Báo cáo khoa học: Simultaneous improvement of catalytic activity and thermal stability of tyrosine phenol-lyase by directed evolution ppt

Simultaneous improvement of catalytic activity and
thermal stability of tyrosine phenol-lyase by directed
evolution
Eugene Rha
1
, Sujin Kim
1
, Su-Lim Choi
1
, Seung-Pyo Hong
2
, Moon-Hee Sung
2
, Jae J. Song
3
and
Seung-Goo Lee
1
1 Industrial Biotechnology & Bioenergy Research Center, KRIBB, Daejeon, South Korea
2 BioLeaders Corp., Daejeon, South Korea
3 Molecular Bioprocess Research Center, KRIBB, JungUp, South Korea
Introduction
Tyrosine phenol-lyase (TPL) (EC4.1.99.2) is a tetra-
meric enzyme that catalyzes the a,b-elimination and
b-replacement of l-tyrosine, with pyridoxal-5¢-phos-
phate (PLP) as the cofactor [1–3]. At high concentra-
tions of ammonium pyruvate, the enzyme catalyzes the
synthesis reaction of aromatic amino acids from
phenolic substrates [4]. The resulting amino acids can
be used as precursors for several neurotransmitters,
such as l-DOPA (DOPA, 3,4-dihydroxyphenylala-

nine), dopamine, epinephrine and norepinephrine [5].
In most living organisms, l-tyrosine is principally syn-
thesized from l-phenylalanine. Yet, for the industrial
production of l-tyrosine and its derivatives, attention
has been focused on enzymatic synthesis using TPL
Keywords
N-terminal arm; protein engineering;
structural relevance; Symbiobacterium
toebii; tyrosine phenol-lyase
Correspondence
S G. Lee and J. J. Song, 111, Gwahangno,
Yuseong, Daejeon 305-806, South Korea
Fax: +82 42 860 4379
Tel: +82 42 860 4373
E-mail: ;
(Received 17 April 2009, revised 11 August
2009, accepted 24 August 2009)
doi:10.1111/j.1742-4658.2009.07322.x
The tyrosine phenol-lyase from Symbiobacterium toebii was engineered to
improve both its stability and catalytic activity by the application of ran-
dom mutagenesis and subsequent reassembly of the acquired mutations.
Activity screening of the random library produced four mutants with a
two-fold improved activity, whereas parallel screening after heat treatment
at 65 °C identified three mutants with half-inactivation temperatures
improved by up to 5.6 °C. The selected mutants were then reassembled
using the staggered extension PCR method, and subsequent screening of
the library produced seven mutants with up to three-fold improved activity
and half-inactivation temperatures improved by up to 11.2 °C. Sequence
analyses revealed that the stability-improved hits included A13V, E83K
and T407A mutations, whereas the activity-improved hits included the

additional T129I or T451A mutation. In particular, the A13V mutation
was propagated in the hits with improved stability during the reassembly–
screening process, indicating the critical nature of the N-terminal moiety
for enzyme stability. Furthermore, homology modeling of the enzyme
structure revealed that most of the stability mutations were located around
the dimer–dimer interface, including the N-terminus, whereas the activity-
improving mutations were located further away, thereby minimizing any
interference that would be detrimental to the co-improvement of the stabil-
ity and catalytic activity of the enzyme.
Abbreviations
DOPA, 3,4-dihydroxyphenylalanine; LB, Luria–Bertani; PLP, pyridoxal-5¢-phosphate; StEP PCR, staggered extension PCR;
T
1 ⁄ 2,
half-inactivation temperature; TPL, tyrosine phenol-lyase.
FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS 6187
from phenolics, such as hydroxylated or halogenated
derivatives of phenol, 4-chlorophenol, 4-nitrophenol
and catechol [1,6].
Although most structure–function studies of TPL
have focused on enzymes from enteric bacteria, includ-
ing Citrobacter freundii and Erwinia herbicola, thermo-
philic enzymes are also considered to provide benefits
as biocatalysts for enzymatic processes. For example, a
thermostable TPL from Symbiobacterium species can
maintain stability with high concentrations of phenolic
substances, whereas enzymes from enteric bacteria are
inactivated under the same conditions [7,8]. Therefore,
a TPL with improved thermal stability or catalytic
activity may be very useful for the development of an
ideal enzymatic process for aromatic amino acids

