Current Protein and Peptide Science, 2010, 11, 91-100 91

Random Mutagenesis Methods for In Vitro Directed Enzyme Evolution

Nikolaos E. Labrou*

Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera
Odos 75, 11855-Athens, Greece

Abstract: Random mutagenesis is a powerful tool for generating enzymes, proteins, entire metabolic pathways, or even
entire genomes with desired or improved properties. This technology is used to evolve genes in vitro through an iterative
process consisting of recombinant generation. Coupled with the development of powerful high-throughput screening or
selection methods, this technique has been successfully used to solve problems in protein engineering. There are many
methods to generate genetic diversity by random mutagenesis and to create combinatorial libraries. This can be achieved
by treating DNA or whole bacteria with various chemical mutagens, by passing cloned genes through mutator strains, by
“error-prone” PCR mutagenesis, by rolling circle error-prone PCR, or by saturation mutagenesis. The next sections of this
review article focus on recent advances in techniques and methods used for in vitro directed evolution of enzymes using
random mutagenesis. Selected examples, highlighting successful applications of these methods, are also presented and
discussed.

1. INTRODUCTION
Natural evolution produces a large number of protein and
enzyme variants. These natural variants exhibit their function
with high specificity and efficiency. However, they are ad-
justed perfectly to perform only their physiological role and
therefore their specificity and stability are usually far away
from what biotechnology industry needs. Directed evolution
is a laboratory in vitro process for generating enzymes, pro-
teins [1-17], entire metabolic pathways [18,19] or even entire
genomes [20-22] with new and desired properties. To create
evolution in vitro in a tube, the process of natural evolution
must be accelerated such that sequence diversity can be cre-
ated and selected in a much shorter timeframe.
The two evolutionary methods which have been used for
in vitro directed evolution are random mutagenesis and gene
recombination [11,13-28]. Gene recombination refers to the
exchange of blocks of sequences among two or more DNA
strands. Stemmer in 1994 introduced DNA shuffling [10],
the first in vitro recombination method. Since that time, nu-
merous other recombination methods have been developed
[1-9,17,20]. Random mutagenesis is a directed evolution
strategy, which introduces random point mutations into
whole genes. Random mutagenesis mechanism includes
modifications which can be divided into the next five catego-
ries: (i) transitions, which involve substitution of a purine
nucleotide by another purine, or a pyrimidine by a second
pyrimidine, (ii) transversions, which involve substitution of a
purine nucleotide by a pyrimidine, or vice versa, (iii) dele-
tions, in which one or more nucleotides are deleted from a
gene, (iv) insertions, in which one or more extra nucleotides
are incorporated into a gene, and (v) inversions, which in-
volve the 180º rotation of a double-stranded DNA segment
*Address correspondence to this author at the Enzyme Technology Labora-
tory, Department of Agricultural Biotechnology, Agricultural University of
Athens, Iera Odos 75, 11855 – Athens, Greece; Tel:/Fax: +30-210-5294308;
E-mail: Lambrou@aua.gr
of two base pairs or longer [25]. There are many methods to
generate genetic diversity by random mutagenesis. This can
be achieved by treating DNA or whole bacteria with various
chemical mutagens [23,29,30], by passing cloned genes
through mutator strains [31,32], by “error-prone” PCR
mutagenesis (epPCR) [33-36], by rolling circle error-prone
PCR (epRCA) [37], or by saturation mutagenesis [13]. How-
ever, no consensus has emerged about which method is most
effective for a particular evolution challenge. This article
shall mostly focus on recent advances in techniques and
strategies for directed evolution using random mutagenesis.
2. METHODS TO GENERATE GENETIC DIVERSITY
BY RANDOM MUTAGENESIS
2.1. The Experimental Cycle of Directed Evolution
The experimental cycle of directed evolution using ran-
dom mutagenesis consists of the following main steps, as
shown in Fig. (1):
1. A parent DNA sequence encoding for a desired protein
is chosen. Sequence diversity is created through a mutagene-
sis step, by introduction of random point mutations.
2. The mutated DNA sequences are ligated into an ex-
pression vector, they are transformed into host cells, and the
protein is expressed.
3. A screening procedure is employed next to select the
transformant(s) containing the encoded sequence(s) for en-
zyme(s) or protein(s) with desired properties.
4. The selected sequence (or sequences), if desirable, can
be further is then amplified and the cycle of mutagenesis,
screening and amplification is repeated (Fig. 2), for further
improvement if necessary.
2.2. Chemical Mutagenesis
Chemical mutagenesis has been used extensively and is
considered ‘food grade’, because it does not involve the in-

