Báo cáo khoa học: Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution doc

Increased amylosucrase activity and specificity, and
identification of regions important for activity, specificity
and stability through molecular evolution
Bart A. van der Veen
1
, Lars K. Skov
2
, Gabrielle Potocki-Ve
´
rone
`
se
1
, Michael Gajhede
2
,
Pierre Monsan
1
and Magali Remaud-Simeon
1
1 Laboratoire Biotechnologie-Bioproce
´
de
´
s, UMR CNRS 5504, UMR INRA 792, Toulouse, France
2 Biostructural Research, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark
Glucansucrases constitute a class of enzymes produ-
cing glucose polymers using sucrose as the sole sub-
strate and are usually members of glycoside hydrolase
(GH) family 70 [1]. Amylosucrase (EC 2.4.1.4) is an
exceptional glucansucrase, because it belongs to GH

family 13, in which many polyglucan-degrading
enzymes (e.g. a-amylase) are found. Furthermore, it
produces a glucan consisting of only a-1,4-linked glu-
cose residues [2,3], which has recently been shown to
be identical to amylose [4]. Unlike amylosucrase, other
enzymes responsible for the synthesis of such amylose-
like polymers require the addition of expensive
activated sugars such as ADP- or UDP-glucose [5].
Amylosucrase can also be used to modify the structure
of polysaccharides such as glycogen by the addition of
a-1,4-linked glucosyl units [6]. These properties make
amylosucrase an interesting enzyme for industrial
applications. This requires, however, improvement of
its catalytic efficiency on sucrose alone (k
cat
¼ 1Æs
)1
)
and its stability (t
½
¼ 21 h at 30 °C), and decrease of
the catalysis of nondesired side reactions resulting in
sucrose isomer formation, which limits the yield of
polymer [6]. Amylosucrase from Neisseria polysaccharea
was the first amylosucrase to be studied as a recombin-
ant enzyme [2,6]. It is the only glucansucrase for which
the structure has been determined [7], and the second
Keywords
amylosucrase; molecular evolution;
polymerase; reaction specificity; sucrose-

binding site
Correspondence
M. Remaud-Simeon, Laboratoire
Biotechnologie-Bioproce
´
de
´
s, UMR CNRS
5504, UMR INRA 792, INSA, 135 avenue de
Rangueil, 31077 Toulouse Cedex 4, France
Fax: +33 561 55 94 00
Tel: +33 561 55 94 46
E-mail:
(Received 12 July 2005, revised 5 October
2005, accepted 24 November 2005)
doi:10.1111/j.1742-4658.2005.05076.x
Amylosucrase is a transglycosidase which belongs to family 13 of the glyco-
side hydrolases and transglycosidases, and catalyses the formation of amy-
lose from sucrose. Its potential use as an industrial tool for the synthesis or
modification of polysaccharides is hampered by its low catalytic efficiency
on sucrose alone, its low stability and the catalysis of side reactions result-
ing in sucrose isomer formation. Therefore, combinatorial engineering
of the enzyme through random mutagenesis, gene shuffling and selective
screening (directed evolution) was applied, in order to generate more effi-
cient variants of the enzyme. This resulted in isolation of the most active
amylosucrase (Asn387Asp) characterized to date, with a 60% increase in
activity and a highly efficient polymerase (Glu227Gly) that produces a lon-
ger polymer than the wild-type enzyme. Furthermore, judged from the
screening results, several variants are expected to be improved concerning
activity and ⁄ or thermostability. Most of the amino acid substitutions

observed in the totality of these improved variants are clustered around
specific regions. The secondary sucrose-binding site and b strand 7, connec-
ted to the important Asp393 residue, are found to be important for amylo-
sucrase activity, whereas a specific loop in the B-domain is involved in
amylosucrase specificity and stability.
Abbreviations
DNS, dinitrosalicylic acid; (EP-)PCR, (error prone-)polymerase chain reaction; GH, glycoside hydrolase; GST, glutathione-S-transferase; IPTG,
isopropyl thio-b-
D-galactoside; OB, oligosaccharide binding site; SB, sucrose-binding site.
FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 673
family 13 enzyme following CGTase [8] for which the
structure of a covalent intermediate is available [9]. A-
mylosucrase possesses the characteristic (b ⁄ a)
8
-barrel
catalytic A domain, a B domain between b strand 3
and a helix 3, and a C-terminal domain consisting of
a sandwich of two Greek key motifs. In addition to
these common structural features, amylosucrase pos-
sesses two unique domains: an a-helical N-terminal
domain and a B¢ domain between b strand 7 and
a helix 7 in the catalytic core, which has been sugges-
ted to be involved in the polymerase activity of this
enzyme. The B and B¢ domains contribute largely to
the formation of an active site pocket, which is closed
on one side by a salt bridge [7]. Several structures of
amylosucrase complexed with substrate and products
[10,11] have indicated the presence of various import-
ant regions inside and outside the active site pocket
characterized by sucrose and oligosaccharide-binding

