
covered in the contribution by Hilvert and co-authors in
this issue [12].
Novel tools for enzyme discoveryThe metagenome approach is nowadays a well-estab-
lished approach to find novel proteins [13]. The limitation
that most microbes cannot be grown under standard
laboratory conditions is overcome here by extracting
the complete genomic DNA from an environmental
sample followed by either first, sequencing and explora-
tion of the genetic information through identification of
open reading frames encoding enzymes and their func-
tional annotation or second, by cloning and functional
expression in metagenome libraries, which are then
screened with high-throughput assays for novel enzyme
activities (Figure 1). In a recent contribution, the com-
pany BRAIN identified a surprisingly large molecular
diversity for the well-known serine protease Subtilisin
Carlsberg [14��]. In only four soil samples they were able
to find 94 sequences of this subtilisin bearing 38
mutations, equivalent to 2–8 amino acid exchanges per
variant. 51 unique protein variants could be functionally
expressed in Bacillus subtilis, which also differed in their
functionality. Hence, their discovery of coexisting gene
variants is a potent source for novel enzyme variants. This
method thus represents an alternative to directed
Current Opinion in Chemical Biology 2013, 17:215–220
216 Biocatalysis and Biotransformation
Figure 1
Starting Material
metagenomic library
gene(s) of interest
information about hotspots
gene of interest
protein structure
structural information andprediction of key motifs
define transition state for thedesired reaction
propose an active site able tostabilize that transition state
QM/MM modeling
accomodate the active siteinto an existing scaffold
ROSETTA algorithm experimental verification
library of novel enzymesnovel protein sequencesdatabase search
gene1: ..HIYQ..PRAHQFgene2: ..YIRP..RMGVRPgene3: ..FVER..GVRGTRgene4: ..FVEL..GVRGSR.
Dire
cted
Evo
lutio
nS
emi-r
atio
nal D
esig
nR
atio
nal D
esig
n
Pro
tein
Eng
inee
ring
Dat
abas
e S
earc
hde
nov
o D
esig
n
cloning and sequencing orfunctional annotation
non-homologous mutagenesis• epPCR• SeSaM
homologous mutagenesis• gene shuffling
saturation mutagenesis• ISM• CASTing
Computational tools• HotSpot Wizard• QSAR• SCHEMA• “small but smart”
library of novel enzymes ornovel protein sequences
screening
gene librarymutagenesis
mutagenesis
computer supportedpredictions and mutagenesis
gene library
specific point mutations
chimeric genes
gene variant
determination of specificactivity, enantioselectivity,
etc.
Sea
rch
for
Exi
stin
g E
nzym
es
Realization Result Screening
Current Opinion in Chemical Biology
Overview of concepts used to identify or create enzymes with desired properties.
Current Opinion in Chemical Biology 2013, 17:215–220 www.sciencedirect.com
Strategies for the discovery and engineering of enzymes for biocatalysis Davids et al. 217
evolution concepts relying only on random mutagenesis
as the metagenome-based microdiversity concept gave a
broad set of well expressed active variants.
The enormous progress in sequencing technology in
combination with metagenome libraries has led to an
exponential increase in the number of sequence data
(currently approx. 20 million sequences). However, the
annotation of the putative protein function is performed
automatically and consequently can lead to misleading
interpretations. In a recent example, we took advantage
of these errors while trying to identify (R)-selective
amine transaminases (ATA) for which no protein
sequence was reported in the literature at the time of
our study. ATA are pyridoxal-50-phosphate (PLP) de-
pendent enzymes catalyzing the synthesis of chiral
amines from ketones (in contrast to amino acid trans-
aminases that convert a-keto acids to a-amino acids)
and only (S)-selective ATA were known. Using a soph-
isticated search algorithm — based on distinct amino
acid motifs of known amino acid transaminases — an
alignment of >5000 protein sequences from public
database of PLP-dependent transaminases identified
21 new protein sequences (equivalent to a hit rate of
0.5%) [15]. For 17 proteins it could be confirmed that
they all are true amine transaminases having the pre-
dicted (R)-enantiopreference and these were then used
in the asymmetric synthesis of a set of 12 chiral (R)-
amines [16]. Another still only scarcely explored source
for novel enzymes is the Brookhaven protein structure
database (www.pdb.org), which contains numerous
proteins, where the 3D-structure has been deposited,
but where the proteins were never biochemically
characterized. We recently identified four (S)-selective
ATA in the pdb and could functionally assign them by
biochemical characterization [17].
Concepts guided by computational toolsMany computational tools such as HotSpot Wizard [18],
ProSAR [19] and SCHEMA have been established to
analyze enzyme 3D-structures or homology models to
guide the design of mutations to alter enzyme properties
[20]. The recently proposed ASRA (adaptive substituent
reordering algorithm) is an alternative to conventional
quantitative structure–activity relationship (QSAR)
methods to enable the identification of enzyme variants
in focused mutant libraries having desired properties. In
combination with iterative saturation mutagenesis (ISM)
[21] the most enantioselective mutants of an epoxide
hydrolase from Aspergillus niger could be predicted [22].