1389-2037/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

92 Current Protein and Peptide Science, 2010, Vol. 11, No. 1 Nikolaos E. Labrou

Fig. (1). The main experimental steps of an in vitro directed evolution process. A parent DNA sequence encoding for a desired enzyme is
chosen. Sequence diversity is created through a random mutagenesis step, (the symbol * represents point mutation). The library of DNA se-
quences is ligated into an expression vector. Recombinant clones are transformed into host cells, and the enzyme variants are expressed. A
screening or selection procedure is employed next to isolate the transformants with the desired activity. Subsequently, the best variant found
after screening, if necessary, is used in another round of mutagenesis.

troduction of heterologous DNA or the manipulation of ex-
isting DNA by recombinant means. Several chemical have
been used [29,30,38]. For example, ethyl methanesulfonate
(EMS) aklylates guanidine residues, causing them to be in-
correctly copied during DNA replication. Since EMS di-
rectly modifies DNA, EMS mutagenesis can be carried out
either in vivo, for example using whole cells or in vitro, us-
ing DNA. Nitrous acid (HNO2) is another chemical mutagen.
It acts by de-aminating adenine and cytosine residues caus-
ing transversion point mutations (A/T to G/C and vice-
versa). Other chemicals that were confirmed as mutagens
and have been used for mutagenesis are: mitomycin C
(MMC), N-methyl-N-nitrosourea (MNU), diepoxybutane
(DEB), 1, 2,7,8-diepoxyoctane (DEO), methyl methane sul-
Engineering Enzymes by Random Mutagenesis
Fig. (2). Random mutagenesis typically starts with a single parental
gene. The cycle of mutagenesis, screening and amplification (evo-
lution round) is repeated until an enzyme variant with the desired
property or function is found.
fonate (MMS), N-methyl-N’-nitro-N-nitrosoguanidine (MN
NG), 4-nitroquinoline 1-oxide (4-NQO), 2-methyloxy-6-
chloro-9(3-[ethyl- 2-chloroethyl]-aminopropylamino)-acrid-
inedihydrochloride (ICR-170), 2-amino purine (2AP), hy-
droxylamine (HA), bisulfate (BS) and methoxylamine (MA).
Table 1 summarizes the characteristic types of DNA damage
and resulting mutations induced by these chemicals.
An efficient procedure for the generation of random GC
to AT transition mutations in a specific DNA segment using
methoxylamine has been described by Kadonaga and
Knowles [39]. By this method a restriction DNA fragment is
inserted in each orientation into an M13 vector. Single-
stranded virion DNA from each recombinant phage is treated
with methoxylamine, and, after reannealing of the mutage-
nized strands, a double-stranded fragment is obtained. Using
this technique, single and double nucleotide substitutions
were generated at a frequency greater than 50% in a 56-base
pair segment of the signal codons of the TEM beta-lactamase
[39]. In another example, Ermakova-Gerdes et al., (1996)
[40] used bisulfite for in vitro random mutagenesis of the
psbDI gene of photosystem II from Synechocystis sp. PCC
6803. Sodium bisulfite reacts specifically with cytosine in
single-stranded regions of DNA and does not attack double-
stranded DNA. Using a hybrid plasmid that was single-
stranded in the region to be mutagenized and double-
stranded elsewhere, mutations were targeted to a specific
psbDI region coding for the lumenal A-B loop of the D2
protein. Using this approach several mutants were isolated
with a total of fifteen different amino acid changes in the
loop region [40].
The advantage of chemical mutagenesis is the simplicity,
economy and the main disadvantage of this method is its
inefficient control of mutation rate and limited amino acid
Current Protein and Peptide Science, 2010, Vol. 11, No. 1 93
substitutions. Recently, Mohan and Banerjee (2008) reported
a novel Dual Approach to Random Chemical Mutagenesis
(DuARCheM) to introduce random mutations in a defined
DNA fragment [41]. DuARCheM involves in vivo chemical
mutagenesis and in vitro genetic manipulations. Amplifica-
tion and mutation in the DuARCheM method leads to a bet-
ter spectrum of mutants because the plasmid is exponentially