sites (SB and OB, respectively, Fig. 1). Combined with
biochemical and mutagenic studies [12–15] this allowed
elucidation of the enzyme’s features implicated in the
amylosucrase reaction mechanism and specificity.
Rational engineering based on these data resulted in
the construction of a highly efficient polymerase [16].
Further rational improvement of catalytic efficiency or
stability would benefit from comparisons of similar
enzymes with different characteristics [17]. Such data
are not available for amylosucrase, because the
only other described amylosucrase, from Deinococcus
radiodurans, has very similar catalytic properties and
stability [18].
This study deals with optimization of the catalytic
properties of amylosucrase to adapt it to industrial
synthesis conditions using directed evolution tech-
niques, describing the positive variants found by
screening of a large variant library.
Results and Discussion
Library construction and screening for improved
variants
Genetic variation was introduced by error prone
polymerase chain reactions (EP-PCR), followed by
shuffling of the PCR products. Cloning and transfor-
mation of the shuffling products to Escherichia coli
TOP10 yielded % 50 000 clones, the plasmid DNA iso-
lated from these clones constituting the variant library.
Transformation of this library to E. coli JM109 cells
yielded over 100 000 colonies, indicating that most of
the 50 000 clones should be represented on the plates.

Ninety clones showing amylase formation after one
day of growth, and thus probably expressing the most
active or efficient polymerases present in the library,
were used for screening. Initial screening rounds for
increased enzymatic activity or stability resulted in the
selection of 39 possibly improved variants, which were
transferred in duplicate to a new microtitre plate.
Screening of these 39 positives was repeated using the
same conditions, finally yielding seven clones improved
for various characteristics, each the result of one or
two amino acid substitutions (Table 1).
Two of the improved clones (E9 and H4) showed
significant amylose production after 3 h incubation
with sucrose at 37 °C, whereas no amylose production
by the wild-type was observed. Variant E9 also showed
Fig. 1. Stereo representation of the structure of the Glu328Gln amylosucrase complexed with sucrose bound in the active site pocket (PDB
code 1JGI). Surface sites binding sucrose (SB) and oligosaccharides (OB) were added from other structures (PDB 1MW3 and 1MW0,
respectively). The central (b ⁄ a)
8
-barrel catalytic domain (A) is flanked by a helical N-terminal domain (N), and a C-terminal domain (C) consist-
ing of b strands. Domains B and B¢ are extended loops 3 and 7, respectively, protruding from the A domain. The active site pocket repre-
sents SB1 (or OB1); alternative sucrose-binding sites are found in the B¢ domain (SB2), in the N-terminal domain (SB3), and in the B domain
(SB4); alternative oligosaccharide binding sites are found in the B¢ domain (OB2), and in the C domain (OB3). Bound sucrose molecules are
shown in green, bound oligosaccharides are shown in cyan. All residues that were mutated in the various clones are marked and represen-
ted as sticks. The figure was produced using
MOLSCRIPT [28] and RENDER3D [29].
Molecular evolution to improve amylosucrase B. A. van der Veen et al.
674 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS
significantly increased activity under all conditions,
including retention of activity after preincubation at 50

or 60 °C. These two variants were selected for more
detailed characterization. They were cloned in pGEX-
6P-3 and the proteins purified to homogeneity as des-
cribed, and verified by electrophoresis followed by
silver staining (results not shown).
Kinetic analysis of the improved variants
The kinetic profile of amylosucrase action on sucrose
does not present a classical Michaelian behaviour, but
it can be modelled by two different Michaelis–Menten
equations, resulting in a high affinity and low V
max
at
low sucrose concentrations (V
max1
and Km
1
) and low
affinity and high(er) V
max
at high sucrose concentra-
tions (V
max2
and Km
2
) [13]. In Table 2 the kinetic data
for the wild-type and the selected variants are shown.
As expected from the screening results, variant E9
shows a general increase in activity and catalytic effi-
ciency. Although activity did not show the threefold
increase found during screening, this variant is the

