To address these issues, we analyzed the process of dextran-independent insoluble glucan formation and examined the enzyme kinetic properties of the nigero-oligosaccharide acceptor react
Trang 1by Streptococcus sobrinus glucosyltransferase I
Hideyuki Komatsu1, Yoshie Abe1, Kazuyuki Eguchi1, Hideki Matsuno1, Yu Matsuoka1, Takayuki Sadakane1, Tetsuyoshi Inoue2, Kazuhiro Fukui2and Takao Kodama1
1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Japan
2 Department of Oral Microbiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Japan
Introduction
Water-insoluble glucan, which is mainly composed of
a-(1fi 3)-glucan, enhances the colonization of
cario-genic bacteria and promotes the formation of dental
plaque on smooth tooth surfaces [1] Two
glucos-yltransferases (GTFs) or glucansucrases (GTF-S and
GTF-I, EC 2.4.1.5) from mutans streptococci are
responsible for the production of this polysaccharide
[1,2] GTF-S and GTF-I catalyze the synthesis of
water-soluble a-(1fi 6)-glucan and water-insoluble
a-(1fi 3)-glucan, respectively They share highly
homologous amino acid sequences and comprise the N-terminal glucansucrase domain (GSd) and the C-terminal glucan-binding domain (GBd) [3,4] The GSd catalyzes glucosyl transfer from sucrose to low molecular mass acceptors such as water, isomaltose, and maltose [5] On the other hand, the GBd binds to glucans such as dextran [6–8]
Unlike GTF-S, pre-existing dextran increases GTF-I activity [9,10] Although truncation of the GBd of GTF-I by genetic engineering results in decreased
Keywords
enzyme kinetics; glucansucrase;
glucosyltransferase; mutans streptococci;
nigerooligosaccharide
Correspondence
H Komatsu, Department of Bioscience and
Bioinformatics, Kyushu Institute of
Technology, Kawazu 680-4, Iizuka 820-8502,
Japan
Fax: +81 948 29 7801
Tel: +81 948 29 7845
E-mail: hide@bio.kyutech.ac.jp
(Received 3 September 2010, revised 18
November 2010, accepted 25 November
2010)
doi:10.1111/j.1742-4658.2010.07973.x
Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elon-gates the a-(1fi 3)-linked glucose polymer branches on the primer dextran bound to the C-terminal glucan-binding domain We investigated the GTF-I-catalyzed glucan synthesis reaction in the absence of the primer dextran The time course of saccharide production during dextran-independent glu-can synthesis from sucrose was analyzed Fructose and glucose were first produced by the sucrose hydrolysis Leucrose was subsequently produced, followed by insoluble glucan [a-(1fi 3)-linked glucose polymers] after a lag phase High levels of intermediate nigerooligosaccharide series accumu-lation were characteristically not observed during the lag phase The results from the enzymatic activity of the acceptor reaction for the nigerooligo-saccharide with a degree of polymerization of 2–6 and methyl a-D -gluco-pyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization The production of insoluble glu-can was numerically simulated using the fourth-order Runge–Kutta method with the kinetic parameters estimated from the enzyme assay The simulated time course provided a profile similar to that of experimental data These results define the relationship between the kinetic properties
of GTF-I and the time course of saccharide production These results are discussed with respect to a mechanism that underlies efficient glucan synthesis
Abbreviations
DP, degree of polymerization; GBd, glucan-binding domain; GS, glucan-binding domain-deficient glucosyltransferase-I; GSd, glucansucrase domain; GSGB, glucosyltransferase-I containing a full-length glucan-binding domain; GTF, glucosyltransferase.
