S.-H. Yoon et al. / Carbohydrate Research 345 (2010) 1730–1735
1731
reaction of glucoamylase and b-glucosidase in a micro-aqueous
reaction system.19,20 The glycosylation was carried out in 100 mL
of diisopropyl ether, containing 0.3–2.2 mL of 0.01 M buffer (pH
for 10 min. The quantitative amounts of the compounds on the
TLC plate were determined by scanning densitometry.28
4–8), and 1 mmol of
as glycosyl donors. Enzymes (0.18–0.137 g) were added, and the
D
-glucose (0.182 g) or other carbohydrates
2.3. Fractionation and purification of
Bio-Gel P-2 column chromatography
L-DOPA a-glycosides by
mixture was incubated for 72 h at 68 °C under reflux.
In the present work, we have synthesized
by enzymatic transglycosylation reactions of
the relatively simplest and cheapest glucosyl donors, sucrose. The
transglycosylation reactions were catalyzed by four different glu-
cansucrases that catalyze the so-called acceptor reactions,21 giving
L
-DOPA
-DOPA with one of
a
-glycosides
Approximately 1 mL of the concentrated reaction products was
added to a Bio-Gel P-2 column (2.5 ꢁ 110 cm) and eluted with
acid–water (pH 3.0).18 The flow rate was 0.4 mL/min and the frac-
tions collected were 3.0 mL each. The amount of carbohydrate con-
tained in each of the fractions was analyzed by TLC28 by the micro
L
L
-DOPA
a
-glycosides.
phenol–sulfuric acid method.29 The
L-DOPA-glycoside fractions
were pooled and concentrated by vacuum rotary evaporation.
2. Experimental
2.1. Materials
2.4. Molecular structure analysis of the reaction products by 1
NMR spectroscopy
H
The reaction products were dried by vacuum rotary evaporation
at ꢀ21 °C and were exchanged three times with 3 mL of D2O. The
final dried products were dissolved in 0.5 mL of pure D2O and
put into 5-mm NMR tubes. All spectra were acquired at 25 °C using
a 500-MHz or a 700-MHz Bruker NMR spectrometer. The 1H chem-
Four different kinds of glucansucrases [EC 2.4.1.5] were ob-
tained by growing constitutive mutants, Leuconostoc mesenteroides
B-512FMC,22,23 B-742CB,23,24 B-1299CA,24,25 and B-1355C,22,24 in a
medium containing
D-glucose instead of sucrose as a carbon
source.26 The four glucansucrases were each concentrated, frac-
tionated, and dialyzed against 20 mM pyridinium acetate buffer
(pH 5.2) by a size-exclusion, hollow-fiber, ultra-filtration system,
with a molecular-weight cut off of 30,000 Da (Amicon, Beverly,
ical shifts of L-DOPA and the L-DOPA-glycosides were compared
with chemical shifts previously reported.18
MA), using the culture supernatants as previously described.26
L
-
3. Results and discussion
DOPA was purchased from Sigma–Aldrich Chemical Co. (St. Louis,
MO, USA). Bio-Gel P-2 (fine) resin was purchased from Bio-Rad
Laboratories (Hercules, CA). Whatman K5 TLC plates were obtained
from Fischer Scientific Co. (USA). All other chemicals were of re-
agent grade. Immobilized yeast was prepared, as previously
described.27
3.1. Synthesis of
of dextransucrases
L-DOPA a-glycosides by the acceptor reactions
Previously, we have synthesized
glucopyranosyl -DOPA and 4-O-
enzymatic transglycosylation reactions of
hexaose ( -cyclodextrin), catalyzed by Bacillus macerans cyclomal-
todextrin glucanyltransferase (CGTase), [EC 2.4.1.19].18 In the
present study, we have synthesized the same two -DOPA -glyco-
L
-DOPA
-glucopyranosyl
-DOPA with cyclomalto-
a
-glycosides (3-O-
a-D-
L
a
-
D
L-DOPA) by
L
2.2. Synthesis of
L-DOPA
a-glycosides by reaction with
a
glucansucrases and sucrose
L
a
Four different glucansucrases (80 U, where 1 U = 1
glucose released from sucrose/min) were added to each 80 mL of
l
mole of
D
-
sides, but with a much simpler and cheaper substrate, sucrose, cat-
alyzed by four different L. mesenteroides B-512FMC, B-742CB, B-
1299A, and B-1355C glucansucrases [EC 2.4.1.5], hereafter referred
to as B-512FMC dextransucrase, B-742CB dextransucrase, B-1299A
dextransucrase, and B-1355C alternansucrase.
