Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor

Article abstract of DOI:10.1016/j.tet.2003.10.113

Cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on Eupergit C and used for the synthesis of maltooligosyl fructofuranosides employing soluble starch as donor and sucrose as acceptor. Using a weight ratio starch-sucrose of 1:2, the conversion of starch into acceptor products catalyzed by soluble and immobilized CGTases was higher than 80% in 48 h. Under these conditions, the reaction was selective for the formation of maltosyl fructofuranoside.

Full text of DOI:10.1016/j.tet.2003.10.113

Tetrahedron 60 (2004) 529–534
Synthesis of maltooligosyl fructofuranosides catalyzed by
immobilized cyclodextrin glucosyltransferase using starch
as donor
a
M. Teresa Martın, M. Angeles Cruces, Miguel Alcalde, Francisco J. Plou, Manuel Bernabe
a
a
a
b
´
´
and Antonio Ballesterosa
,
*
a
´
´
Departamento de Biocatalisis, Instituto de Catalisis, C.S.I.C., Cantoblanco, 28049 Madrid, Spain
´ ´
Instituto de Quımica Organica, C.S.I.C., 28006 Madrid, Spain
b
Received 18 August 2003; revised 16 September 2003; accepted 17 October 2003
Abstract—Cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on Eupergit C and used
for the synthesis of maltooligosyl fructofuranosides employing soluble starch as donor and sucrose as acceptor. Using a weight ratio starch–
sucrose of 1:2, the conversion of starch into acceptor products catalyzed by soluble and immobilized CGTases was higher than 80% in 48 h.
Under these conditions, the reaction was selective for the formation of maltosyl fructofuranoside.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
improved functionality compared with the parent acceptor
molecules, e.g. increased water solubility or stability,
bifidogenic properties, etc.6
Cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) is
a bacterial enzyme belonging to the a-amylase family (or
glycoside hydrolase family 13)1 that catalyzes four reac-
tions.2,3 Cyclization is an intramolecular transglycosylation
in which the CGTase converts starch into cyclodextrins
(CDs). Coupling is an intermolecular transglycosylation
between a CD molecule and a linear oligosaccharide.
Disproportionation is an intermolecular transglycosylation
between two carbohydrates. In addition, CGTase displays a
weak starch hydrolysis activity (where water acts as
acceptor).
In this context, when sucrose is employed as acceptor,
maltosyl fructofuranoside ([a-D-Glu-(1!4)-a-D-Glu-
(1!2)-b-D-Fru) and higher polymerization products are
obtained.7 A mixture of maltooligosyl fructofuranosides
(‘maltooligosyl fructose’) with a degree of polymerization
of 3–7 is currently being commercialized as coupling sugar,
and is obtained using starch as donor and sucrose as
acceptor, in a process catalyzed by CGTase.8 Coupling
sugar, a non-cariogenic sweetener, is also used as a viscosity
modifier, being more stable than other reducing sugar
mixtures.7
Either via coupling or disproportionation, CGTases are able
to perform the so-called acceptor reaction allowing the
synthesis of products of industrial interest. The main
requirement for strong acceptor properties is the presence
of a D-glucopyranose structure (chair form) with equatorial
hydroxyl groups at C-2, C-3 and C-4.4 Different carbo-
hydrates (such as xylose, sucrose, glucose, maltose and
lactose), glycosides, sugar alcohols, vitamins and flavo-
noids, have been successfully used as CGTase acceptors.5
The resulting transglycosylated products usually exhibit
Thermoanaerobacter sp. (a thermophilic anaerobic bacter-
ium) produces a highly thermostable CGTase that displays a
high disproportionation activity.9– 11 This enables the
enzyme to be used in several acceptor reactions.12
In this work, we have studied the synthesis of maltooligosyl
fructose employing soluble starch as donor and sucrose as
acceptor, catalyzed by CGTase from Thermoanaerobacter
sp. immobilized on Eupergit C. The reaction rates using the
soluble and immobilized CGTases were compared. The
main reaction products were isolated by preparative column
chromatography, and characterized using NMR and MS
analysis.