Trang 2glucan synthesis [11–13], GBd-deficient GSd can still
synthesize a-(1fi 3)-glucan from sucrose in the
absence of the pre-existing dextran (i.e in a
dextran-independent manner) [14], albeit at a lower rate This
suggests that the glucan synthesis reaction catalyzed by
the GSd is a basic function of GTF-I
Although knowledge of the dextran-independent
reaction is important for a comprehensive
understand-ing of GTF-I function, its underlyunderstand-ing mechanism
remains incompletely defined In this respect, two
con-trasting mechanisms have been proposed for
glucansuc-rases One mechanism is proposed by Robyt et al from
pulse⁄ chase experiments using [14C]sucrose [15,16] This
mechanism involves processive elongation by which the
enzyme continuously transfers glucose residues to only
one acceptor molecule until the release of the final
product Another mechanism is suggested by Mooser
et al.on the basis of the identification of the active site
and the similarity with established glycosidase
mecha-nisms [5,17] The mechanism occurs via nonprocessive
elongation, whereby the enzyme releases the products
after each transfer of a glucose residue to the acceptor
Steady-state kinetic studies have been performed for
other glucansucrases as well as fructansucrase [18–22]
In addition, the processes of insoluble glucan synthesis
from sucrose, investigated with high-performance anion
exchange chromatography analysis, are also reported
[23,24] However, it is not clear how changes in the
intermediate oligosaccharides during the glucan
syn-thesis can be interpreted with respect to the kinetic
properties of enzymes In other words, the relationship
between the enzyme kinetic properties and the process
of insoluble glucan synthesis remains unknown
To address these issues, we analyzed the process of
dextran-independent insoluble glucan formation and
examined the enzyme kinetic properties of the
nigero-oligosaccharide acceptor reaction by using GBd-deficient
GTF-I (GS) and GTF-I containing a full-length GBd
(GSGB) from Streptococcus sobrinus 6715 (serotype g)
(Fig 1) A numerical simulation based on the enzyme
kinetic parameters is in good agreement with the time
course of dextran-independent insoluble glucan
forma-tion These results suggest that the dextran-independent
synthesis of insoluble glucan by GTF-I proceeds via
the nonprocessive elongation of a-(1fi 3)-linked
glu-cose polymers (nigerooligosaccharides)
Results
Functional characterization of GSGB
We previously investigated the functional role of the
GBd in dextran-dependent a-(1fi 3)-glucan synthesis
using GTF-I¢ (Asp85–Ile1256), which contains the GSd and the 246 N-terminal residues of the GBd (approxi-mately 50% of the GBd) The results indicate that the GBd enhances a-(1fi 3)-branch formation on GBd-bound dextran [14] In the present study, we character-ized newly prepared GSGB (Asp85–Asn1592) as an enzyme possessing the full-length GBd (Fig 1)
Figure 2A shows the dextran-dependent synthesis of insoluble glucan as monitored by light scattering GSGB rapidly produced insoluble glucan in the pres-ence of dextran (filled squares in Fig 2A), whereas GS did not (open squares in Fig 2A) When glucosyl transfer activity (initial velocity) was measured as a function of dextran concentration, the activity of GSGB was highly dependent on dextran (filled circles
in Fig 2B); however, that of GS was nearly zero, inde-pendently of dextran concentration (open circles in Fig 2B) On the other hand, there was no significant difference in sucrose hydrolysis activity in the absence
of dextran between GS and GSGB In addition, the sucrose hydrolysis activities of GS and GSGB as a function of sucrose concentration obeyed simple Michaelis–Menten kinetics, with similar kcat and Km values (data not shown) The kcat and Km values were
as follows: GS, 11.4 ± 0.4 s)1 and 0.37 ± 0.07 mm; GSGB, 12.0 ± 0.4 s)1 and 0.29 ± 0.04 mm These values are corroborated by our previous report [14]
Time course of dextran-independent glucan synthesis
We examined the kinetics of insoluble glucan synthesis from sucrose by GS and GSGB in the absence of dex-tran (Fig 3A,B) For this purpose, we incubated sucrose (50 mm) with GS or GSGB (0.4 lm) in the absence of dextran, and analyzed the products at arbi-trary time intervals The time courses of the produc-tion of insoluble glucan and other sugars by GSGB were essentially the same as those for GS (Fig 3)
Fig 1 Schematic structures of GTF-I and its variant proteins The proteins used start at Asp85 of GTF-I and terminate at Ser1085 and Asn1592 for GS and GSGB, respectively Both proteins contain an extra peptide, TMITNSSSVPG, from the multiple cloning site of pUC18 at their N-terminal ends The black bars in the GBd indicate the ‘A’ repeat [6,7].