[9–11].
On a molecular level, protein stabilization is related
to increased rigidity in an unstable structural unit,
whereas improved enzyme activity is related to
increased flexibility of the catalytic residues in the
active site [12,13]. Therefore, many studies have
reported the concomitant occurrence of increased sta-
bility and compromised catalytic activity. Nonetheless,
several recent studies have been successful in simulta-
neously improving both activity and stability using
directed evolution technology [14–16]. Thus, it would
appear that such co-improvements will most probably
occur when the two properties are combined with
weak structural and functional interference [17].
Accordingly, to improve the potential of TPL as a
biocatalyst, this study used random mutagenesis, fol-
lowed by a staggered extension PCR (StEP PCR) to
reassemble beneficial mutations. StEP PCR is an alter-
native DNA shuffling technology that is based on a
short-cycle PCR [12]. Three-dimensional modeling
analysis of the consequent hits was also performed to
investigate the simultaneous improvement of the activ-
ity and stability of Symbiobacterium toebii TPL, as a
better structural understanding of how proteins
respond to mutations and recombination can help in
the development of more ambitious enzyme engineer-
ing strategies, such as increasing the probability of
mutant sequences having the desired properties [18,19].
Results and Discussion
Mutagenesis and characterization of mutant

enzymes
The error-prone PCR using S. toebii TPL as the
template produced a library of TPL mutants, each
containing two to six mutations distributed over the
entire sequence (approximately 2.7 mutations in
1377 bp). The mutation frequency was determined by
sequencing 10 clones randomly picked from the naive
library (1.2 · 10
5
colonies). When cultivating 12 000
colonies from the mutagenesis library in Luria–Bertani
(LB) medium and assaying them on microtiter plates
at 37 °C, four ‘activity’ mutants, A1–A4, were identi-
fied (Table 1). Meanwhile, parallel screening after heat
treatment at 65 °C for 10 min highlighted three other
‘stability’ mutants, S1–S3 (Table 1).
To quantify the stability of the selected mutants, the
remaining activity was measured after incubation for
30 min at various temperatures between 37 and 75 °C.
Table 1 shows a comparison of the apparent half-
inactivation temperatures (T
1 ⁄ 2
), which were up to
5.6 °C higher for the stability mutants than for the wild-
type enzyme. Meanwhile, the activities of A1 and A2
were two-fold greater than that of the wild-type enzyme,
whereas the activity of the stability mutants was rela-
tively unchanged. The specific mutations in the activity
and stability mutants are summarized in Table 1.
Co-improvement of activity and thermal stability

To reassemble the acquired mutations, StEP PCR was
conducted using the hits from the random mutagenesis
as a mixed template. From the resulting library
(1.4 · 10
6
colonies), 10 colonies were randomly
selected and their sequences analyzed. The number of
crossovers was approximated to be 2.2 times along the
gene size (1377 bp) when counting the crossovers as
the recombination of mutations from different tem-
plates.
Next, after examining 1200 colonies from the Esc-
herichia coli library for the remaining activity, mutants
AS1–AS7 were selected on the basis of a co-improve-
ment in stability and activity. For example, the T
1 ⁄ 2
values for AS4 and AS6 were 6.7 and 11.2 °C higher,
respectively, and the catalytic activities were improved
by 2.8- and 2.0-fold, respectively (Table 1).
Isolates AS1–AS7 were analyzed to have fewer
mutations (1.2 per gene, in contrast with 2.7 per gene
in the A and S mutants), implying that many of the
original mutations had been deleted during the reas-
sembly process. Aligning each mutation against the
sequences of A1–A4 and S1–S3 allowed the parental
origin of the mutations to be estimated. For example,
AS4, composed of A13V, E83K and T451A, was ana-
lyzed to be a reassembly of S3 (harboring the A13V
mutation), S2 (harboring the E83K mutation) and A2
(harboring the T451A mutation). A more comprehen-