Keywords: Cyclodextrin glucanotransferase; Glycosyltransferases;
Transglycosylation; Thermoanaerobacter sp; Coupling sugar; Glucosyl
sucrose.
*
Corresponding author. Tel.: þ34-91-5854808; fax: þ34-91-5854760;
e-mail address: a.ballesteros@icp.csic.es
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.113

´
M. T. Martın et al. / Tetrahedron 60 (2004) 529–534
530
2. Results and discussion
major products. Spectral data correlated well with those
reported for maltosyl fructose by Munksgaard,17 and for
maltotriosyl and maltotetraosyl fructose by Monthieu.13
However, small amounts of maltooligosyl fructosides with a
degree of polymerization up to 7–8 were also observed by
TLC and HPLC (Fig. 1).
CGTase is able to use starch12 or cyclodextrins13 as glucose
donor in transglycosylation processes. Starch is a cheap,
abundant and renewable agroproduct, therefore it is
preferred to expensive cyclodextrins for industrial purposes.
CGTases do not require sugar nucleotides (as all the
glycosyltransferases of the Leloir pathway) nor sugar–
phosphates (as most glycosyltransferases of the non-Leloir
pathway) as donors, since it uses the free energy of cleavage
of the readily available starch.3
In the absence of sucrose, the consumption of starch led to
the formation of CDs.12 The presence of co-substrate
molecules such as sucrose that are strong acceptors (and,
consequently, strong promoters of the formation of linear
transfer products) inhibited CD formation. As shown in
Table 1, in the presence of the same weight of starch and
sucrose, the formation of CDs was almost negligible.
Reaction selectivity can be modulated by varying the donor/
acceptor ratio. Thus, using a starch/sucrose ratio 2:1 (w/w)
the yield of maltooligosyl fructose was 72% and minor
amounts of CDs (7–10%) were formed. Under the above
conditions, the selectivity to maltosyl fructose was quite
low. In contrast, using a starch/sucrose ratio of 1:2 (w/w) the
yield of oligosaccharides was 80%, and the formation of
maltosyl fructose was enhanced (60%).
For these acceptor reactions, the immobilization of
glucosyltransferases may allow for their stabilization as
well as enabling the separation of the biocatalyst. Different
approaches have been applied to the immobilization of
CGTases, based on adsorption or covalent coupling.14 We
have recently reported CGTase immobilization by covalent
binding to aminated silica, activated Sepharose 4B and
epoxy-activated acrylic polymers such as Eupergit C.15 The
latter support represents one of the best candidates for
CGTase immobilization displaying a half life at 95 8C five
times higher that that of the soluble enzyme, as was
demonstrated elsewhere.15b
No significant differences were found concerning yield and
product distribution when analysing the effect of enzyme
immobilization on the reaction (Table 1). However, the
reaction rate was higher with the immobilized CGTase (Fig.
2). The initial rate of maltosyl fructose formation with the
Eupergit C-immobilized CGTase (14.6 g/l h) was about 3-
fold higher as compared with the soluble enzyme (5.6 g/l h).
This could be due to improved thermostability of CGTase
immobilized on Eupergit C compared to the soluble
enzyme.15
The acceptor reaction with soluble starch (Paselli SA2, with
an average degree of polymerization of 50) as donor and
sucrose as acceptor was studied with soluble and CGTase
immobilized on Eupergit C. This enzyme has a higher
affinity for disaccharides as acceptors compared with
monosaccharides.3,6,16 When sucrose is employed as
acceptor, maltosyl fructose (1) is initially formed (Scheme
1). Maltosyl fructose also acts as acceptor, yielding
maltotriosyl fructose (2). A series of maltooligosyl fructo-
sides is subsequently formed.