Using SCHEMA, a structure-guided computational
algorithm, it was possible to recombine multiple low
identity proteins to obtain chimeric enzymes with
improved thermostability or substrate specificity [23].
In a very recent example this technique was used to
recombine human arginase I and II to catalytically active
chimeras [24].
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Semirational design/focused directedevolutionSemirational methods, based on the knowledge derived
from biochemical and structural data, combine the advan-
tages of rational and random tools to create small focused
libraries, which is especially advantageous if no high-
throughput assay system is available. Methods of choice
for the semirational approach are site-saturation mutagen-
esis methods like ISM or CASTing [25]. Using these
techniques the substrate specificity of an amylosucrase
from Neisseria polysaccharea (a-transglucosidase) was
recently altered after screening 20,000 variants. The best
mutant showed 400-fold enhanced catalytic efficiency
toward the donor substrate sucrose and the non-natural
acceptor substrate allyl 2-acetamido-2-deoxy-alpha-D-
glucopyranoside [26]. The regioselectivity and enantios-
electivity of a P450-BM3 monooxygenase toward cyclo-
hexene-1-carboxylic acid methyl ester was altered via
saturation mutagenesis at 24 single-residue sites and
some two-residue sites. Only the two-residue sites
yielded (R)-selective mutants with >95%ee [27].
Saturation mutagenesis at more than one amino acid
residue simultaneously creates a huge number of variants
and due to the degeneracy of the genetic code the amino
acid distribution in the resulting library is unbalanced.
Several concepts were developed to reduce the library
size: one can decrease it by using NDT or NDK codons
with the disadvantage that not all proteinogenic amino
acids are covered. An alternative is to use chemically
synthesized dinucleotide or trinucleotide phosphorami-
dites [28] to allow a fully controlled randomization, but
this method is still not generally used due to costs and
availability of starting materials. The codon search algor-
ithm CoFinder [29��] allows to determine the optimal
primer mixture for the desired amino acid distribution. A
very recent example for reducing codon redundancy has
been called 22c-trick [30]. Here, a mixture of three
oligonucleotides (two degenerate) with NDT (12 codons)
VHG (9 codons) and one TGG primer resulted in a codon
to amino acid ratio of 22:20. This mixture contains no stop
codons and only two redundant codons for valine and
leucine. A quite similar approach using four primers (with
NDT, VMA, ATG and TGG codons) was also reported
[31].
Structure-guided approachesThis concept combines structural information with
protein sequences. The well established ‘consensus
approach’ is based on the theory that most abundant
amino acids at each position in a set of homologous
enzymes contribute more than average to protein per-
formance than the nonconsensus amino acids [32]. Com-
parison of sequences within large enzyme families can
then identify conserved amino acids followed by their
insertion into the starting protein. The Bommarius group
combined this method with the B-FIT analysis (B-Factor
Current Opinion in Chemical Biology 2013, 17:215–220
218 Biocatalysis and Biotransformation
Iterative Test) to improve the thermostability of an a-
amino ester hydrolase from Xanthomonas campestris and
obtained a quadruple mutant, which was 7 8C more
thermostable and 1.3-fold more active than the wild-type
[33]. In another example the thermostability of an endo-
glucanase from Clostridium thermocellum could be
improved by this method too. The sequence alignment
of 18 glycoside hydrolases with 30–60% homology yielded
three more thermostable mutants with similar or higher
activity compared to the wild-type [34].
In another approach, the 3DM-software was successfully
used to guide protein engineering of enzymes from the a/
b-hydrolase fold superfamily [35]. 3DM is a commercial
structure-based sequence alignment and analysis tool,
which goes beyond the consensus approach as it not only
enables to identify the distribution of amino acids within
a protein superfamily, but can also analyze connectivity
between residues as it is constructed from protein struc-
ture alignments. In a subset of the a/b-hydrolase fold
enzyme superfamily, 1751 structurally related esterases
served as basis to create ‘small, but smart’ libraries [36].
The 3DM analysis enabled to design libraries to cover
only amino acids frequently occurring at a given position
in this superfamily and hence led to a substantial
reduction in the library size to be created and screened
in the laboratory. Targeting four residues in the active site
region of an esterase from Pseudomonas fluorescens (PFE)
improved the enantioselectivity toward a chiral carboxylic
acid from E = 3 to E = 80 and also improved activity up to
240-fold [37]. In combination with the B-FIT method,
the 3DM-analysis was also used to increase the thermo-
activity of PFE by 8 8C [38]. Rational design in combi-
nation with the B-FIT method and the 3DM database for
the glucosidase family 13 containing 5092 sequences
helped to create mutants of a sucrose phosphorylase from
Bifidobacterium adolescentis with more than doubled half-
life at 60 8C [39]. In the 3DM database for methyltrans-
ferases 140 sequences of the subfamily were aligned
and — supported by a homology model — the analysis
led to the identification of the putative cofactor binding
site and catalytic residues, which could be confirmed by
site-directed mutagenesis [40].