substrate solution composed of 20 mM -DOPA and 40 mM sucrose
L
in 20 mM pyridinium acetate buffer (pH 5.2), which was degassed
with a vacuum pump for 20 min and then incubated at 23 °C for
24 h under vacuum, with slow stirring. The pH of the digest was
then decreased to 3.0 by addition of 1.0 M HCl to stop the reaction.
The reaction digest was concentrated to 10 mL with a vacuum
rotary evaporator at 23 °C. Ten milliliters (one volume ratio) of
EtOH was added slowly to 10 mL of the concentrated reaction di-
gest to precipitate the dextran that was produced as a by-product,
and then it was kept at 4 °C for 24 h. The resulting precipitate was
removed by centrifugation at 10,000g for 30 min, and the superna-
tant was concentrated again to 10 mL by vacuum rotary evapora-
tion to remove the EtOH. Immobilized yeast (8 g, wet weight)
was added to the enzyme digests and incubated at 23 °C for 24 h
under vacuum to remove fermentable carbohydrates. The insolu-
ble materials and immobilized yeast were removed by centrifuga-
tion at 10,000g for 30 min, followed by glass filtration. The filtrate
was concentrated to ꢀ4 mL by vacuum rotary evaporation, the pH
was adjusted to 3, and then the mixture was placed under nitro-
gen. The reaction digests that were obtained at each reaction step
Three of the dextransucrases synthesize three structurally dif-
ferent dextrans and show different specificities: B-512FMC dex-
transucrase synthesizes dextran with
residues in the main chains with 5-6%
cose residues and long -(1?6)-linked
onto the main -(1?6) chains; B-742CB dextransucrase synthe-
sizes -(1?6)-linked -glucose residues in the main chains, with
single -glucose residues -(1?3)-linked branches on every -glu-
cose residues in the main chains and a few main chains with
(1?3)-linked branches onto main chains; B-1299A dextransucrase
synthesizes -(1?6)-linked -glucose residues in the main chains
with 33% -(1?2)-linked single branched -glucose residues and
1% main chains linked to main chains by -(1?2) branch linkages;
B-1355C alternansucrase synthesizes alternating -(1?6) and
(1?3)-linked -glucose residues in the main chains with 7% -glu-
cose and main chains branched by -(1?3) linkages onto the main
chains.21 All four of the glucansucrases also carry out acceptor
reactions that transfer -glucose residues to other carbohydrates
a
-(1?6)-linked
-(1?3)-linked single
-glucose chains branched
D
-glucose
a
D-glu-
a
D
a
a
D
D
a
D
a
-
a
D
a
D
a
a
a-
D
D
a
were analyzed by TLC.28 An appropriate amount (2–5
l
L) of sample
D
was added to a Whatman K5 silica gel TLC plate and irrigated 2–3
times over ꢀ18-cm path length with 3:1 volume proportions of
CH3CN and H2O. The carbohydrates on the TLC plate were visual-
ized by dipping the plate into a solution containing 0.3% (w/v) N-
(1-naphthyl)ethylenediamine and 5% (v/v) sulfuric acid in MeOH,
followed by drying and subsequent heating in an oven at 130 °C
and to some noncarbohydrate compounds containing hydroxyl
groups.21
In the presence of
duced -fructose, -glucose, isomaltulose, leucrose (
anosyl-(1?5)- -fructopyranose), isomaltodextrins (isomaltose,
isomaltotriose, and isomaltotetraose), and new products
L-DOPA, all four of the glucansucrases pro-
D
D
a-D
-glucopyr-
D