Simultaneous improvement of catalytic activity andthermal stability of tyrosine phenol-lyase by directed evolution Eugene Rha1, Sujin Kim1, Su-Lim Choi1, Seung-Pyo Hong2, Moon-Hee Sung2,
Trang 1Simultaneous improvement of catalytic activity and
thermal stability of tyrosine phenol-lyase by directed
evolution
Eugene Rha1, Sujin Kim1, Su-Lim Choi1, Seung-Pyo Hong2, Moon-Hee Sung2, Jae J Song3and Seung-Goo Lee1
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: sglee@kribb.re.kr; jjsong@kribb.re.kr
(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 65C identified three mutants with half-inactivation temperatures improved by up to 5.6C 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 11.2C 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 activstabil-ity of the enzyme
Abbreviations
DOPA, 3,4-dihydroxyphenylalanine; LB, Luria–Bertani; PLP, pyridoxal-5¢-phosphate; StEP PCR, staggered extension PCR;
T1⁄ 2,half-inactivation temperature; TPL, tyrosine phenol-lyase.
Trang 2from 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 stabilactiv-ity 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 increasengineer-ing 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· 105 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 (T1⁄ 2), which were up to 5.6C 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· 106 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 T1 ⁄ 2 values for AS4 and AS6 were 6.7 and 11.2C 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
Trang 3no 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 80C 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 10C 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.
Activity mutations
Stability mutations
Relative activitya(fold)
Stability changeb(C) Random mutation
(first screening)
Reassembly
(second screening)
High activity and stability
a Fold increase in activity of mutant enzymes at 37 C compared with that of wild-type TPL b Increase in T1⁄ 2when 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
Activity change (fold)
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.
Trang 4beneficial 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 4C,
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 usmodel-ing 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 Ca 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)
pH
–1 )
0
1
2
3
pH
40 50 60 70 80
40 50 60 70 80
20
40
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 stabilactiv-ity 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.
Trang 5l-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.
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.
Trang 6T407A 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,
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
(Stratagene, La Jolla, CA, USA) with the following primers: 5¢-CTCAAGACCCGTTTAGAGGCCC-3¢ (forward)
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
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
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
pHCE IIB and transformed into E coli JM83 cells
Expression and screening of mutant 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.,
expres-sion from the constitutive expresexpres-sion 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
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.
Trang 7Cellytic B (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 condisolu-tions below) After
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
Purification and characterization
mixed with an equal volume of Cellytic B, treated for
USA) and sonicated Thereafter, the solution was
centri-fuged at 24 000 g for 30 min, and the supernatant was
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
column (Pharmacia) and eluted using a reverse gradient of
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
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 mm l-tyrosine and 10 lm
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 lmol of
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
References
1 Kumagai H, Yamada H, Matsui H, Ohkishi H & Ogata
K (1970) Tyrosine phenol lyase I Purification, crystalli-zation, and properties J Biol Chem 245, 1767–1772
2 Phillips RS, Demidkina TV & Faleev NG (2003) Struc-ture and mechanism of tryptophan indole-lyase and tyro-sine phenol-lyase Biochim Biophys Acta 1647, 167–172
3 Milic´ D, Matkovic´-Cˇalogovic´ D, Demidkina TV, Kuli-kova VV, Sinitzina NI & Antson AA (2006) Structures