amplified in the presence of mutagen pressure, unlike in the
in vitro chemical mutagenesis system in which the template
molecule does not replicate.
2.3. Mutator Strains
A simple and efficient approach for introducing random
point mutations, without mutation bias, into whole genes is
based on utilization of mutator strains lacking DNA repair
mechanisms [31,42]. For example, the Escherichia coli de-
rivative Epicurian coli XL1-Red is an E. coli strain that
lacks three of the primary DNA repair pathways, MutS,
MutD and MutT, resulting in a random mutation rate 5000-
fold higher than in the wild type strain. Introduction of a
plasmid bearing the gene encoding the protein of interest
leads to mutations during replication. The protocol for using
the mutator strain is composed of two steps: transformation
of the mutator strain and recovery of the mutated plasmids
from the transformant. The advantage of this method is that
involves simple protocol and a ligation step is unnecessary.
In addition, a wide variety of mutations can be incorporated
including substitutions, deletions and frame-shifts. The
drawback with this method is that the strain becomes pro-
gressively sick as it accumulates mutations in it’s own ge-
nome. This is because several steps of growth, plasmid isola-
tion, transformation and re-growth are normally required to
obtain diversity. In addition, the mutation frequency is low
under the standard conditions (0.5 mutations per kilobase),
and a cultivation period longer than 24 h is often required for
introducing multiple mutations.
Mutants of an esterase from Pseudomonas fluorescens
were generated using the mutator strain, Epicurian coli ®
XL1-Red [43]. One variant (Ala209Asp/Leu181Val) stereo-
selectively hydrolyzed a sterically-hindered 3-hydroxy ester,
which was not accepted as substrate by the wild type en-
zyme. After several mutation cycles, mutants were assayed
by plating the esterase-producing colonies onto minimal-
media agar plates containing the 3-hydroxy ethyl or glyceryl
ester and indicators. In another example Callanan et al., [44]
accomplished engineering of the pH-dependence of Lacto-
bacillus gasseri ADH beta-glucuronidase. Lactobacillus gas-
seri ADH beta-glucuronidase exhibits high activity in acidic
pH range (~pH 5.0) whereas, its activity is low in (pH 6-7).
In an effort to improve the activity of this enzyme in neutral
pH ranges, its gene was subjected to random mutagenesis by
passage through Epicurian coli mutator strain XL1-Red.
Two enzyme variants (D524G and D573A) were selected.
One of these, the D573A variant, was significantly more
active in the pH range of 4-8. This enzyme variant may be
used as a reporter enzyme in expression hosts that are not
acidophilic.
2.4. “Error-Prone” PCR
“Error-prone” PCR (epPCR) is based on the substantially
increasing of the overall error frequency of Taq DNA po-
94 Current Protein and Peptide Science, 2010, Vol. 11, No. 1
Table 1. Frequently Used Chemical Mutagens and Mutation Type Caused
Chemical
ICR-170
MMC, DEO
4-NQO, DEB
MNNG, EMS, MNU
NA, HA
2AP
MMS Alkylation/strand breaks
MA
BS Reacts specifically with C in single-stranded
lymerase. This enzyme lacks 3’-5’ exonuclease activity and
exhibits high error rate (0,8-1,1 x 10-4 base substitutions/bp
of product) under standard condition [45,46]. By changing
some PCR conditions one can increase this error rate. These
conditions include: (1) the addition of Mn2+ for reducing the
base pairing specificity [47], [Fig. (3)]; (2) unbalanced dNTP
stoichiometry in order to achieve misincorporation [16], (3)
increased Mg2+ concentration for stabilizing no complemen-
tary base pairs [48] and (4) increased polymerase concentra-
tion for enhancing the probability of elongation of mis-
primed termini. After PCR, the library of mutated genes
must be cloned into a suitable plasmid. The disadvantage of
this approach is that the size of the library is limited by the
efficiency of the cloning step since a ligation step is neces-
sary. In addition, the success of mutagenesis depends on
mutational bias of DNA polymerase. Although point muta-
tions are the most common types of mutation in epPCR, de-