In conclusion, CGTase immobilized on Eupergit C is able to
use starch as donor and sucrose as acceptor, resulting in
excellent yields of maltooligosyl fructose (92%). The
starch/sucrose ratio is of great importance for inhibiting
cyclodextrin synthesis and for modulating the selectivity of
the process. Two industrial processes for the CGTase-
catalyzed synthesis of coupling sugar have been patented
using the enzyme from Bacillus macerans8 and Thermo-
anaerobacter.18 Our Eupergit-CGTase can become an
attractive alternative to the soluble enzyme for these
processes. In addition, new applications may emerge from
employing distinct acceptors in reactions catalyzed by
CGTase from Thermoanaerobacter sp.
The effect of the weight ratio starch–sucrose (from 2:1 to
1:2) on product distribution, using 10% (w/v) soluble starch
and variable amounts of sucrose, was tested (Table 1). The
maximum conversion to oligosaccharides, regardless of the
donor/acceptor ratio, was achieved at 48 h. There are no
commercially available standards of maltosyl fructofurano-
sides. Thus, the main reaction products were isolated by
column chromatography (purities higher than 95%) and
characterized using NMR and MALDI-TOF mass spec-
trometry. Maltosyl fructose (1), maltotriosyl fructose (2)
and maltotetraosyl fructose (3) were confirmed to be the
Table 1. Maltooligosyl fructosides and cyclodextrin production using different starch/sucrose (w/w) ratios catalyzed by soluble and immobilized CGTase.
Conditions: 10% (w/v) soluble starch, 60 8C, 10 mM sodium citrate buffer (pH 5.5) containing 0.15 mM CaCl2, 48 h reaction. The conversion of starch into
maltooligosyl fructose and cyclodextrins is also presented
Ratio starch/sucrose (w/w)
Enzyme
Products formed (g/l)a
Starch conversion (%)b
Maltooligosyl-fructose Cyclodextrins
1
2
3
aCD
b-CD
gCD
2:1
1:1
Soluble
Immobilized
28
35
21
26
11
13
3.3
2
6.8
5.2
—
—
72
72
10.1
7.2
Soluble
Immobilized
51
51
35
34
15
14
0.6
—
2.5
—
—
—
78
79
3.1
—
1:2
Soluble
Immobilized
88
95
48
50
15
16
—
—
—
—
—
—
83
92
—
—
a
Nomenclature of the different maltooligosyl fructosides: 1, maltosyl fructose; 2, maltotriosyl fructose; 3, maltotetraosyl fructose.
Based on starch consumption.
b

´
M. T. Martın et al. / Tetrahedron 60 (2004) 529–534
531
Scheme 1. Acceptor reaction using soluble starch as donor and sucrose as acceptor catalyzed by Thermoanaerobacter CGTase. Nomenclature used for
maltoolygosyl fructofuranosides: maltosyl fructose (1) where n¼1; maltotriosyl fructose (2) where n¼2; maltotetraosyl fructose (3) where n¼3. Reaction
conditions given in Section 3.
3. Experimental
NMR spectra were recorded on a Varian UNITY spec-
trometer at 500 MHz (1H) and 125 MHz (13C) at 30 8C for
solutions in D2O. The identity of the protons and carbons of
the different units were obtained through 2D- homo-
(DQCOSY and TOCSY) and hetero- (HMQC and HMBC)
experiments. Chemical shifts are referred to the residual
signal of HOD at 4.71 ppm for 1H NMR, and that of acetone
as external reference at 31.07 ppm for 13C NMR. Mass
spectral analyses were obtained on a a Bruker (Bremen,
3.1. General
CGTase from Thermoanaerobacter sp. was kindly provided
by Novo Nordisk (Toruzyme 3.0 L, batch ACN00019), and
purified as described.15 Cyclodextrins (a-, b- and g-) were
purchased from Sigma. Potato soluble starch (Paselli SA2)
was kindly donated by Avebe (Foxhol, The Netherlands).
Figure 1. HPLC chromatogram of the reaction mixture at 48 h using soluble starch as donor and sucrose as acceptor catalyzed by CGTase immobilized on
Eupergit C. Conditions: 10% soluble starch (w/v), 20% (w/v) sucrose, 10 mM sodium citrate buffer (pH 5.5) containing 0.15 mM CaCl2, 60 8C. The sucrose
peak appeared split in two peaks. 1 to 5 represents the homologous series of acceptor products where 1 is maltosyl fructose.