most active amylosucrase found to date. In contrast,
variant H4, selected for improved polymer formation,
showed a general decrease in catalytic efficiency. The
improvement of this variant compared with the wild-
type is found in the significantly increased polymeriza-
tion activity at high sucrose concentrations, and the
twofold increased ratio of polymerization over hydro-
lysis at both low and high sucrose concentrations.
Polymerase efficiency of the improved variants
The results of the iodine staining of polymer formed
by the variants are shown in Table 3. Contrary to the
wild-type, variant H4 produces polymer from low con-
centrations of sucrose (5 mm) and under all conditions
this variant produces longer amylose chains than the
wild-type, as judged by the increase in k
max
. These
findings can be related to the increased ratio of poly-
merization over hydrolysis activity. Thus, in this
variant the different reactions (hydrolysis and poly-
merization) are affected differently, in which case the
Table 1. Screening and sequence results of the improved variants. Act., activity based on DNS response; Tstab, improved thermostability;
Pol., improved polymerase.
Characteristics screening Purified enzyme Mutations DNA Protein
A9 Act. 1.5· C45T, A226G N76D
A10 Tstab. C469G, G691T,C1441T P157A, D231Y
D1 Tstab. C698T,C1239T,G1597A P234L, G554S
D8 Act. 1.5· G184A, C1317T, G1516A E62K, D506N
E9 Act. 3·⁄Tstab. Improved Act. ⁄ Tstab. G99A, G495A, A1159G N387D
F9 Act. 1.5· G1221T, G1839T Q613H

H4 Pol. + Improved Pol. A680G E227G
D2
a
Act. 2· Improved Act. ⁄ Pol. C123T, G1165C, A1509T V389L, N503I
G1
a
Act. 2· Improved Act. ⁄ Pol. C58T, G444T, T1793C R20C, F598S
a
Variants described previously [19].
Table 2. Kinetics of the action on sucrose of (variant) enzymes. Kin-
etic values that reflect the improved properties suggested by the
screening results [improved activity (E9) or enhanced polymer for-
mation (H4)] are indicated in bold.
Km
1
(mM)
k
cat1
(s
)1
)
k
cat1
⁄ Km
1
(s
)1
ÆmM
)1
)

Km
2
(mM)
k
cat2
(s
)1
)
k
cat2
⁄ Km
2
(s
)1
ÆmM
)1
)
Total activity
Wild-type 4.0 0.74 0.19 71 1.4 0.020
H4 4.4 0.60 0.14 165 1.7 0.010
E9 4.2 1.19 0.28 82 2.2 0.026
Hydrolysis
Wild-type 2.5 0.35 0.14 29 0.52 0.018
H4 0.8 0.18 0.23 48 0.43 0.009
E9 2.3 0.54 0.23 62 0.86 0.014
Polymerization
Wild-type 8.1 0.43 (1.2)
a
0.05 112 0.90 (1.7) 0.008
H4 9.6 0.36 (2.0) 0.04 300 1.43 (3.3) 0.005

E9 5.6 0.64 (1.2) 0.11 102 1.30 (1.5) 0.013
a
Values between brackets indicate the ratio polymerization ⁄ hydro-
lysis.
Table 3. k
max
of the iodine-stained reaction products from different
concentrations of sucrose after 24 h incubation at 30 °C with (vari-
ant) enzymes. The average DP of the amylose products, calculated
using the formula in the methods section, is shown between brack-
ets. nd, not detectable.
[Suc] (m
M)
5 1020 50100200
Wild-type nd nd 560 (45) 575 (57) 570 (52) 555 (42)
H4 580 (62) 595 (84) 605 (108) 600 (94) 605 (108) 585 (68)
E9 nd nd 570 (52) 580 (62) 570 (52) 560 (45)
B. A. van der Veen et al. Molecular evolution to improve amylosucrase
FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 675
nature of the produced polymer is affected. Similarly,
a general increase in catalytic efficiency, as observed
for variant E9, does not significantly affect polymer
synthesis. Furthermore, polymer formation occurs in
the later stages of the reaction (initially polymerization
consists of oligosaccharide formation), and also
depends on the affinity for the oligosaccharides pro-
duced to be used as acceptors. This appears to be
improved for variant H4, as has been shown previ-
ously for mutant Arg226Ala [16].
Temperature dependency of (variant)