letions and frameshift mutations are also possible. epPCR is
by far the most popular random mutagenesis method.
Fig. (3). The dependence of error-prone rate on Mn+2 concentra-
tions for the aq DNA polymerase.
Nikolaos E. Labrou
Type of DNA Lesion Mutation Type
Intercalation Frameshift
Interstrand cross-linking Deletion
DNA adducts Base-pair substitution
Alkylation Base-pair substitution
Modification of bases Base-pair substitution
Base analogue Base-pair substitution
Several different types
Transition mutations GC to AT
regions of DNA
Current low-fidelity DNA polymerases that are com-
monly used during random mutagenesis in vitro, display
strong mutational preferences, favoring the substitution of
certain nucleotides over others. The result is a biased and
reduced diversity in the library [45,46]. In an effort to reduce
mutational bias, a commercial available product, termed Mu-
tazyme® DNA polymerase, was launched. This enzyme pro-
duces a more uniform mutational spectrum compared to
other epPCR methods. Recently, Vanhercke et al., [49] com-
bined the two different low-fidelity DNA polymerases, Taq
and Mutazyme®, which have opposite mutational spectra. As
a first step, random mutants of the Bacillus thuringiensis
cry9Ca1 gene were generated by separate error-prone PCRs
with each of the two polymerases. Subsequent shuffling by
staggered extension process (StEP) [5] of the PCR products
resulted in intermediate numbers of AT and GC substitu-
tions, compared to the Taq or Mutazyme® epPCR libraries.
Benjamin and Connolly, [50] have reported a mutant
variant of DNA polymerase from Pyrococcus furiosus (Pfu
DNA polymerase) with superb performance in error-prone
PCR. Mutations in Pfu DNA polymerase were inserted in a
short loop linking two long -helices that comprise the ‘fin-
gers’ sub-domain of the protein. This region is responsible
for binding the incoming dNTPs and ensuring that only cor-
rect bases are inserted opposite the complementary base in
the template strand. Mutations in the short loop, when com-
bined with an additional mutation that abolishes the 3–5
proof-reading exonuclease activity, convert the extremely
accurate wild-type polymerase into a variant with low fidel-
ity.
epPCR has been successfully used in the directed evolu-
tion of CYP102A2, which is a P450 monoxygenase from the
B. subtilis [51]. After a single round of mutagenesis by
epPCR on the CYP102A2 gene and screening, the variant
Pro15Ser was found (Fig. 4). This variant showed approxi-
mately 6- to 9-fold increased activity with SDS, lauric acid
and 1,4-naphtho-quinone, and enhanced activity for other
substrates such as ethacrynic acid and -amino-n-caproic
acid. Examination of molecular model of the enzyme sug-
gested that Pro15 positioned away from the substrate binding
Engineering Enzymes by Random Mutagenesis
site. Thus, this study illustrates the significant effects of resi-
dues distal to the active site on catalysis and binding. In an-
other example, a mutant library of AppA2 phytase was gen-
erated by epPCR [52]. Enzyme variants were expressed ini-
tially in Saccharomyces cerevisiae for screening and then in
Pichia pastoris for characterizing thermostability. Two vari-
ants (K46E and K65E/K97M/S209G) were selected which
showed over 20% improvement in thermostability, and 6-7
°C increases in melting temperatures.
Fig. (4). Structural representation of the heme domain of
CYP102A2 (residues 1-472) from B. subtilus. The bound heme and
Pro15 are shown in a stick representation. The figure was produced
using PyMol (DeLano scientific).
Recently engineering of the large subunit (HycE, 569
amino acids) of E. coli hydrogenase-3 for enhanced hydro-
gen production by an epPCR was accomplished [53]. After
selection, seven enhanced enzyme variants were obtained
with a novel chemochromic membrane screen that directly
detected hydrogen from individual colonies. The best epPCR
variant contained eight mutations (S2T, Y50F, I171T,
A291V, T366S, V433L, M444I, and L523Q) and had 17-
fold higher hydrogen-producing activity compared to the
wild-type enzyme.
The in vitro MutaGen procedure was recently introduced
by Emond et al., [54]. This method is based on the use of