´
M. T. Martın et al. / Tetrahedron 60 (2004) 529–534
532
Figure 2. Maltosyl fructose (W) and maltotriosyl fructose (X) production employing a starch:sucrose (w/w) ratio of 1:2 catalyzed by soluble
Thermoanaerobacter CGTase and immobilized CGTase on Eupergit C. Reaction conditions described in Section 3.
Germany) Reflex III MALDI-TOF mass spectrometer
equipped with an ion source with visualization optics and
a N2 laser (337 nm). Mass spectra were recorded using a
dihydroxybenzoic acid matrix and methanol as eluent.
42A columns (300£7.8 mm, Bio-Rad) connected in series.
Two deashing cartridges (30£4.6 mm, Bio-Rad) were used
prior to the column. Water was used as mobile phase
(0.7 ml/min). The column temperature was maintained at
85 8C using a Timberline oven. A refractive-index detector
9040 (Varian) was employed. Integration was carried out
using the Varian Star 4.0 software.
Cyclization assays. The production of cyclodextrins was
detected spectrophotometrically via the formation of
inclusion complexes with several organic compounds.
Paselli SA2 (partially hydrolyzed potato starch with an
average degree of polymerization of 50) was used as
substrate at final concentrations of 5% (w/v) for b- and g-
CD, and 2% (w/v) for a-CD. a-CD was determined at
490 nm on the basis of its ability to form a stable, colorless
inclusion complex with methyl orange.19 b-CD was
determined at 552 nm on the basis of its ability to form a
stable, colorless inclusion complex with phenolphthalein.20
g-CD was determined measuring the color increase at
630 nm due to the formation of an inclusion complex with
bromocresol green.21 The CGTase activity was measured at
85 8C by incubating the enzyme (0.03–0.4 U/ml) in the
presence of soluble starch in 10 mM sodium citrate buffer
(pH 5.5) containing 0.15 mM CaCl2. The formation of the
corresponding CD was followed for 5 min. One unit of
activity was defined as that catalyzing the production of
1 mmol of CD per min under the above conditions.
Thin-layer chromatography (TLC) was carried out on
aminated silica gel plates (Lichroprep-NH2, 25–40 mm,
Merck) using water/butanol/ethanol 2:3:5 (v:v.v) as elu-
ent.22 Spots were detected by immersion of plates into a
mixture formed by 45 ml of 1 g urea dissolved in 85%
H3PO4 aqueous, 48 ml butanol and 50 ml ethanol,23 drying
and heating at 120 8C for 5 min.
Immobilization on Eupergit C was carried out at 25 8C with
gentle orbital shaking (150 rpm) in 1.2 M potassium
phosphate buffer pH 7.0. A 1:100 (w/w) enzyme–support
ratio was employed. After incubation for 72 h, the beads
were collected by vacuum filtration using a porous glass
filter. The beads were rinsed thoroughly on the same filter
with approximately 3£20 ml of diluted potassium phos-
phate and 2£20 ml of 10 mM sodium citrate buffer, pH
5.5.15
The progress of the acceptor reaction was analyzed by high-
performance liquid chromatography (HPLC). 1.7 b-CD
units of soluble (140 U/mg protein) or immobilized CGTase
(147 U/g support) were incubated at 60 8C with 10% (w/v)
soluble starch in 10 mM sodium citrate buffer (pH 5.5)
containing 0.15 mM CaCl2 in the presence of different
amounts of sucrose -studied ratio starch–sucrose (w/w):
2:1, 1:1 and 1:2. The final volume was 10 ml. At different
times, aliquots were taken and mixed with the same volume
of 0.4 M NaOH to quench the reaction. Samples were
centrifuged during 15 min at 8500g. HPLC was performed
using a ternary pump 9012 (Varian) and two Aminex HPX-
3.2. Synthesis and purification of maltooligosyl fructo-
furanosides
1.7 b-CD units of immobilized CGTase (147 U/g support)
were incubated at 60 8C with 10% (w/v) soluble starch in
10 mM sodium citrate buffer (pH 5.5) containing 0.15 mM
CaCl2 in the presence of 20% (w/v) sucrose. The final
volume was 10 ml. The reaction was maintained for 48 h.