Trang 3In the initial phase ( 60 min), the concentration of
fructose increased (open squares in Fig 3) and the
concentration of released glucose was lower than that
of fructose (open circles in Fig 3) As a result, the
difference between the concentrations of fructose and
glucose increased with time, suggesting that glucosyl
transfer occurred Leucrose, a known GTF-I product
[25,26], was detected after 30 min, and its
concentra-tion increased over time (open triangles in Fig 3)
Finally, the concentration of insoluble glucan increased
with a lag time of 60 min (filled squares in Fig 3) The
glucosidic linkages of the insoluble glucan produced by
GS or GSGB were analyzed with 13C-NMR
spectros-copy (Fig S1) The spectra confirm that both of the glucans were a-(1fi 3)-linked glucose polymers The release of the sugars flattened off after 100 min, indi-cating sucrose depletion
After all of the sucrose (50 mm) had been consumed,
35 mm fructose, 12 mm glucose, 12 mm leucrose and
12 mm (glucose equivalent) insoluble glucan were pro-duced The total concentrations of fructose and glu-cose were approximately 50 mm (resulting from 35 mm fructose and 12 mm leucrose) and 35–40 mm (resulting from 12 mm glucose, 12 mm leucrose, and 12 mm insoluble glucan), respectively The fructose yield was equivalent to the initial amount of sucrose, whereas the glucose yield was significantly less The 10–15 mm glucose that went undetected appears to have been incorporated into soluble nigerooligosaccharides
A
B
Fig 2 Effect of dextran on the synthesis of insoluble glucan and
glucosyl transfer activity by GS and GSGB (A) Dextran-dependent
insoluble glucan synthesis The insoluble glucan synthesis reaction
was initiated by adding GSGB (filled squares) or GS (open squares)
to the sucrose solution in the presence of dextran The amount of
insoluble glucan was monitored by light scattering The reaction
mixture contained 50 n M GTFs, 50 lgÆmL)1 dextran T2000 and
100 m M sucrose in 10 m M Mops (pH 7.0) (B) Glucosyl transfer
activity as a function of dextran concentration The reaction mixture
contained 10 n M GTFs, 50 m M sucrose, 100 m M NaCl, and 10 m M
Mops (pH 6.8) Glucosyl transfer velocity was estimated as
described in Experimental procedures Filled and open circles
indi-cate the GTF activity of GSGB and GS, respectively.
A
B
Fig 3 Saccharide production kinetics during the dextran-indepen-dent GTF-I reaction (A) and (B) show the production of saccharide
by GS and GSGB, respectively Open squares, open circles, open triangles and filled squares indicate the production of fructose, glucose, leucrose, and insoluble glucan, respectively The produc-tion of insoluble glucan is plotted as the glucose equivalent molar concentration The starting solution contained 0.4 l M enzyme (GS
or GSGB) and 50 m M sucrose in 10 m M potassium phosphate (pH 6.8).