low-fidelity DNA polymerases pol beta and pol eta. This
technique was applied on a 2 kb gene encoding amylosu-
crase, an attractive enzyme for the industrial synthesis of
amylose-like polymers. Mutations were introduced during a
single replicating step performed by mutagenic polymerases
pol beta and pol eta. The authors found that variants gener-
ated by pol beta were 4 to 7-fold less mutated than those
created with pol eta. In addition, pol beta and pol eta provide
different and complementary mutation spectra. Some of the
mutants that were generated by pol eta displayed unusual
modifications, including combinations of base substitutions
and codon deletions which are rarely generated using other
methods [54].
Current Protein and Peptide Science, 2010, Vol. 11, No. 1 95
Asano et al., used recently epPCR to construct a mutant
library of E. coli aspartase [55]. Aspartase, catalyzes the re-
versible deamination of L-aspartic acid to fumaric acid and
ammonia, and is highly selective towards L-aspartic acid.
The mutant library was introduced to E. coli and the trans-
formants were screened for production of fumaric acid-mono
amide from L-aspartic acid-alpha-amide. One aspartase vari-
ant (Lys327Asn) with altered substrate specificity was iso-
lated. This enzyme variant showed improved ability to cata-
lyze the deamination of L-aspartic acid-alpha-amide.
A novel method, ‘frame shuffling’, for preparing protein
libraries was recently reported by Kashiwagi et al., [56].
With this method, a Y-family DNA polymerase was used.
This DNA polymerase introduces frame shift mutations at
high rates and the resultant progeny produce mutant proteins
having segmental sequence changes.
2.5. Rolling Circle Error-Prone PCR
Rolling circle amplification (RCA) is an isothermal
method that amplifies circular DNA by a rolling circle
mechanism, yielding linear DNA composed of tandem re-
peats of the circular DNA sequence, as illustrated in Fig. (5)
[57]. Error-prone RCA (epRCA) is a variant of epPCR in
which wild-type sequence is first cloned into a plasmid, and
then the whole plasmid is amplified under error-prone condi-
tions [37]. In comparison to other methods, epRCA consists
of only one DNA amplification step followed by transforma-
tion of the host strain, without treatment with any restriction
enzymes or DNA ligase, and results in a randomly mutated
plasmid library with 3–4 mutations per kilobase.
2.6. Site-Saturating Mutagenesis.
Once a specific amino acid position within a polypeptide
has been identified to be critical for the protein's function, it
is of interest to identify the ideal amino acid for this position
[58,59]. Site-saturation mutagenesis (SSM) allows the sub-
stitution of specific sites against all 20 possible amino acids
at once. SSM may be efficient carried out by PCR as shown
in Figs. (6) and (7). The first of these is termed whole plas-
mid, single-round PCR [60] (Fig. 6). In this technique, two
oligonucleotide primers containing mutant codon with a
mismatched sequence, are complementary to the opposite
strands of a double-stranded DNA plasmid template are ex-
tended using PCR and DNA polymerase. By this PCR the
entire plasmid containing breaks that do not overlap is pro-
duced. The wild type plasmid selectively eliminated using
DpnI endonuclease digestion since it is derived from E. coli
and is thus methylated. As a result a circular, nicked vector
containing the mutated gene is produced. This nicked vector
may be transformed directly into competent E. coli cells and
the nick in the DNA is repaired by the cell machinery to give
a mutated, circular plasmid.
The second method is termed the overlap extension
method [61,62] and is shown in Fig. (7). This technique is
carried out employing two PCRs and four primers, comple-
mentary to a section of the gene of interest. The primer pairs
for the two PCRs are 1/3 and 2/4, respectively. The primers
2 and 3 contained the mutant codon with a mismatched se-
quence. Two double-stranded DNA products are obtained.
When these double stranded duplexes are denatured and then
96 Current Protein and Peptide Science, 2010, Vol. 11, No. 1 Nikolaos E. Labrou