sive representation of the correlation between the
mutations is shown in Fig. 1 (redrawn from the data
in Table 1). Interestingly, mutants AS1–AS3, showing
Directed evolution of tyrosine phenol-lyase E. Rha et al.
6188 FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS
no significant improvement in activity, only contained
mutations originating from the S1–S3 mutants (the
gray-shaded ellipse in Fig. 1). Meanwhile, the four
other mutants (AS4–AS7) included mutations from
both the stability and activity mutants, and demon-
strated a significant improvement in both stability and
catalytic activity (Fig. 1). As such, the activity and sta-
bility mutation combinations in Table 1 represent the
synergistic recruitment of the original characteristics of
the parental mutants.
The wild-type protein and two mutants, AS4 and
AS7, were purified to homogeneity with purification
yields of over 40%, and investigated for their stability
and activity at temperatures between 40 and 80 °C. As
a result, the specific activities of the mutants were at
least two-fold higher than that of the wild-type across
a broad temperature range (Fig. 2A). When heated for
30 min in 0.1 m potassium phosphate buffer (pH 8.0),
the thermal stability of both mutants was confirmed to
be up to 10 °C higher than that of the wild-type
(Fig. 2B). When the pH properties of the purified
enzymes were investigated, the AS4 and AS7 enzymes
displayed an alkaline shift for their maximum activity
(Fig. 2C). In addition, the mutant enzymes exhibited a
higher remaining activity than the wild-type after incu-

bation for 36 h at alkaline pH (Fig. 2D).
Extraction of structural information from the
evolutionary process
Directed evolution has instigated a new enzyme engi-
neering paradigm for improving enzyme properties
without reliance on structural data [18,20]. This tech-
nology takes advantage of the natural process in which
Table 1. Genetic and catalytic changes of S. toebii TPL during evolutionary engineering.
Library Screening Name
Activity
mutations
Stability
mutations
Relative
activity
a
(fold)
Stability
change
b
(°C)
Random mutation
(first screening)
High activity A1 E42D
c
, T129I 2.04 )1.3
A2 A196T, T451A 1.99 )1.3
A3 T129I 1.69 )2.8
A4 T129I, V262A 1.47 +1.5
High stability S1 T407A 0.88 +5.6

S2 E83K 1.05 +3.1
S3 A13V, I457F 1.27 +5.5
Reassembly
(second screening)
High activity
and stability
AS1 A13V, T407A 1.12 +10.1
AS2 A13V, E83K, I457F 1.42 +5.6
AS3 A13V, E83K 1.42 +6.7
AS4 T451A A13V, E83K 2.84 +6.7
AS5 T451A A13V, E83K, T407A 1.87 +11.2
AS6 T129I A13V, E83K, T407A 1.99 +11.2
AS7 T129I A13V 2.10 +7.5
a
Fold increase in activity of mutant enzymes at 37 °C compared with that of wild-type TPL.
b
Increase in T
1 ⁄ 2
when heated for 30 min under
standard assay conditions compared with that of wild-type TPL (T
1 ⁄ 2
=63°C).
c
Italic letters indicate mutations that were erased during the
reassembly process.
15
1
5
10
2

3
5 6
2
3
1
4
7
4
1
–5
0
0.5 1 2 3
Activity change (fold)
Stability change (ºC)
3
2
Fig. 1. Schematic map of the activity and thermal stability during
the evolutionary engineering of S. toebii TPL. Open symbols (
)
indicate activity-related mutants and filled symbols (
) indicate
stability mutants obtained during the screening of random muta-
genesis libraries. Gray symbols (
) indicate reassembled hits
obtained during subsequent shuffling experiments, where the full
and broken arrows show the trajectory of stability and activity
mutations, respectively. The gray-shaded ellipse encircles S1–S3
and AS1–AS3 hits that only include stability mutations.
E. Rha et al. Directed evolution of tyrosine phenol-lyase
FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS 6189

beneficial mutations accumulate, whereas deleterious
mutations are simultaneously removed through the
recombination of homologous DNA fragments [18,19].
As such, certain mutations tend to appear more often
with the progression of the evolutionary engineering
process. In this study, the A13V, E83K and T407A
mutations originating from the S1–S3 hits were repeat-
edly detected during the screening of the reassembly
library (Table 1) and, of these mutations, A13V was
detected most frequently, indicating its critical nature
for stability. This mutation has already been shown to
increase the thermal stability of the enzyme by 4 °C,
with a slight compromise in enzyme activity [21].
Meanwhile, the four hits AS4–AS7 that exhibited a
co-improvement of activity and stability included the
additional T129I or T451A mutation (Table 1).
Therefore, to understand the structure and function
of the mutated residues, hypothetical structures of
S. toebii TPL were generated by comparative model-
ing using the open and closed structures of the TPLs
from E. herbicola [1C7G in Protein Data Bank (PDB)
entries] and C. freundii as templates [2,22–24]. The
open and closed models were deposited in PMDB under
accession numbers PM0075863 and PM0075934,
respectively. When the open conformation was super-
imposed on the open and closed templates (1C7G
and 2VLH, respectively), the rmsd values were 0.37
and 1.97 A
˚
for the C