The immobilized CGTase and the remaining starch were
removed by centrifugation. The aqueous supernatant was
evaporated azeotropically with methanol under reduced
pressure. After evaporation, the resulting residue was

´
M. T. Martın et al. / Tetrahedron 60 (2004) 529–534
533
fractionated by column chromatography using Lichroprep-
NH2 silica gel (25–40 mm, Merck) as adsorbent and an
adsorbent–sample ratio of approx. 30:1 (w/w). The reaction
products were eluted with a mixture H2O–ethanol–1-
butanol 2:5:3 (v/v/v). The order of elution was maltosyl
fructose (1), maltotriosyl fructose (2), maltotetraosyl
fructose (3) and higher polymerization products, as shown
by TLC and HPLC. Solvents were removed under vacuum
from collected fractions of the respective oligosaccharides
and characterized.
(MALDI-TOF): calcd for C30H52O26Na 851.4, found 851.3;
calcd for C30H52O26K 867.5, found 867.2.
Acknowledgements
We thank Ms. Satoko Endo (Hayashibara Co. Ltd,
Okayama, Japan) for providing us with a sample of
commercial coupling sugar and for technical help. We
thank Dr. Iqbal S. Gill (BioSynTech, USA) for a critical
reading of the manuscript. This research was supported by
the Spanish CICYT (project PPQ2001-2294). We thank
Instituto Danone, and Ministerio de Educacion y Cultura for
research fellowships.
3.2.1. Maltosyl fructose (1) [a-D-glucopyranosyl-(1!4)-
a-D-glucopyranosyl-(1!2)-b-D-f0ructofuranoside].
1H
0
0
NMR (d, ppm): 5.40 (d, 1H, H-1 , J1 2 ¼3.7 Hz), 5.39 (d,
1H, H-100, J1 2 ¼3.7 Hz), 4.20 (d, 1H, H-3, J3,4¼8.7 Hz),
00 00
4.02 (m, 2H, H-4þH-30), 3.98 (m, 1H, H-50), 3.088 (m, 1H,
H-5), 3.88–3.76 (m, 6H, H-6aþH-6b0þ0 H-6 aþ60bþH-
600aþ600b), 3.76–3.64 (m, 2H, H-300þH-5 ), 3.69 0(m, 1H,
H-40), 3.67 (s, 2H, H-1aþH-1b), 3.58 (m, 2H, H-2 þH-200),
3.40 (t, 1H, H-400, J3,4¼J4,5¼9.2 Hz); 13C NMR (d, ppm):
104.5 (C-2), 100.7 (C-100), 92.3 (C-10), 82.2 (C-5), 77.6 (C-
4000), 77.3 (C-3), 74.9 (C-4), 73.9þ73.8 (C-30þC-300), 73.6 (C-
5 ), 72.6 (C-200), 71.8 (C-50),0071.7 (C-20), 70.2 (C-400), 63.2
(C-6), 62.2 (C-1), 61.3 (C-6 ), 61.0 (C-60). MS (MALDI-
TOF): calcd for C18H32O16Na 527.3, found 527.1; calcd for
C18H32O16K 543.4, found 543.1.
References and Notes
1. Henrissat, B.; Bairoch, A. Biochem. J. 1993, 293, 781–788.
2. Tonkova, A. Enzyme Microb. Technol. 1998, 22, 678–686.
´
´
3. Plou, F. J.; Martın, M. T.; Gomez de Segura, A.; Alcalde, M.;
Ballesteros, A. Can. J. Chem. 2002, 80, 743–752.
4. (a) Kobayashi, S. Cyclodextrin Producing Enzyme (CGTase).