Trang 4To detect the soluble nigerooligosaccharides, soluble
products were analyzed with HPLC, but no peak
cor-responding to nigerooligosaccharides was detected
(data not shown) In addition, TLC detected major
spots of fructose, glucose, and leucrose; immobile spots
and diffuse smears corresponding to a degree of
poly-merization (DP)‡ 4 were observed after 50 min
(Fig S2) These results suggest that the
nigerooligo-saccharides were marginally accumulated and that they
constituted a small proportion of the oligosaccharides
with a wide range of DP values
Glucosyl transfer activity of GS and GSGB for
nigerooligosaccharide acceptors
Next, we estimated the glucosyl transfer activity of
acceptor reactions for methyl a-d-glucopyranoside,
nigerooligosaccharides (DP = 2–6), and leucrose
(Fig 4) Methyl a-d-glucopyranoside was used as a
glucose analog that is not a substrate of hexokinase in a
glucose assay system The data were analyzed on the
basis of the Michaelis–Menten equation; the maximum
activity (kcat) and Michaelis constant for acceptor
(KmAcc) values are listed in Table 1 Methyl
a-d-glucopyr-anoside and nigerooligosaccharides served as the
gluco-syl acceptors for GS and GSGB; in contrast, leucrose
was not a glucosyl acceptor The activity for
nigerooligosaccharides was higher than that for methyl
a-d-glucopyranoside Furthermore, the
nigerooligo-saccharides with higher DPs exhibited higher activity
The rank order of glucosyl transfer efficiencies (kcat⁄
KmAcc) was as follows: nigerohexaose >
nigeropen-taose = nigerotetraose > nigerotriose = nigerose >
methyl a-d-glucopyranoside In addition, the kinetics of
the acceptor reactions appeared to be similar between
GS and GSGB
Kinetic simulation of GTF glucan synthesis
To explain the time course of saccharide production
during the synthesis of insoluble glucan, we assumed
Scheme 1, which shows the intermediate products that
are the probable acceptors for the GTF-I reaction in
the absence of dextran GTF-I was assumed to be
capa-ble of catalyzing the following reactions: (a) the
hydro-lysis of sucrose to glucose and fructose (reaction 1);
(b) the transfer of glucose residues from sucrose to
glucose and nigerooligosaccharide to produce
higher-DP nigerooligosaccharide (reactions 2–7); and (c) the
transfer of glucose residues from sucrose to fructose to
produce leucrose (reaction 8) Here, we assumed that
nigerooligosaccharides with DP‡ 7 were insoluble,
because the solubility of nigeroheptaose (DP = 7)
was extraordinarily low (submillimolar level) at neu-tral pH
For numerical simulation, we made two assump-tions: (a) Michaelis–Menten kinetics for sucrose hydrolysis, as described above and indicated by Kraij
et al [21] for reuteransucrase; and (b) ping-pong bi-bi
A
B
Fig 4 GTF activity as a function of acceptor concentrations The initial velocity of glucosyl transfer by GS (A) and GSGB (B) was measured in the presence of leucrose (open diamonds), methyl a- D -glucopyranoside (filled circles), nigerose (open circles), nigerotri-ose (filled squares), nigerotetranigerotri-ose (open squares), nigeropentanigerotri-ose (filled triangles), and nigerohexaose (open triangles) The lines indi-cate the curve fitted to the Michaelis–Menten equation to obtain
kcat and KmAcc (Table 1), with the nonlinear regression program in the ORIGIN software package (v 6.1J) The data from the GS nigero-pentaose reaction were analyzed by nonlinear least-squares analy-sis, but the parameters were not obtained with reasonable accuracy Because the solubility of nigerooligosaccharides (DP ‡ 4) was very low, GTF activity could not be measured at higher con-centrations.
Trang 5kinetics for glucosyl transfer from sucrose to an
acceptor sugar, as indicated for other glucansucrases
[5,18,19] and fructansucrase [22], and confirmed for
the nigerotriose acceptor reaction by GS (Fig S3)
With these assumptions, the glucan synthesis process
was numerically simulated on the basis of Scheme 1,
with the experimentally estimated kinetic parameters
(Table 2) The characteristic time course obtained
from the experimental data was reproduced by this
simulation (Fig 5): (a) the fructose concentration
increased at an appreciably higher rate than the
glu-cose concentration, until all of the sucrose was
con-sumed – the difference between the concentrations of
fructose and glucose subsequently increased with time;
(b) insoluble glucan (DP‡ 7) production increased after a lag phase ( 50 min); (c) small quantities of each nigerooligosaccharide (DP = 2–6) were pro-duced (Fig 5B); (d) leucrose production increased with a delay time ( 30 min); and (e) the final yield
of saccharide was similar to that of the experimental data, even in terms of the total concentration of soluble nigerooligosaccharides (DP = 2–6) In other words, the difference between the total concentrations
of fructose and glucose (50 mm and 35–40 mm, respectively) can be explained by this simulation The simulation also indicates that the difference is attributable to the production of 10–15 mm soluble nigerooligosaccharides
Table 1 Kinetic parameters of the acceptor reaction The parameters were obtained by nonlinear least-squares curve-fitting analysis for the data presented in Fig 4 The k cat and K mAccwere estimated from the maximum activity and the Michaelis constant for the acceptor, respec-tively ND, not determined.