a
traces, respectively. The
homology model consisted of four identical subunits,
with PLP molecules near the catalytic lysine residue
(K258). Each subunit comprised an N-terminal arm
(M1–T21), a small domain (R22–S58, D312–I457)
and a PLP-binding large domain (D59–V311). The
active sites were located in clefts between the two
domains, each constituting a catalytic dimer with the
adjacent subunit. The two dimers were then tightly
coupled via intertwined N-terminal arms near a
hydrophobic cluster (M57–E70) at the center of the
tetramer (Fig. 3A).
When marking the stability mutations (A13V, E83K
and T407A) in the three-dimensional model of S. toebii
TPL, shown in Fig. 3A, they were all distributed
around the dimer–dimer interface of the tetrameric
assembly (red spheres). Meanwhile, the activity muta-
tions (T129I and T451A) that survived the reassembly
process were positioned far away from the dimer–
dimer interface (cyan spheres). Interestingly, when the
activity mutations that were deleted during the reas-
sembly process (E42D, A126T and V262A) were
marked in the three-dimensional model (Fig. 3), they
were located near the dimer–dimer interface of the
three-dimensional structure (gray spheres).
To further investigate the stabilizing effect of Ala13,
various N-terminal homologs were retrieved from the
blast website and aligned, as shown in Table 2. The
position corresponding to Ala13 was found to vary in

sequence and identified as a serine in many known
microbial species, including C. freundii and E. herbicola,
yet was a branched amino acid (Val, Thr or Met) in
b-tyrosinases (TPL) from anaerobic bacteria and
120 4
0
20
40
60
80
100
1
0
2
3
4
5
6
Temperature (ºC)
60
80
100
120
Temperature (ºC)
p
H
Specific activity (units·mg
–1
)
Specific activity (units·mg

–1
)
Relative activity (%) Relative activity (%)
0
1
2
3
p
H
40 50 60 70 80
40 50 60 70
80
6 7 8 9 10 11
5 6
7 8 9 10 11
0
20
40
AB
CD
Fig. 2. Effect of temperature and pH on
stability and catalytic activity of purified TPL
proteins. (A) Specific activity assayed at
various temperatures in 100 m
M potassium
phosphate buffer (pH 8.0). (B) Relative activ-
ity assayed to evaluate thermal stability.
Enzymes were pre-incubated in 100 m
M
potassium phosphate buffer for 30 min at

various temperatures. (C) Specific activity
assayed in 50 m
M potassium phos-
phate ⁄ glycine buffer at the indicated pH
values. (D) Relative activity assayed to
evaluate pH stability. Enzymes were
pre-incubated in 50 m
M potassium phos-
phate ⁄ glycine buffer for 36 h at ambient
temperature. Filled circles indicate S. toebii
TPL, and open circles and triangles indicate
AS4 and AS7, respectively.
Directed evolution of tyrosine phenol-lyase E. Rha et al.
6190 FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS
l-tryptophanases. Notwithstanding, the flanking
sequences of Ala13 were highly conserved and symmet-
rical in each N-terminal homolog examined (Table 2).
Together with this sequence observation, a magnified
picture of the subunit interfaces (Fig. 4) revealed that
the symmetric sequence consisted of two possible elec-
trostatic interactions, K10–E15
*
–K12, that flanked the
contact between the Ala13 residues and the interacting
subunits, where the asterisked E15 indicates the inter-
acting residue contributed by the other intertwined
subunit. Therefore, the stabilizing effect of the Ala13
to Val mutation could be related to a tighter assembly
with the interacting subunits.
In a previous study, the current authors have shown