In Enzymes for Carbohydrate Engineering; Park, K. H.,
Robyt, J. F., Choi, D., Eds.; Elsevier: Amsterdam, 1996; pp
23–41. (b) Kitahata, S. Denpun Kagaku 1990, 37, 59–67.
5. (a) Shibuya, T.; Miwa, Y.; Nakano, M.; Yamauchi, T.; Chaen,
H.; Sakai, S.; Kurimoto, M. Biosci. Biotechnol. Biochem.
1993, 57, 56–60. (b) Aga, H.; Yoneyama, M.; Sakai, S.;
Yamamoto, I. Agric. Biol. Chem. 1991, 55, 1751–1756. (c)
Kometani, T.; Terada, Y.; Nishimura, T.; Takh, H.; Okada, S.
Biosci. Biotechnol. Biochem. 1994, 58, 1990–1994. (d)
Rendleman, J. A. Biotechnol. Appl. Biochem. 1996, 24,
121–127.
3.2.2. Maltotriosyl fructose (2) [a-D-glucopyranosyl-
(1!4)-a-D-glucopyranosyl-(1!4)-a-D-glucopyranosyl-
(1!2)-b-D-fructofuranoside]. 1H NMR (d, ppm): 5.38 (d,
00
000
1H, H-10, J1 2 ¼3.7 Hz), 5.36þ5.35 (2d, H-1 þH-1 ,
J¼3.7 Hz), 4.18 (d, 1H, H-3, J3,4¼8.6 Hz), 4.05 (t, 1H, H-
4, J4,5¼8.6 Hz), 4.00 (dd, 1H, H-30, J¼8.9þ10 Hz), 3.94
(m, 1H, H-50), 3.86 (m,1H,H-5), 3.86–3.70 (m, 8H, H-
6aþ6bþH-60aþ60bþH-600aþ600bþH-6000aþ6000b), 3.68 (m,
2H, H-500þH-5000), 3.64 (s, 2H, H-1aþH-1b),0 3.70–3.60
(m, 2H, H-40þH-400), 3.60–3.50 (m, 3H, H-2 þH-200þH-
2000), 3.38 (t, 1H, H-4000, J¼9.6þ9.2 Hz); 13C NMR (d0, ppm):
104.6 (C-2), 100.7þ100.6 (C-100þC-1000), 92.9 (C-1 ), 82.3
(C-5), 77.9þ77.7 (C-40þC-400), 77.5 (C-030 ), 750.000 (C-4),
0
0
6. Park, D. C.; Kim, T. K.; Lee, Y. H. Enzyme Microb. Technol.
1998, 22, 217–222.
7. Crittenden, R. G.; Playne, M. J. Trends Food Sci. Technol.
1996, 7, 353–361.
8. Shigetaka, N.; Naoto, O.; Masakazu, O.; Junsuke, O. US
Patent 3819484, 1974..
74.3þ73.9þ73.8þ73.7
(C-30þC-3 þC-3 þC-500),
72.7þ72.5þ72.2 (C-20þC-200þC-200), 71.9 (C-50), 71.8 (C-
50000), 70.3 (C-4000), 63.3 (C-6), 62.3 (C-1), 61.5 (2)þ61.1 (C-
9. Starnes, R. L.; Hoffman, C. L.; Flint, V. M.; Trackman, P. C.;
Duhart, D. J.; Katkocin, D. M. Starch Liquefaction with a
Highly Thermostable Cyclodextrin Glycosyl Transferase from
Thermoanaerobacter species. In Enzymes in Biomass Con-
version. Leathman, G. F., Himmel, M. E., Eds.; American
Chemical Society: Washington, DC, 1991; pp 384–393.
10. Norman, B. E.; Jorgensen, S. T. Denpun Kagaku 1992, 39,
101–108.
6 þC-600þC-6000). MS
(MALDI-TOF):
calcd for
C24H42O21Na 689.3, found 689.2; calcd for C24H42O21K
705.4, found 705.2.