Acceptor
k cat (s)1)
K mAcc (m M )
k cat ⁄ K mAcc (s)1Æm M )1)
k cat (s)1)
K mAcc (m M )
k cat ⁄ K mAcc (s)1Æm M )1)
a Errors of the estimates are indicated.
Scheme 1 Kinetic model of dextran-independent insoluble glucan synthesis.
Trang 6No significant difference was observed with respect to
dextran-independent insoluble glucan synthesis and the
nigerooligosaccharide acceptor reaction between GS
and GSGB (Figs 3 and 4); therefore, the
dextran-independent synthesis of a-(1fi 3)-glucan is not an
artificial reaction catalyzed by the deficient protein,
but rather a basal reaction of GTF-I In contrast, in the
presence of dextran, the enzymatic activity of GSGB
was much higher than that of GS (Fig 2)
Dextran-dependent glucan synthesis probably takes place via
enhancement of the basal reaction of the GSd by the
binding of dextran to the GBd This result is consistent
with the idea that the GBd plays an important role in
dextran-dependent glucan synthesis [2,11–13]
The experimental time course of saccharide
produc-tion agreed well with the simulation based on
Scheme 1, using experimentally determined kinetic
parameters This agreement, in turn, supports the
appropriateness of the model in Scheme 1; thus, our
results provide a reasonable explanation for the
dex-tran-independent glucan synthesis reaction At the
beginning of the reaction, sucrose is hydrolyzed to
glu-cose and fructose The released gluglu-cose subsequently
serves as the glucosyl acceptor to form nigerose The
subsequent glucosyl transfers take place to
nigerooligo-saccharides to form higher-DP nigerooligonigerooligo-saccharides
In this manner, insoluble glucan is formed by the
elon-gation of nigerooligosaccharides
Efficient glucan synthesis, such that the accumulation
of intermediate nigerooligosaccharides (DP = 2)6) is
minimized, is explainable by the single-chain
mecha-nism, whereby the enzyme ‘continuously’ elongates only
one acceptor molecule until the release of the resulting
glucan [15,16] However, because the KmAccvalues were
relatively high (5–80 mm; Table 1) as compared with the
concentrations of acceptors (glucose and
nigerooligo-saccharides) produced under the experimental
condi-tions, the enzyme tends to release the nigerooligo-saccharide during the reaction This mode is consistent with nonprocessive elongation, whereby the enzyme releases the products after each transfer of a glucose residue to the acceptor Hence, efficient insoluble glucan synthesis with minimal accumulation of intermediate nigerooligosaccharides is not achieved by continuous (or processive) elongation; it is, rather, achieved mainly through the enzymatic kinetic properties of GTF-I, the catalytic efficiency of which increases with an increase in the DP of nigerooligosaccharide (Table 1) In fact, we demonstrated that the enzyme kinetic model can simu-late the time course of insoluble glucan synthesis Non-processive elongation was previously demonstrated for the formation of insoluble amylase-like polymers from sucrose by Neisseria polysaccharea amylosucrase [23]
Table 2 Kinetic parameters for the numerical simulation
Simula-tion parameters were estimated as described in Doc S1.