amylosucrases
Under screening conditions, variant E9 also showed
some increased thermostability, hence the temperature
dependency of amylosucrase activity was investigated
(Fig. 2). The wild-type enzyme is very rapidly dena-
tured at temperatures over 50 °C, thus no activity can
be measured at these temperatures (manuscript in
preparation). Compared with the wild-type, activity at
elevated temperatures had increased drastically for
variant E9, which indicates increased stability. In con-
trast, variant H4, which was not selected for increased
thermostability, appears to have a decreased stability
and the temperature optimum is decreased compared
with that of the wild-type.
Structural analysis of the mutations
The effects of the mutations on enzyme properties are
given in Table 1, and the positions of the mutated resi-
dues in the crystal structure of amylosucrase are shown
in Fig. 1, which also shows the binding sites of sucrose
[10] and oligosaccharides [11]. It is immediately obvi-
ous that the mutations are grouped in certain regions
of the structure. Although several mutations are found
in the vicinity of the sucrose-binding site SB2, which is
separated from the active site pocket by a salt bridge
formed by residues Asp144 and Arg509 (Fig. 1), few
mutations are found at the other binding sites, and
none in the amylosucrase-specific B¢ domain, or at the
substrate access channel.
Regions involved in activity
In each of the two variants described in more detail

here, only one amino acid substitution was found. In
the first variant, E9, which is the most active amylo-
sucrase found to date, Asn387 in b strand 7 is replaced
by an aspartate. A positive effect on activity by muta-
tions in b strand 7 is also shown by variant D2 in
which Val389 at the end of b strand 7 is replaced by
leucine [19]. These mutations probably affect the first
part of loop 7 (B¢ domain) and consequently the
important Asp393 residue (Fig. 3), which is conserved
in all GH family 13 enzymes, and plays an essential
role in catalysis by stabilizing the glucose residue
bound at subsite )1 in the various reaction stages [8].
Interestingly, a second mutation in variant D2,
Asn503Ile, is situated in the group of mutations close
to SB2 (Fig. 4). It is found in the part of loop 8 that
also contains Arg509, and interacts with a sucrose
bound at SB2 via the backbone nitrogen of Ser508.
Another mutation found in this loop is Asp506Asn in
variant D8, which also shows increased activity under
screening conditions. Such mutations probably influ-
ence the properties of loop 8 in this region, thus affect-
ing SB2 and Arg509 forming the salt bridge, indicating
that these specific amylosucrase features are involved
in catalysis.
Regions involved in reaction specificity
A very interesting region containing mutations near
SB2 is the loop in the B domain including residue
Glu227 which has been mutated in variant H4 (Fig. 5).
Variant Glu227Gly found in the shuffling library and
site-directed mutant Arg226Ala [16] both result in

highly efficient polymerases. Thus via this loop the
B domain is very important for reaction specificity via
0
20
40
60
80
100
120
15 25 35 45 55 65
Temperature (°C)
Relative activity (%)
Fig. 2. Temperature optima of the variants. Wild-type (s), H4 (n),
and E9 (d) amylosucrase activity was measured at different tem-
peratures, and the values recalculated as the percentage of the
maximal activity for the enzyme concerned.
Molecular evolution to improve amylosucrase B. A. van der Veen et al.
676 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS
oligosaccharide binding in the active site (OB1;
Fig. 5A), which is also observed in other family 13
enzymes such as cyclodextrin glycosyltransferase, in
which several residues of the B domain are essential
for the catalysis of the characteristic cyclization reac-
tion [20,21].
Regions involved in thermostability
Both variant enzymes Arg226Ala and Glu227Gly show
reduced thermostability, indicating that this loop in
the B domain is also involved in the thermostability of
amylosucrase. In fact, two variants that were positive
when screening for improved thermostability have