that the mutation of Thr15 to Ala in the vicinity of
the hydrophobic core induces a tighter binding of the
cofactor in C. freundii TPL, thereby reducing the
decomposition rate of the cofactor by a Pictet–Spen-
gler reaction [9]. In the S. toebii enzyme, Pro16 occu-
pies the Thr15 position (Table 2, Fig. 4) and is located
close to Ala13, which is very important for the stabil-
ity of the enzyme in this study.
In addition, the structural rationale for the other
stability (E83K and T407A) and activity (T129I and
T451A) mutations was investigated on the basis of the
open and closed conformation models. As a result, the
Table 2. Comparison of N-terminal sequences between homologous proteins and S. toebii TPL. Italic letters highlight residues correspond-
ing to Ala13 in S. toebii TPL, and bold letters mark highly conserved residues in homologous N-termini.
Strains (% identity) N-terminal sequence Source
SC1(100) -MQRPWAEPYKIKAVEPIRMTT Symbiobacterium toebii gi:1805293 b-tyrosinase
Sth (99) 1-MQRPWAEPYKIKAVEPIRMTT S. thermophilum gi:55977750 b-tyrosinase
Fnu (67) 8-AEPFRIKSVETVKMID Fusobacterium nucleatum gi:27887209 b-tyrosinase
Dno (66) 13-AEPFKIKSVEPVKMIS Dichelobacter nodosus gi:146233320 b-tyrosinase
Cte (66) 11-AEPFKIKSVEPVKMIS Clostridium tetani gi:28202972 b-tyrosinase
Pag (63) 5-AEPFRIKSVETVSMIS Pantoea agglomerans gi:260188 b-tyrosinase
Cfr (63) 5-AEPFRIKSVETVSMIP Citrobacter freundii gi:401201 b-tyrosinase
Ehe (63) 5-AEPFRIKSVETVSMIS Erwinia herbicola
gi:1351283 b-tyrosinase
Cag (69) 17-RRSWAEPWKIKTVEPLRIIS Chloroflexus aggregans gi:117996139 b-tyrosinase
Dha (68) 6-AEPFRIKVVEPVRSMK Desulfitobacterium hafniense gi:109642283 b-tyrosinase
Rca (70) 15-RRSWAEPWKIKMVEPLRVTT Roseiflexus castenholzii gi:156232199 b-tyrosinase
Cau (68) 16-RRSWAEPWKIKMVEPLRVTS Chloroflexus aurantiacus gi:76167194 b-tyrosinase
Tde (66) 8-AEPFRIKVVETVKMID Treponema denticola gi:42526628 b-tyrosinase
Tte (58) 9-AEPYKIKMVEPLKITT T. tengcongensis gi:20808031 tryptophanase

Dre (54) 5-QPKAEPFRIKMVEPIKMIS Desulfotomaculum reducens gi:134052564 tryptophanase
Cno (55) 6-EPFKIKMVEPLTITT Clostridium novyi gi:118442982 tryptophanase
Stt (54) 2-PKGEPFKIKMVEPIRLIP
S. thermophilum gi:2842553 tryptophanase
Pvu (49) 1-MAKRIVEPFRIKMVEKIRVPS Proteus vulgaris gi:2914379 tryptophanase
AB
Glu83
Thr129
Thr451
Thr407
Ala13
N
Thr451
Thr407
Ala13
Val262
Glu42
Ala13
N
Glu83
Thr129
Ala196
*
Fig. 3. Structural assignments of stability-
and activity-improving mutations in homo-
logy model framework (A) and subunit
structure (B) of S. toebii TPL. Red and cyan
letters indicate stability and activity
mutations, respectively, whereas black
letters indicate the activity mutations

deleted during the reassembly process. PLP
was adopted from the 1C7G PDB file and is
indicated by yellow sticks.
E. Rha et al. Directed evolution of tyrosine phenol-lyase
FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS 6191
T407A mutation in the small domain was correlated
with the stability of the substrate-binding site, as T407
was in van der Waals’ contact with important
substrate-binding residues, T50 and R405, in the same
domain. Meanwhile, E83 was located on the surface of
the large domain near the hydrophobic core, but no
direct interaction with other residues was detected in
this study. By contrast, the T129I and T451A muta-
tions were located in the large and small domains,
respectively, constituting the active site cleft between
the domains (Fig. 3B). In the closed conformation, it
has been proposed that the small domain undergoes
an extraordinary motion towards the large domain,
closing the active site cleft and bringing the cataly-
tically important residues R382 and F449 into the
active site [3,24].
Consequently, the modeling studies revealed that
most of the stability mutations were located around
the dimer–dimer interface, including the N-terminus.
Meanwhile, the activity-improving mutations were
found further away from the interface, as the activity-
related mutations near the interface were seemingly
deleted during the reassembly–screening steps, thereby
allowing the directed co-evolution of the stability and
catalytic activity of S. toebii TPL.