Reaction
k cat (s)1)
K mSuc (m M )
K mAcc (m M )
k (s)1Æm M )1)
Acceptor reaction
A
B
Fig 5 Kinetic simulation of dextran-independent glucan synthesis
by GTF-I The time course of saccharide production was simulated
on the basis of the enzyme kinetic parameters listed in Table 2 (A) Time course of sucrose and major products Molar concentra-tions of sucrose (filled circles), fructose (open squares), glucose (open circles) and leucrose (open triangles) are indicated The con-centrations of soluble nigerooligosaccharides (open diamonds) and insoluble glucan (filled squares) are converted into glucose equivalent molar concentrations Nigerooligosaccharides with DP ‡ 7 were defined as insoluble glucan (B) Nigerooligosaccharide production The amounts of nigerose (circles), nigerotriose (squares), nigerotetra-ose (diamonds), nigeropentanigerotetra-ose (triangles) and nigerohexanigerotetra-ose (inverted triangles) are indicated as molar concentrations.
Trang 7The improved catalysis in the presence of increasing DP
may be a common feature of glucansucrase
Additional studies are required to elucidate the
dex-tran-dependent synthesis of glucan As the affinity of
the GBd of GTF-I for dextran is extraordinarily high
(Km= 4.88· 107m)1) [27], the enzyme can hardly be
dissociated from dextran GTF-I may elongate the
a-(1fi 3)-glucose branches of dextran in a
processive-like manner Moulis et al propose a semiprocessive
mechanism of polymerization for Leuconostoc
mesen-teroides NRRL B-512F dextransucrase and L
mesen-teroidesNRRL B-1355 alternansucrase [24] According
to this mechanism, the glucan-binding domains at the
C-terminal end of GTFs act as mediators of the shift
between the processive and nonprocessive processes;
GTF-I may otherwise randomly migrate on the dextran
without dissociating during the reaction Recently,
high-speed atomic force microscopy demonstrated that
Trichoderma reesei cellobiohydrolase I moves along
crystalline cellulose in a processive manner [28];
there-fore, to understand the mechanism underlying the
dextran-dependent GTF-I reaction, the dynamics of
binding of GTF-I to dextran should be investigated with
the use of single-molecule measurements, surface
plas-mon resonance, and⁄ or quartz crystal microbalances
This study defines, for the first time, the relationship
between the kinetic properties of GTF and the time
course of saccharide production Owing to GTF’s
kinetic property of improving activity in the presence
of nigerooligosaccharides with increasing DPs,
inter-mediate nigerooligosaccharides minimally accumulate
during production, and insoluble glucan is efficiently
produced This feature may be favorable for dental
plaque formation by mutans streptococci, because the
efflux of the intermediate product may be diminished
in an oral environment Moreover, such kinetic
analy-sis could also provide useful information regarding the
GTF-I-catalyzed synthesis of a-(1fi 3)-linked glucose
polymers for industrial production of foods,
pharma-ceuticals, and cosmetics [4,29]
Experimental procedures
Plasmids
Plasmid pGS [14] was used for the expression of GS
(Asp85–Ser1085) To construct the GSGB (Asp85–
Asn1592)-encoding plasmid pGSGB6R, an EcoRI–BglII
fragment (1.5 kbp) encoding a part of the multiple cloning
site and Asp85–Glu596 was isolated from pAB2 [6] and
inserted into a BglII–EcoRI-digested pAB1 [6] fragment
(3.4 kbp) encompassing the Asp597–Asn1592 coding
region Both plasmids contained pUC18-derived DNA
(containing the lac promoter and the ampicillin resistance gene) and encoded an extra peptide, TMITNSSSVPG, from the multiple cloning site at the N-terminal end
Enzymes
GS was prepared from Escherichia coli JM109 transformed with pGS Protein expression and purification were carried out as described previously [14] GSGB was expressed in