amino acid substitutions in this loop. In variant A10
an Asp231Tyr mutation occurs, which is actually the
only mutation that directly affects a sucrose-binding
residue (Fig. 5B). Furthermore, Asp231 has been des-
cribed as the most important ‘geometric lock’ respon-
sible for a closed conformation of a highly flexible
loop in the B¢ domain. Removal of the Asp231 side
chain allowed simulation of large movements of this
loop using geometric techniques [22]. The Asp231Tyr
mutation probably improves interactions with hydro-
phobic residues of this neighbouring loop in the
B¢ domain, further stabilizing it. In variant A10, this
mutation is combined with a Pro157Ala mutation in
loop 2, a substitution which is not expected when
looking for thermostability. In another variant, D1,
such a contradictory mutation is Pro234Leu in the
connection of the Glu227 loop to a b sheet in the
B domain. However, in this variant a second mutation
is Gly554Ser in the loop connecting the catalytic
domain and the C domain, which may be another
important area for protein stability.
Mutations in the N-terminal domain
Besides the remarkable cluster close to SB2, also sev-
eral mutations are found in the N-terminal domain.
In variant D8, containing the Asp506Asn mutation, a
Glu62Lys mutation is found in an a helix in the N-ter-
minal domain, which does not provide a logical
explanation for the increase in activity. Also in variant
G1 a mutation (Arg20Cys) is found in the N-terminal
domain, however, in this case, the mutated residue

(Arg20) participates in the SB3 site and may in this
way affect the enzymatic activity. Another mutation in
Fig. 4. Detail of the structure of amylosucrase complexed with
sucrose (PDB code 1MW3), showing the positions of the mutated
residues Asn503 and Asp506. In this structure a Tris molecule is
bound at the catalytic site, indicated by the three catalytic residues
(Asp286, Glu328 and Asp393), and sucrose (Suc) is bound at SB2,
close to the salt bridge formed by Asp144 and Arg509. Mutated
residues Asn503 and Asp506 are located in a flexible loop connect-
ing two helical parts of loop 8 (purple). Besides Arg509 the second
helix contains residues Ser508, hydrogen bonding to the sucrose
with its backbone nitrogen. The central b-barrel is shown as solid
strands depicted in yellow. The figure was produced using
PYMOL
(W. L. DeLano, DeLano Scientific, San Carlos, CA).
Fig. 3. Detail of the structure of the Glu328Gln amylosucrase com-
plexed with maltoheptaose (PDB code 1MW0), showing the posi-
tions of the mutated residues Asn387 in b strand 7, and Val389 in
the first part of loop 7 (purple). Only the two glucose residues (G2)
around the cleavage site are shown and represented as sticks, as
are the three catalytic residues (Asp286, Gln328, and Asp393), and
the residues forming the salt bridge that closes the active site
(Asp144 and Arg509). The central b-barrel is shown as solid strands
depicted in yellow. The figure was produced using
PYMOL (W. L.
DeLano, DeLano Scientific, San Carlos, CA)
B. A. van der Veen et al. Molecular evolution to improve amylosucrase
FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 677
the N-terminal domain that appears to have a positive
effect on activity is found in variant A9. Here, the only

substitution is Asn76Asp, situated in a bend connect-
ing two a helices and no obvious structurally based
reason for the improvement can be found.
Mutations in the C-terminal domain
A second mutation in variant G1 is Phe598Ser in the
C-terminal domain, which may have some effect,
because it replaces a solvent-exposed hydrophobic resi-
due with a hydrophilic residue. In the C domain
another mutation found is Gln613His, in variant F9,
which shows a slight increase in activity under screen-
ing conditions. Also for these substitutions no direct
explanation for a positive effect on enzyme activity
can be derived from the structure.
In conclusion, screening and analysis of a large amy-
losucrase variant library resulted in the isolation of a
very efficient polymerase and the most active amylo-
sucrase enzyme characterized to date, both resulting
from mutations that would not be chosen rationally.
Furthermore, regions could be identified in the enzyme
that are clearly important for amylosucrase activity, as
b strand 7, connecting to the important Asp393 resi-
due, and the region close to the salt bridge and the
secondary sucrose-binding site SB2. Other regions are
involved in specificity and thermostability, as the loop
containing Glu227 in the B domain. These findings
provide new perspectives for engineering improved
amylosucrase enzymes for industrial applications by
site-directed or massive mutagenesis in the identified
regions.
Experimental procedures