Fig. (5). Representation of the RCA method. Random hexamers are hybridized with template plasmid, and the resulting double-strand
segments function as primers in the polymerization reaction carried out by
29 DNA polymerase. As the 'front' of the extending strand of the
plasmid encounters double-stranded portions of DNA, the advancing new strand displaces the old one from the template. This extension
process covers the entire length of the plasmid multiple times, leading to the formation of repeated sequences of the template (concatemers).

annealed, two heteroduplexes are produced wherein each
strand of the heteroduplex contains the desired mutagenic
codon. The overlapping ends of each heteroduplex are then
filled in using DNA polymerase. In the second PCR the
primers 1 and 4 are used to amplify the entire gene.
The advantages of whole plasmid, single-round PCR are
that only one PCR needs to be performed and two, compared
to four in overlap extension method, primers are required.
The drawbacks of this technique relative to overlap exten-
sion are that it does not work well with large plasmids (>10
kB) and typically only two nucleotides can be replaced at a
time.
Other methods of SSM that have been developed, includ-
ing combinatorial cassette mutagenesis [63], recursive en-
semble mutagenesis [64], scanning saturation mutagenesis
[65], codon cassette mutagenesis [66], iterative saturation
mutagenesis (ISM) [67,68], and sequence saturation mutage-
nesis (SeSaM) [69]. This later method is able to randomize a
DNA sequence at every nucleotide position through use of a
universal base.
Conventional methods for codon randomization employ
NNN-, NNB-, NNK- or NNS-containing oligonucleotides (N
= A/C/G/T; B = C/G/T; K = G/T; S = G/C), as these combi-
nations each encode all 20 amino acids. Incorporating the
full degeneracy of the genetic code into a library of genes by
using NNN codons has severe limitations. For example, a
significant percentage of the genes will contain premature
termination codons. This is even more important if multiple
codons are randomized. In addition, the most common pro-
tein variants (containing combinations of Arg, Leu and Ser,
each of which are encoded by six codons) will be very abun-
dant compared to the rarest (with Met or Trp at each random-
ized position). This usually complicates the screening or
selection procedures [36].
These problems can be improved by reducing codon sets:
NNB codons have the smallest probability of encoding a
stop codon (one in 48), while NNK and NNS codons mini-
mize the overrepresentation of the commonest variants [36].
Several interesting examples of SSM have been recently
published as for example an approach for redesigning coen-
zyme specificity of NAD+-dependent formate dehydrogenase
[58]. NADP+-dependent dehydrogenases have many diag-
nostic, analytical and industrial applications, but their com-
mercial exploitation is hindered by the high cost of the corre-
sponding coenzyme. Therefore, protein engineering studies
of NAD+-dependent formate dehydrogenase (FDH) aiming
at redesigning coenzyme specificity (NADP+ vs. NAD+), for
the development of a NADP+ regeneration system is of prac-
tical importance. Structural analysis of the 3D-model of
FDH from C. boidinii showed that Asp195 which is located
at the nucleotide binding site is the main structural determi-
Engineering Enzymes by Random Mutagenesis Current Protein and Peptide Science, 2010, Vol. 11, No. 1 97