E coli JM109 transformed with pGSGB6R, and prepared with the same procedure as that used for GS GSGB was further purified by gel filtration chromatography on a Toyopearl HW-55 column (TOSOH) (2.5· 90 cm) pre-equilibrated in 0.5 m NaCl and 10 mm potassium phos-phate (pH 6.8) The protein concentration was determined from the absorbance at 280 nm, with a molar extinction coefficient calculated from the amino acid composition [30]
Preparation of a-(1fi 3)-glucan
GS was added to 300 mm sucrose solution containing 0.02% NaN3 and 10 mm potassium phosphate (pH 6.8), giving a final concentration of 100 nm The mixture was kept at room temperature until no more reducing sugars were produced (approximately 1 week) The resulting insol-uble material was collected by decanting and centrifugation
at 600 g for 4 min, and was subsequently washed with water by suction filtration The structure of the product was confirmed by13C-NMR spectroscopy
Preparation of nigerooligosaccharides The a-(1fi 3)-glucan described above was subjected to mild acid hydrolysis (0.05 m H2SO4at 80C for 2 h) to release the small amount of fructose ( 1%) contained in the glucan The fructose-free glucan was then subjected to limited acid hydrolysis (0.1 m H2SO4at 100C for 100 min) to produce nigerooligosaccharides After the hydrolysis was stopped by neutralization with NaHCO3, the insoluble material was removed by centrifugation at 500 g for 4 min Finally, the soluble fraction was applied to a column (4· 22 cm) con-taining equivalent weights of activated carbon (Wako Pure Chemical, Osaka, Japan) and Celite (Wako Pure Chemical) (Fig S4) The column was maintained at 15C, and was then washed with water until the glucose was completely eluted The bound oligosaccharides were eluted with a gradi-ent of 0–8% butanol The sugars were detected by use of the phenol⁄ H2SO4method [31], and characterized by TLC
Measurement of glucosyl transfer velocity The glucosyl transfer rates were measured as described
by Konishi et al [14] The initial velocities of all GTF reactions were measured for the first 4–8 min at 25C The
Trang 8reaction mixtures contained dextran,
nigerooligosaccha-rides, methyl a-d-glucopyranoside and leucrose at various
concentrations in 10 nm enzyme, 50 mm sucrose, 100 mm
NaCl, and 10 mm Mops (pH 6.8) After the concentrations
of the resultant glucose and fructose were measured, the
extent of sucrose splitting was determined from the amount
of fructose released, and the extent of glucosyl transfer was
calculated by subtracting the amount of free glucose from
the amount of free fructose
For kinetic analysis, the assay was carried out with at
least four different concentrations of each acceptor [methyl
a-d-glucopyranoside and nigerooligosaccharide (DP = 2–6)]
The kinetic parameters (velocity constant, k; Michaelis
constant, Km) were determined with the nonlinear
regres-sion program in the origin software package (v 6.1J)
(OriginLab, Northampton, MA, USA)
Light scattering
The formation of insoluble glucan was monitored by light
scattering at 90 in a thermostated cell (25 C) at a
wave-length of 350 nm The reaction mixture contained 50 nm
GTFs, 50 lgÆmL)1 dextran T2000 and 100 mm sucrose in
10 mm Mops (pH 7.0)
Analysis of products during the glucan synthesis
reaction
GS or GSGB (0.4 lm) and 50 mm sucrose were incubated
in 10 mm potassium phosphate (pH 6.8) at 25C A
suit-able volume of mixture was sampled at various time points,
and the reaction was stopped by addition of NaOH at a
final concentration of 10 mm After the samples had been
immediately centrifuged at 18,000 g for 10 min, the
insolu-ble glucan was recovered as a precipitate and washed with
water The supernatants were neutralized with HCl The
precipitates were completely hydrolyzed in 1.2 m HCl at
approximately 100C for 50 min, and this was followed by
neutralization with 1.2 m NaHCO3 The hydrolysate was
then subjected to the dinitrosalicylic acid method [32] to
esti-mate the amount of insoluble glucan in terms of glucose For
the analysis of soluble sugars in the supernatants, the
follow-ing methods were employed: (a) glucose and fructose