Bacterial strains and plasmids ⁄ growth conditions
One ShotÒ E. coli TOP10 (Invitrogen, Carlsbad, CA) was
used for transformation of ligation mixtures. E. coli JM109
(Promega, Madison, WI) was used to screen amylosucrase
variants and large-scale production of the selected mutants.
Plasmid pZErO-2 (Invitrogen) was used for subcloning of
PCR products and screening, and plasmid pGEX-6P-3
(Amersham Pharmacia Biotech, Piscataway, NJ) was used
for production of glutathione S-transferase (GST)–amylo-
sucrase fusion proteins. Bacterial cells were grown on
Luria–Bertani (agar) containing 50 lgÆmL
)1
kanamycin
(when harbouring plasmid pCEASE01S01F), or
100 lgÆmL
)1
ampicillin (when harbouring a pGEX-6P-3-
derived plasmid). To express amylosucrase in E. coli JM109
media were supplemented with isopropyl thio-b-d-galacto-
side (IPTG; 1 mm). When appropriate, Luria–Bertani agar
plates contained 50 gÆL
)1
sucrose for visualization of amy-
losucrase activity, by halos formed through formation of
amylose in the agar.
A
B
Fig. 5. The Glu227 loop in (A) the structure of the Glu328Gln amy-
losucrase complexed with maltoheptaose (B) the structure of amy-
losucrase complexed with sucrose. This flexible loop (purple) is

situated between an a helix and a b strand in the B domain. Unlike
Asp226, none of the mutated residues in this loop interact with
maltotheptaose bound in the active site in (A). However, Asp231
has hydrogen bonding interactions with the sucrose bound at SB2
in (B). Further, highlighted are the three catalytic residues (Asp286,
Gln ⁄ Glu328 and Asp393), the residues forming the salt bridge that
closes the active site (Asp144 and Arg509), and the Tris molecule
bound in the active site (B). The central b-barrel is shown as solid
yellow strands. Figure produced using
PYSMOL (W. L. DeLano,
DeLano Scientific, San Carlos, CA).
Molecular evolution to improve amylosucrase B. A. van der Veen et al.
678 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS
DNA manipulations
Restriction endonucleases and DNA-modifying enzymes
were purchased from New England Biolabs (Ipswich, MA)
and used according to the manufacturer’s instructions.
DNA purification was performed using QIAQuick (gel
extraction) and QIASpin (miniprep; Qiagen, Valencia, CA).
DNA sequencing was carried out using the di-deoxy chain-
termination procedure [23] by MilleGen (Labe
`
ge, France).
Generation of variant libraries
EP-PCR using two different enzymes, Mutazyme (Strata-
gene, La Jolla, CA) and Taq DNA-polymerase (New Eng-
land Biolabs), was applied to introduce random mutations
and the PCR products shuffled as described previously [19].
The shuffling products were digested with HindIII and Xho I
and ligated with pZErO digested with the same enzymes.

The resulting constructs were transformed to E. coli TOP10
cells and plated on Luria–Bertani agar plates containing
sucrose. The colonies were scraped from these plates for
isolation of the plasmids, constituting the shuffling library.
Selection of positive clones
The shuffling gene library pCEASE01S01F was trans-
formed to E. coli JM109 and plated on Luria–Bertani agar
containing sucrose. From these plates, clones showing for-
mation of amylose after one day of growth, thus expressing
highly active or efficient polymerases, were identified visu-
ally due to the precipitation of the polymer. These were
selected and grown in microtitre plates containing 200 lL
Luria–Bertani per well, supplemented with 1 mm IPTG and
50 lgÆmL
)1
kanamycin. These mini-cultures were horizon-
tally shaken at 250 r.p.m., for 15 h at 30 ° C.
Screening for improved amylosucrases
Because amylosucrase is produced intracellularly, lysozyme
was added to a final concentration of 0.5 gÆL
)1
and the cells
were frozen at )20 °C. After thawing for 30 min at room
temperature, several screening conditions were applied to
select improved amylosucrases.
The screen for increased enzymatic activity was carried
out with sucrose alone as substrate, at a final concentration
of 150 mm. Reactions were performed at combinations of
temperature and incubation time that resulted in only slight
product formation for the wild-type. Incubations at 30 °C