Materials and methods
Materials
Sodium pyruvate and PLP were obtained from Musashino
Shoji (Tokyo, Japan) and Fluka (Seelze, Germany), respec-
tively, and yeast extract and bacto-casitone were purchased
from BD (Franklin Lakes, NJ, USA). The other chemicals,
including l-tyrosine, phenol and NH
4
Cl, were all purchased
from Sigma-Aldrich (St Louis, MO, USA). The restriction
endonucleases, T4 DNA ligase and Vent DNA polymerase
were purchased from New England Biolabs (Beverly, MA,
USA), and the Taq DNA polymerase was obtained from
Takara (Otsu, Japan). The oligonucleotides were synthe-
sized at Bioneer Co. (Daejeon, South Korea), and the
DNA sequencing was performed by Solgent Co. (Daejeon,
South Korea).
Random mutagenesis and DNA shuffling
The plasmid pHCE IIB-TPL, harboring the S. toebii TPL
gene [7], was used as template for an error-prone PCR
employing a Genemorph II Random Mutagenesis kit
(Stratagene, La Jolla, CA, USA) with the following
primers: 5¢-CTCAAGACCCGTTTAGAGGCCC-3¢ (forward)
and 5¢-ATGCGTCCGGCGTAGAGGAT-3¢ (reverse).
Thermal cycling was performed using a DNA Thermal
Cycler (Bio-Rad, Hercules, CA, USA). The amplified PCR
products were digested with NdeI and HindIII to yield a
1.377 kb DNA fragment. The plasmid pHCE IIB (Biolead-
ers, Daejeon, South Korea) was also digested with NdeI
and HindIII, and dephosphorylated with shrimp alkaline

phosphatase (Roche, Mannheim, Germany). The plasmid
and insert were then ligated at 4 °C for 24 h with a T4
DNA ligase, following which the products were electro-
transformed into E. coli JM83 (ATCC #35607) and spread
on LB–ampicillin plates. After incubation overnight at
37 °C, the ampicillin-resistant colonies were transferred to
fresh LB–ampicillin plates using toothpicks.
A mixture of plasmids selected from the random muta-
genesis library was utilized as the DNA template for StEP
PCR [16,25] with Vent DNA polymerase and the following
primers: 5¢-AACGGCGGCATGTCTTTCTATA-3¢ (for-
ward) and 5¢-ATGCCTGGCAGTTCCCTACTCT-3¢
(reverse). PCR was carried out at 95 °C for 5 min, followed
by 40 cycles of 30 s at 94 °C and 5 s at 55 °C, plus an addi-
tional 15 cycles of 30 s at 95 °C, 30 s at 55 °C and 1.5 min
at 72 °C. The amplified DNA was then cloned into
pHCE IIB and transformed into E. coli JM83 cells.
Expression and screening of mutant library
Escherichia coli JM83 cells harboring the TPL library
within the constitutive expression system pHCE IIB were
inoculated manually using toothpicks into 96 deep-well
plates containing an LB–ampicillin medium (500 lL) and
cultivated in a well plate culture system (Bioneer Co.,
Daejeon, South Korea) for 20 h at 37 °C. Protein expres-
sion from the constitutive expression system did not require
the addition of an inducer [26]. The cultivated cells
(450 lL) were centrifuged for 20 min using a well plate cen-
trifuge (Hanil Sci., Incheon, South Korea), washed in
50 mm Tris ⁄ HCl buffer (pH 8.0) and treated with 200 lL
P16