of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a
45, 7544–7552
4 Yamada H & Kumagai H (1975) Synthesis of l-tyrosine related amino acids by b-tyrosinase Adv Appl Microbiol
19, 249–288
5 Gelenberg AJ & Gibson CJ (1984) Tyrosine for the treatment of depression Nutr Health 3, 163–173
6 Lu¨tke-Eversloh T, Santos CN & Stephanopoulos G (2007) Perspectives of biotechnological production of
Trang 8l-tyrosine and its applications Appl Microbiol Biotechnol
77, 751–762
7 Lee SG, Hong SP, Choi YH, Chung YJ & Sung MH
(1997) Thermostable tyrosine phenol-lyase of
determina-tion, and overproduction in Escherichia coli Protein
Expr Purif 11, 263–270
8 Suzuki S, Horinouchi S & Beppu T (1988) Growth of a
tryptophanase-producing thermophile, Symbiobacterium
cocul-ture with a Bacillus sp J Gen Microbiol 134, 2353–2362
9 Lee SG, Hong SP, Kim do Y, Song JJ, Ro HS & Sung
MH (2006) Inactivation of tyrosine phenol-lyase by
Pictet–Spengler reaction and alleviation by T15A
muta-tion on intertwined N-terminal arm FEBS J 273, 5564–
5573
10 Burton SG, Cowan DA & Woodley JM (2002) The
search for the ideal biocatalyst Nat Biotechnol 20,
37–45
11 Kim DY, Rha E, Choi SL, Hong SP, Sung MH & Lee
SG (2007) Development of bioreactor system for
l-tyro-sine synthesis using thermostable tyrol-tyro-sine phenol-lyase
J Microbiol Biotechnol 17, 116–122
12 Lee SG, Hong SP, Song JJ, Kim SJ, Kwak MS & Sung
MH (2006) Functional and structural characterization
of thermostable d-amino acid aminotransferases from
13 Hoseki J, Okamoto A, Takada N, Suenaga A, Futatsugi
N, Konagaya A, Taiji M, Yano T, Kuramitsu S &
Kagamiyama H (2003) Increased rigidity of domain
structures enhances the stability of a mutant enzyme
created by directed evolution Biochemistry 42, 14469–
14475
14 Song JK & Rhee JS (2000) Simultaneous enhancement
of thermostability and catalytic activity of
phospholi-pase A(1) by evolutionary molecular engineering Appl
Environ Microbiol 66, 890–894
15 Bae E & Phillips GN Jr (2006) Roles of static and
dynamic domains in stability and catalysis of adenylate
kinase Proc Natl Acad Sci USA 103, 2132–2137
16 Zhao H, Giver L, Shao Z, Affholter JA & Arnold FH
(1998) Molecular evolution by staggered extension
process (StEP) in vitro recombination Nat Biotechnol
16, 258–261
17 Moore JC, Jin HM, Kuchner O & Arnold FH (1997)
Strategies for the in vitro evolution of protein function:
enzyme evolution by random recombination of
improved sequences J Mol Biol 272, 336–347
18 Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D & Arnold FH (2005) Evolving strategies for enzyme engineering Curr Opin Struct Biol 15, 447– 452
19 Voigt CA, Mayo SL, Arnold FH & Wang ZG (2001) Computational method to reduce the search space for directed protein evolution Proc Natl Acad Sci USA 98, 3778–3783
20 Olsen M, Iverson B & Georgiou G (2000) High-throughput screening of enzyme libraries Curr Opin Biotechnol 11, 331–337
21 Kim J-H, Song JJ, Kim B-G, Sung MH & Lee SC (2004) Enhanced stability of tyrosine phenol-lyase from
Biotechnol 14, 153–157
22 Antson AA, Demidkina TV, Gollnick P, Dauter Z, von Tersch RL, Long J, Berezhnoy SN, Phillips RS, Haru-tyunyan EH & Wilson KS (1993) Three-dimensional structure of tyrosine phenol-lyase Biochemistry 32, 4195–4206
23 Sundararaju B, Antson AA, Phillips RS, Demidkina
TV, Barbolina MV, Gollnick P, Dodson GG & Wilson
KS (1997) The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4¢-hydroxyphe-nyl)propionic acid, together with site-directed mutagen-esis and kinetic analysis, demonstrates that arginine 381
is required for substrate specificity Biochemistry 36, 6502–6510
24 Milic´ D, Demidkina TV, Faleev NG, Matkovic´-Cˇalog-ovic´ D & Antson AA (2008) Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray struc-tures of quinonoid intermediates J Biol Chem 283, 29206–29214
25 Zhao H & Arnold FH (1997) Optimization of DNA shuffling for high fidelity recombination Nucleic Acids Res 25, 1307–1308
26 Poo H, Song JJ, Hong SP, Choi YH, Yun SW, Kim JH, Lee SC, Lee SG & Sung MH (2002) Novel high-level con-stitutive expression system, pHCE vector, for a conve-nient and cost-effective soluble production of human tumor necrosis factor-a Biotechnol Lett 24, 1185–1189
27 Schwede T, Kopp J, Guex N & Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server Nucleic Acids Res 31, 3381–3385
28 Otey CR & Joern JM (2003) High throughput screen for aromatic hydroxylation In Directed Enzyme Evolution:
G, eds), pp 141–148 Humana Press, Totowa, NJ