saturation mutagenesis and screening. Two P450 BM-3 vari-

ants with an up to six-fold increase in catalytic efficiency
(kcat/Km) were identified and isolated.

Fig. (7). The overlap extension method for site-saturation mutage-

nesis. The symbol ‘*’ represents point mutation(s).
Fig. (6). Whole plasmid, site-saturation mutagenesis using single-
round PCR. The symbol ‘*’ represents point mutation(s).

nant that modulates coenzyme specificity. This residue was

subjected to directed evolution employing saturation
mutagenesis. Activity screening of several mutant clones
allowed the isolation of an enzyme variant (Asp195Gln)
(Fig. 8) which displayed about 5 x107-fold change in coen-
zyme specificity towards NADP+ [58].
In another example, Ang et al., [70] applying one round
of saturation mutagenesis at position 205 (Val205) in aniline
dioxygenase, followed by a round of random mutagenesis
and selection, indentified a mutant of the enzyme with high
activity towards aniline, 2,4-dimethylaniline, and 2-
isopropylaniline. Li et al., [71] have recently reported the
creation of cytochrome P450 BM-3 variant with enhanced
ability to produce indigo and indirubin [71] using the combi-
nation of random and saturation mutagenesis. Cytochrome
P450 BM-3 with the mutations A74G, F87V, and L188Q
could catalyze indole to produce indigo and indirubin. To
Fig. (8). Structural representations of Candida boidinii FDH. The
further enhance this capability, random mutagenesis on the
bound NAD+ and Asp196 are shown in a stick representation and
monooxygenase domain of P450 BM-3 mutant were per-
labelled. Bound azide (is considered as a formate ion analogue) is
formed. Three mutants exhibited higher hydroxylation activ-
shown as dotted-spheres and labelled. The figure was produced
ity toward indole, in comparison to parent enzyme, were
using PyMol (DeLano scientific).
isolated. The mutated sites were further subjected to site-
98 Current Protein and Peptide Science, 2010, Vol. 11, No. 1
More recently, site-saturation mutagenesis was used to
create a new L-asparaginase variant with improved stability
[13]. L-Asparaginase catalyses the hydrolysis of L-Asn, pro-
ducing L-Asp and ammonia. This enzyme is an anti-
neoplastic agent; it is used extensively in the chemotherapy
of acute lymphoblastic leukaemia. Enhancing the stability of
L-ASNase by protein engineering improve its body-
residence time, thereby minimizing immunosuppressive ef-
fects by lowering therapeutic dose. Site-saturation mutage-
nesis was carried out at position 133 (Asp133). Asp133 is
located at a neutral region on the enzyme surface and makes
a significant and unfavourable electrostatic contribution to
overall stability. Screening of a library of random Asp133
mutants established that that this position is involved in de-
termining thermostability and showed that the Asp133Leu
mutation confers optimal thermostability [13].
2.7. Structure-Guided Enzyme Evolution
The in vitro-generated randomized libraries (i.e. < 1015
variants) represent a minute proportion of the possible se-
quence-, structure- or function-space. For example, for a
100-residue protein the different sequences are 20100. This
highlights the need to target the diversity in such a manner
that the probability of finding the desired clone is increased.
Recent progress in biocomputing has allowed the develop-
ment of algorithms to characterize and guide library diversity
and design [36,72-74].
For example, Web-based tools such as ConSurf-HSSP
and SCHEMA serve to guide library design. ConSurf-HSSP
web server [75] calculates the evolutionary conservation at
each amino acid position of a target protein using the pre-
calculated multiple sequence alignments of the HSSP (Ho-
mology-derived Secondary Structure of Proteins) database
[73]. The result is projected onto the protein’s structure, gen-
erating an interactive, three-dimensional map that empha-
sizes conserved and variable regions. Mapping functionally