A13
A13
K10
K12
E15
V14
P16
K12
E15
V14
[39–49]
N–TER
Hydrophobic
Core [57–69]
[310–322]
[411–420]
K10
K12
Fig. 4. Structural symmetry and putative electrostatic interactions
within intertwined N-terminal arm. Broken lines represent electro-
static interactions in the vicinity of A13 in the homology model of
S. toebii TPL.
Directed evolution of tyrosine phenol-lyase E. Rha et al.
6192 FEBS Journal 276 (2009) 6187–6194 ª 2009 The Authors Journal compilation ª 2009 FEBS
Cellytic BÔ (Sigma-Aldrich) for 30 min at 37 °C. The cell
lysate (100 lL) was then transferred using a multichannel
pipette (Eppendorf, Hamburg, Germany) into 96-well PCR
plates and mixed with an equal volume of a substrate solu-
tion (described in the assay conditions below). After incu-
bation at 37 °C, the reaction solutions were heated for

3 min at 94 °C, centrifuged to remove any insoluble aggre-
gates and analyzed for phenol production. Following the
activity analysis, the remaining cells with positive hits
(50 lL) were transferred to fresh LB–ampicillin medium
and preserved as glycerol stocks at )20 °C.
Purification and characterization
The mutant cells were cultivated at 37 °C for 24 h in
200 mL of LB medium containing 100 lgÆmL
)1
of ampicil-
lin. The cells were then suspended in 5 mL of NaCl ⁄ P
i
,
mixed with an equal volume of Cellytic BÔ, treated for
30 min with 20 lL of DNase (10 UÆmL
)1
; Roche) and
100 lL of lysozyme (100 mgÆmL
)1
; Sigma, St Louis, MO,
USA) and sonicated. Thereafter, the solution was centri-
fuged at 24 000 g for 30 min, and the supernatant was incu-
bated at 48 °C for 30 min to precipitate heat-labile E. coli
proteins. The solution was then centrifuged again, loaded
onto a Resource Q ion exchange column (Pharmacia,
Uppsala, Sweden), washed with a standard buffer and
eluted using a 0–0.5 m KCl gradient. Most of the active
fractions were pooled, adjusted to include 1.7 m
(NH
4

)
2
SO
4
, loaded onto a hydrophobic Phenyl Superose
column (Pharmacia) and eluted using a reverse gradient of
(NH
4
)
2
SO
4
from 1.7 to 0 m. The active fractions were then
dialyzed against 100 mm Tris ⁄ HCl (pH 8.0) and stored at
4 °C. All the column procedures were carried out using an
AKTA system (Amersham Bioscience, Uppsala, Sweden) at
room temperature.
Homology modeling and structural analysis
To examine the structural and functional effects of the
detected mutations, three-dimensional models were gener-
ated of both the wild-type and mutant S. toebii TPL. The
wild-type model of S. toebii was produced using ProModII
and optimized using Gromos96 from SWISS-MODEL
[27], an automated comparative protein modeling server.
The tetrameric structure of TPL from E. herbicola (1C7G
in PDB entry), sharing a 63% sequence identity with
S. toebii TPL, was used as the template for comparative
modeling. The coenzyme PLP was adopted from the
1C7G PDB file and fitted into the PLP-binding site of
each monomer. Meanwhile, an open conformation model

and mutant models of S. toebii TPL were constructed
using the Build Model module from Discovery Studio
(Accelrys, San Diego, CA, USA). The model with the best
loop conformations was then selected using the Profiles-3-
D verification method, and the structure was optimized on
the basis of energy minimization in the DS CHARM
module employing the steepest descent method followed
by the conjugate gradient method. During the minimiza-
tion process, the protein backbone was restrained using an
harmonic constraint.
Database
Model data are available in the PMDB database under the
accession numbers PM0075863, PM0075934, PM0075854
and PM0075847.
Enzyme assay
The enzyme activity in solution was measured by incubat-
ing 5 lg of the enzyme with 1 mml-tyrosine and 10 lm
PLP in 50 mm Tris ⁄ HCl buffer (200 lL, pH 8) for 15 min
at 37 °C. After heating at 94 °C for 3 min, the amount of
phenol in solution was measured colorimetrically using a
microplate reader (Bio-Rad) based on the 4-aminoantipyrin
method [28]. One unit of enzyme was defined as the amount
of enzyme able to catalyze the formation of 1 l mol of
phenol in 1 min at 37 °C. The protein concentration was
determined using the Bradford assay (Bio-Rad) with BSA
as the standard.
Acknowledgements
This project was supported by the Bio R&D program
and the pioneer research program through the KOSEF
(Korea Science and Engineering Foundation) and a

grant from the KRIBB Research Initiative Program.
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