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
Trang 1identification of regions important for activity, specificity and stability through molecular evolution
Bart A van der Veen1, Lars K Skov2, Gabrielle Potocki-Ve´rone`se1, Michael Gajhede2,
Pierre Monsan1and Magali Remaud-Simeon1
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 (kcat¼ 1Æs)1) and its stability (t½¼ 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: remaud@insa-tlse.fr
(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 engineerresult-ing
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.
Trang 2family 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 37C, 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, protrudconsist-ing 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 RENDER 3 D [29].
Trang 3significantly increased activity under all conditions,
including retention of activity after preincubation at 50
or 60C 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 Vmax at
low sucrose concentrations (Vmax1 and Km1) and low
affinity and high(er) Vmax at high sucrose
concentra-tions (Vmax2and Km2) [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 concentrapolymeriza-tions, 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 kmax 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.
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.
Km1
(m M )
kcat1
(s)1)
kcat1⁄ Km 1 (s)1Æm M )1)
Km2 (m M )
kcat2 (s)1)
kcat2⁄ Km 2 (s)1Æm M )1) Total activity
Hydrolysis
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 )
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)
Trang 4nature 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 50C, 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 formaffect-ing the salt bridge, indicataffect-ing 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
Temperature (°C)
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.
Trang 5oligosaccharide 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)
Trang 6the 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 resiresi-due 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 Shot 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
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).
Trang 7DNA 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 XhoI
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 30C
Screening for improved amylosucrases
Because amylosucrase is produced intracellularly, lysozyme
was added to a final concentration of 0.5 gÆL)1and 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 30C
for 6 h or 37C 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 95C for 7 min, adding 60 lL of this
mixture to 180 lL H2O, 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 I2, 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 50C, 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 30C, 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 PhastGeltm
gradient 8–25 (Amersham Pharmacia Biotech) under dena-turing conditions, followed by staining with 0.5% (w⁄ v) AgNO3 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)1of pure enzyme at 30C
At regular time intervals (5 min) 20 lL samples were taken and the amylosucrase was immediately inactivated by heat-ing (3 min 90C) The formation of glucose and fructose was analysed using the d-glucose⁄ d-fructose UV-method
Trang 8(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 consumpforma-tion of sucrose, and thus the
total activity The fructose formation minus glucose
forma-tion then reflects the polymerizaforma-tion 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 kmax¼ longer polymer) For shorter amylose chains
(< 120 glucose residues) such as produced by amylosucrase
[4] an increase in kmaxwith increase in the average degree
of polymerization is observed according to the following
formula [27]:
average degree of polymerization¼ 1:025e2=ð1k1max 1:558e3Þ:
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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