important regions tends to identify those parts of the protein
that are most likely to abrogate parental function when mu-
tated. Other suite of programs – GLUE, PEDEL and
DRIVeR– for estimating various library statistics have been
described [76,77]. Programs such as GLUE, PEDEL and
DRIVeR estimate the number of distinct sequence variants in
a library, or provide a library size to aim for a specified pro-
portion of all possible variants. More recently, Firth and Pat-
rick [78] have introduced the program GLUE-Including
Translation (GLUE-IT), which finds the expected amino acid
completeness of libraries in which up to six codons have
been independently varied. In addition, the same authors
have also presented the program PEDEL-AA, which calcu-
lates amino acid statistics for libraries generated by epPCR
[78]. Input in this program includes the parent sequence,
overall mutation rate, library size, indel rates and a nucleo-
tide mutation matrix. Output includes amino acid complete-
ness and diversity statistics, and the number and length dis-
tribution of sequences truncated by premature termination
codons.
CONCLUSION
Directed evolution using random mutagenesis is a versa-
tile tool for developing novel enzymes. It has been used to
Nikolaos E. Labrou
improve enzyme catalytic activity and efficiency, modulate
(limit or expand) substrate specificity, alter cofactor specific-
ity, and improve stability over a wider range of conditions.
Although random mutagenesis uncovers diversity in a very
small region of sequence space, has been proven to be suffi-
cient efficient to allow the wholesale changes required, for
example, to evolve a novel activity in a target gene [79-82].
Several engineered proteins are currently being marketed.
For example, Proleukin® IL-2 (interleukin-2) that is used for
cancer treatment has a mutated free cysteine that leads to
decreased aggregation and improved bioavailability [83].
Other examples include: a mutant of NAD +-dependent for-
mate dehydrogenase with altered cofactor specificity towards
NADP+ [84], a mutant of Taq DNA polymerase (Muta-
zyme®) that exhibits increased misinsertion and misexten-
sion frequencies compared to wild type Taq (Stratagene,
USA), a mutant of HIV protease that resist autoproteolysis,
resulting in an enzyme with improved stability and substan-
tially higher activity levels over extended time periods
(Sigma-Aldrich, USA).
Recent developments in molecular biology have resulted
in more sophisticated and improved methods for creating
sequence diversity using random mutagenesis that are appli-
cable on different molecular levels, ranging from the optimi-
zation of single enzymes to entire metabolic pathways
[81,82]. Such methods continue to expand the range of pro-
tein and enzyme variants that can be obtained with properties
suitable for commercial and academic applications.
ACKNOWLEDGEMENT
This work was supported by the Hellenic General Secre-
tariat for Research and Technology: Operational Program for
Competitiveness, Joint Research and Technology Program
and by the NATO LST.CLG.979818 grand.
ABBREVIATIONS
2AP = 2-Amino purine
BS = Bisulfate
DEB = Diepoxybutane
DEO = 1,2,7,8-Diepoxyoctane
DuARCheM = Dual Approach to Random Chemical
Mutagenesis
EMS = Ethyl methanesulfonate
epPCR = Error-prone PCR
epRCA = Error-prone rolling circle amplification
FDH = Formate dehydrogenase
HA = Hydroxylamine
ICR-170 = 2-Methyloxy-6-chloro-9(3-[ethyl-2-
chloroethyl]-aminopropylamino) acridin-
edihydrochloride
ISM = Iterative saturation mutagenesis
MA = Methoxylamine
MMC = Mitomycin C
MMS = Methyl methane sulfonate
Engineering Enzymes by Random Mutagenesis
MNU = N-methyl-N-nitrosourea
MNNG = N-methyl-N’-nitro-N-nitrosoguanidine
4-NQO = 4-Nitroquinoline 1-oxide
RCA = Rolling circle amplification
SeSaM = Sequence saturation mutagenesis
SSM = Site saturation mutagenesis.
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