concen-trations were determined with the enzymatic assay [33]; and
(b) leucrose and nigerooligosaccharide were detected by
TLC, and their concentrations were determined by HPLC
Simulation of the glucan synthesis reaction
The synthesis of insoluble glucan was numerically analyzed
on the basis of Scheme 1 We assumed Michaelis–Menten
kinetics for sucrose hydrolysis [14,21], and ping-pong
bi-bi kinetics for the glucosyl transfer from sucrose to an
acceptor sugar [5,18,19,22] Using the quasi-steady-state
assumption, we obtained a set of 10 differential equations that describe the time course of saccharide production The fourth-order Runge)Kutta method with a 3.6-min step was used to solve the 10 differential equations, with the kinetic parameters estimated from the experimental data using visual basic for applications in Microsoft Excel (http:// chemeng.on.coocan.jp) All parameters used are listed in Table 2 The details of these differential equations and the simulation parameter are described in Doc S1
13C-NMR analysis
To inhibit base-catalyzed reactions, the insoluble glucan was dissolved at 60 mgÆmL)1in 0.5 m NaOD in D2O con-taining 3.5 mgÆmL)1NaBD4 The 13C-NMR analyses were performed on a JEOL JNM-A500 spectrometer (Center for Instrumental Analysis, Kyushu Institute of Technology) Spectra were recorded at 125.65 MHz at room temperature, with an acquisition time of 0.9667 s and 8000 scan accumu-lations Chemical shifts are expressed in parts per million, with 3-trimethylsilyl-1-propanesulfonic acid sodium salt as
a reference Peaks were assigned according to the method outlined in Colson et al [34]
HPLC Leucrose and nigerooligosaccharide concentrations were measured by HPLC with a YMC-pack polyamine II column (4.6· 250 mm) (YMC, Kyoto, Japan) The column was maintained at 30C, and the flow rate was 0.5 mLÆmin)1 The eluents were water⁄ acetonitrile (25 : 75, v ⁄ v) and water⁄ acetonitrile (30 : 70, v ⁄ v) for the analysis of disaccha-rides (leucrose and nigerose) and nigerooligosacchadisaccha-rides (DP = 3–5), respectively Carbohydrates were detected with
a differential refractometer Concentrations were calculated from the peak area of the refractive index, using a calibration curve The sample for leucrose analysis was incubated with yeast invertase (0.2 mgÆmL)1) at 35C for 30 min to con-sume sucrose before injection, because leucrose was eluted at 7.7 min, which was relatively close to sucrose (7.4 min)
TLC After the samples were concentrated on a centrifugal evap-orator, they were separated by TLC, with three ascents on Silica Gel 70 plates (Wako Pure Chemical) in 15 : 3 : 4 (v⁄ v ⁄ v) 1-butanol ⁄ pyridine ⁄ water [35] The sugars were detected by spraying the plates with H2SO4, and then heat-ing at 120C for 5 min
Acknowledgements
We thank H Sakamoto (Kyushu Institute of Technol-ogy) for providing access to the apparatus for
Trang 9biochemical experiments We appreciate the assistance
provided by M Ikeguchi, Y Fujita and T Kawauchi
for nigerooligosaccharide preparation and separation
This work was supported in part by grants-in-aid for
Scientific Research (B) 13557161 and (C) 13671904 (to
K Fukui), (C) 13680744 (to T Kodama) and a
grant-in-aid for Young Scientists (B) 13080537 (to H
Koma-tsu) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan The initial parts of
this work were performed at the Institute for Materials
Chemistry and Engineering, Kyushu University
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Supporting information
The following supplementary material is available: Fig S1 13C-NMR analysis of the insoluble glucan produced by GS and GSGB
Fig S2 TLC analysis of saccharide production by GS and GSGB
Fig S3 Double reciprocal plot of nigerotriose accep-tor reaction of GS
Fig S4 Separation of nigerooligosaccharides by acti-vated carbon chromatography
Doc S1 Simulation of the glucan synthesis reaction This supplementary material can be found in the online version of this article
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