3.2.3. Maltotetraosyl fructose (3) [a-D-glucopyranosyl-
(1!4)-a-D-glucopyranosyl-(1!4)-a-D-glucopyranosyl-
(1!4)-a-D-glucopyranosyl-(1!2)-b-D-fructofur0ano-
side]. 1H NMR (d, ppm): 5.40–5.38 (m, 4H, H-1 þH-
100þH-1000þH-1lV), 4.20 (d, 1H, H-3, J3,4¼8.7 Hz), 4.03 (m,
2H, H-4þH-30), 3.96 (m, 3H, H-5þH-50þH-3lV), 3.86–3.70
´
11. Alcalde, M.; Plou, F. J.; Andersen, C.; Martın, M. T.;
Pedersen, S.; Ballesteros, A. FEBS Lett. 1999, 445, 333–337.
´
12. Martın, M. T.; Alcalde, M.; Plou, F. J.; Dijkhuizen, L.;
Ballesteros, A. Biocatal. Biotransform. 2001, 19, 21–35.
13. Monthieu, C.; Guibert, A.; Taravel, F. R.; Nardin, R.; Combes,
D. Biocatal. Biotransform. 2003, 21, 7–15.
(m,
10H,
H-6aþ6bþH-60aþ60bþH-600aþ600bþH-
6000aþ6000bþH-6lVaþ6lVb), 3.67 (s, 2H, H-1aþH-1b),
3.65–3.55 (m, 4H, H-20þH-200þH-2000þH-2lV), 3.42 (t, 1H,
14. (a) Kim, P. S.; Shin, H. D.; Park, J. K.; Lee, Y. H. Biotechnol.
Bioprocess 2000, 5, 174–180. (b) Tardioli, P. W.; Zanin,
G. M.; Moraes, F. F. Appl. Biochem. Biotechnol. 2000, 84,
1003–1019.
H-4lV, J¼9.5þ9.7 Hz); C NMR (d, ppm): 104.50 (C-2),
13
00
000
lV
100.6þ100.4þ100.3 (C-1 þC-1 00þC-1 ), 92.8 (C-1 ), 82.2
(C-5), 77.8þ77.4 (2) (C-40þC-4 þC-4 ), 76.8 (C-3), 74.9
000
(C-4), 74.2 (2)þ73.8þ73.7þ73.6 (C-30þC-300þC-3000þC-
´
15. (a) Martın, M. T.; Alcalde, M.; Plou, F. J.; Ballesteros, A.
0
00
lV
3 ), 72.6þ72.4 (2)þ72.1þ71.8þ71.7 (2) (C-2 þC-2 þC-
´
Indian J. Biochem. Biophys. 2002, 39, 229–234. (b) Martın,
M. T.; Plou, F. J.; Alcalde, M.; Ballesteros, A. J. Mol. Catal.
B: Enzym. 2003, 21, 299–308.
000
2 þC-2lVþC-50þC-500þC-5000), 0 70.2 (C-4lV), 63.2 (C-6),
62.3 (C-1), 61.4 (3)þ61.0 (C-6 þC-600þC-6000þC-6lV). MS

´
M. T. Martın et al. / Tetrahedron 60 (2004) 529–534
534
16. Nakamura, A.; Haga, K.; Yamame, K. FEBS Lett. 1994, 337,
66–70.
C. L.; Dijkstra, B. W.; Bergsma, J.; Dijkhuizen, L.
Biochemistry 1995, 34, 3368–3376.
17. Munksgaard, V. PhD Thesis, Danmarks Farmaceutiske
Højskole, 1981.
21. Kato, T.; Horikoshi, K. Anal. Chem. 1984, 56, 1738–1740.
22. Del Rio, G.; Morett, E.; Soberon, X. FEBS Lett. 1997, 416,
221–224.
18. Pedersen, S. WO Patent 92/13962, 1992.
19. Hirai, H.; Toshima, N.; Uenoyama, S. Polym. J. 1981, 13,
607–610.
23. Weiss, T. J.; Brown, M.; Zeringue, H. J.; Fenge, R. O. J. Am.
Oil Chem. Soc. 1971, 48, 145–148.
20. Penninga, D.; Strokopytov, B.; Rozeboom, H. H.; Lawson,