for 6 h or 37 °C for 3 h were used in this study. Reducing-
sugar production was measured by adding 50 lL of the
reaction mixture to 50 lL of dinitrosalicylic acid (DNS)
[24], incubating at 95 °C for 7 min, adding 60 lL of this
mixture to 180 lLH
2
O, and measuring the absorbance at
540 nm. The formation of the amylose-type polymer was
analysed by adding 10 lL of iodine solution (100 mm
KI, 6 mm I
2
, 0.02 m HCl) to the remaining reaction mix-
ture, the positive clones being revealed by development of a
blue colour. Changes in ratios of these separate measure-
ments are indicative of changes in polymerization efficiency
[19].
Screening for thermostability was carried out by preincu-
bation of the microtitre plates at elevated temperatures
(20 min 50 °C, 10 min 60 °C), which inactivates the wild-
type enzyme. After cooling, sucrose and glycogen (final
concentrations 150 mm and 5 gÆl
)1
, respectively) were added
as substrate, glycogen being a strong activator of amylo-
sucrase activity [25]. After overnight incubation at 30 °C,
iodine staining was used to detect polymer formation by
variants that remained active.
Production and purification of improved variants
Selected clones were grown in 4 mL Luria–Bertani cultures
for plasmid isolation. After sequencing, the genes of the

most promising variants were subcloned in vector pGEX-
6P-3, using the EcoRI and XhoI restriction sites, for GST
fusion protein expression. Variant GST–amylosucrases were
produced in 100 mL cultures using E. coli JM109 as host
and the proteins were extracted as described previously [6].
Purification of the variant amylosucrases was carried out as
described by the provider of plasmid pGEX-6P-3 (Amer-
sham Pharmacia Biotech), using the on column cleavage
protocol to elute GST-free enzyme. The purity of the
enzymes was analysed by electrophoresis on the PHAST
system (Amersham Pharmacia Biotech), using PhastGel
tm
gradient 8–25 (Amersham Pharmacia Biotech) under dena-
turing conditions, followed by staining with 0.5% (w ⁄ v)
AgNO
3
. Previously purified wild-type GST–amylosucrase
[19] was used as reference in characterization of the vari-
ants; the GST-fusion having been reported as not influen-
cing the catalytic properties of the enzyme [16].
Protein concentration determination
Protein concentrations were determined with the Bradford
method [26] using the Bio-Rad reagent (Bio-Rad Laborat-
ories, Hercules, CA) and bovine serum albumin as a
standard.
Kinetic analysis of the improved variants
Kinetic parameters of the action on sucrose were deter-
mined by incubating various substrate concentrations
(5 mm)1 m) with % 0.1 mgÆmL
)1

of pure enzyme at 30 °C.
At regular time intervals (5 min) 20 lL samples were taken
and the amylosucrase was immediately inactivated by heat-
ing (3 min 90 °C). The formation of glucose and fructose
was analysed using the d-glucose ⁄ d-fructose UV-method
B. A. van der Veen et al. Molecular evolution to improve amylosucrase
FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 679
(Boehringer Mannheim ⁄ R-Biopharm, Mannheim, Germany)
according to the manufacturer’s procedure, but scaled
down to be used in microtitre plates. The glucose formation
reflects the hydrolysing activity, because it can only be
formed when water is used as acceptor. The fructose forma-
tion reflects the total consumption of sucrose, and thus the
total activity. The fructose formation minus glucose forma-
tion then reflects the polymerization activity [19].
Polymerase efficiency of the improved variants
Polymer formation was analysed by iodine staining of a
sample taken after 24 h incubation; the comparative length
of produced polymer was judged by the optimal wavelength
(higher k
max
¼ longer polymer). For shorter amylose chains
(< 120 glucose residues) such as produced by amylosucrase
[4] an increase in k
max
with increase in the average degree
of polymerization is observed according to the following
formula [27]:
average degree of polymerization ¼1:025e
À2

=ð1k
À1
max
À1:558e
À3
Þ:
Temperature dependency of (variant)
amylosucrases
The optimal reaction temperature was determined by meas-
uring the standard activity at different temperatures. Stand-
ard activity is determined by incubating the enzyme with
sucrose and glycogen at final concentrations of 146 mm and
0.1 gÆL
)1
, respectively [6], and measuring the fructose for-
mation using the DNS method.
All assays were performed in duplicate at least, and devi-
ations were < 10%.
Acknowledgements
This work was supported by the EU project N°
QLK3-CT-2001–00149; Combinatorial Engineering of
GLYCoside hydrolases from the a-amylase superfamily
(CEGLYC).
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