Tunnel software toolsAs many enzymes have buried active sites, predictions for
changing substrate specificity, enantioselectivity or
regioselectivity are difficult without detailed knowledge
about the shape and size of the tunnels enabling substrate
and product entry or release. Also the solvent access can
be important. Nowadays geometry-based softwares like
CAVER [41], MOLE [42] and POREWALKER [43] help
to understand these relationships, but have the limitation
to calculate primarily static structures. An extensive over-
view about these different software tools can be found in a
current excellent review [44��]. CAVER could be suc-
cessfully applied to engineer the tunnel in a haloalkane
Current Opinion in Chemical Biology 2013, 17:215–220
dehalogenase from Sphingobium japonicum leading to a
mechanism change in product release [45]. The same
software was used to gain insights into the role of sub-
strate protonation and the inhibitor interactions in the
human monoamine oxidase A [46]. With the latest version
CAVER 3.0 it is now also possible to include protein
dynamics into these calculations and to analyze multiple
different access pathways using large collections of
protein structures [47]. Analysis of tunnel residues and
their mutation enabled to substantially improve the sol-
vent stability of the haloalkane dehalogenase DhaA from
Rhodococcus rhodochrous [48].
New methods for mutagenesisIn the past decades numerous mutagenesis methods
beside error-prone PCR and gene shuffling have been
described [49], but still few new tools are reported. Most
molecular biology methods only insert point mutations,
but the Tawfik group recently described a method
dubbed TRINS (tandem repeat insertions), in which they
describe how defined sequences of variables length (3–150 bp) can be randomly introduced into the gene of
interest. Although TRINS is limited to insertion-by-
duplication using distinct short sequences, it allows to
identify regions in a protein, which are not accessible by
standard protocols as shown experimentally for variants of
a TEM-1 b-lactamase [50].
Tools for high-throughput screeningOnce a mutant library has been created, the desired
variants must be identified for which various high-
throughput screening (HTS) methods have been pub-
lished, usually tailor-designed for the specific problem of
interest [51]. By far the highest throughput is possible by
fluorescence activated cell sorting (FACS) [52,53] or
using microfluidic devices [54].
Agresti et al. presented an ultra-HTS platform in which
aqueous droplets dispersed in oil as picoliter-volume
reaction vessels are used and thousands can be screened
per second. After two rounds of evolution 108 variants of a
horseradish peroxidase were analyzed within 10 h leading
to a variant with 10-fold higher catalytic efficiency com-
pared to the wild-type. The authors claimed that their
device reduced the assay costs 1-million-fold since the
total reaction volume was <150 ml and the assay was
1000-fold faster than a FACS system [55].
A flow cytometry-based screening system for directed
evolution of a protease applying in vitro compartmenta-
lization (IVC) was developed by the Schwaneberg group.
A high mutational load error-prone PCR-based mutant
library of Subtilisin Carlsberg was expressed in an extra-
cellular protease-deficient Bacillus subtilis strain and
screened in double emulsions using a fluorogenic peptide
substrate for increased resistance toward a protease
inhibitor. After three rounds of iterative sorting an
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Strategies for the discovery and engineering of enzymes for biocatalysis Davids et al. 219
enzyme variant containing six mutations was found exhi-
biting a 160% increased resistance compare to the wild-
type [56].
Another example for protease engineering using FACS
uses a so-called counter-selection approach to eliminate
unwanted enzyme variant and this enabled to alter the
substrate specificity of the outer membrane endopepti-
dase OmpT [53]. We have recently developed a FACS-
based system in which highly enantioselective variants of
the esterase PFE were selected from a mutant library in
Escherichia coli by coupling one enantiomer of the sub-
strate to the carbon source glycerol and the other enan-
tiomer to a toxic compound. An E. coli showing better
viability could hence be sorted out by the FACS and
analyzed for PFE mutants having higher selectivities
[52].
ConclusionsThe various novel tools covered in this review substan-
tially facilitate the discovery and engineering of enzymes.
Especially the combination of bioinformatic tools to
guide library design helps to achieve the engineering
target much faster and with less effort in terms of labor
and consumables reducing development times from years
to months. These achievements are not only useful for
biocatalyst development, but also facilitate synthetic
biology and metabolic engineering projects.
AcknowledgementWe thank the German Research Foundation (DFG, Grant Bo1862/6-1) forfinancial support.
References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:
� of special interest�� of outstanding interest
1.��
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44.��
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