Enzymatic synthesis of 2-aminoethyl β-d-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase

Article abstract of DOI:10.1016/j.carres.2012.12.009

Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl β-d-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, K_ic = 0.31 ± 0.04 M and K_iu = 0.604 ± 0.035 M.

Full text of DOI:10.1016/j.carres.2012.12.009

Carbohydrate Research 368 (2013) 104–110
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Enzymatic synthesis of 2-aminoethyl b-D-galactopyranoside
catalyzed by Aspergillus oryzae b-galactosidase
a
a
b
b
a
Cecilia Porciúncula González , Agustín Castilla , Lucía Garófalo , Silvia Soule , Gabriela Irazoqui ,
Cecilia Giacomini
a
a,
⇑
Cátedra de Bioquímica, Dpto. de Biociencias, Facultad de Química, UdelaR, Gral. Flores 2124, CC 1157, Montevideo, Uruguay
Laboratorio de Carbohidratos, DQO, Facultad de Ciencias/Facultad de Química, UdelaR, Alfredo Navarro 3051, Montevideo, Uruguay
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides.
Generation of new low molecular weight galactosides is of interest since they are potential galectin inhib-
itors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor
Received 7 September 2012
Received in revised form 29 November 2012
Accepted 7 December 2012
Available online 19 December 2012
agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl b-D-
galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized
enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose,
and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol
Keywords:
Transglycosylation
Galactosides
b-galactosidase
Glycosidases
proved to be an enzyme inhibitor, fitting
a
mixed inhibition model with inhibition constants,
K
ic = 0.31 ± 0.04 M and Kiu = 0.604 ± 0.035 M.
Ó 2012 Elsevier Ltd. All rights reserved.
Enzyme inhibition
1
. Introduction
Recent advances in glycobiology have significantly increased re-
In nature glycosidases catalyze hydrolysis of glycosidic bonds,
however these enzymes have proven to be extremely useful for
the synthesis of new glycosides. Transglycosylation reaction mech-
anism of these enzymes is stereoselective and result in the forma-
search in carbohydrate chemistry. Galactosides have aroused con-
siderable attention due to their involvement in several biological
processes, and especially as galectin inhibitors.
tion of anomerically pure glycosides in a single step, which is an
1–7
13–15
Galectins are a
interesting alternative to the complex chemical synthesis.
b-
family of highly conserved carbohydrate binding proteins which
contain conserved carbohydrate recognition domains (CRDs) capa-
ble of binding specific carbohydrate structural motifs. When bound
to specific ligands they can regulate a number of biological pro-
cesses including inflammatory responses, infectious processes,
and tumor invasion. Galectins are generally overexpressed in can-
cer cells and contribute to tumor progression by mechanisms such
as: suppression of cytotoxic drug-induced apoptosis of tumor cells,
galactosidases are widely used to catalyze the transfer of a galacto-
syl moiety from a donor molecule to an acceptor one, preserving
the anomeric center configuration.
1
3–16
Transglycosylation catalyzed by b-galactosidases takes place in
two steps. First a galactosyl–enzyme complex is formed between
the enzyme and the galactosyl moiety of the donor, while the agly-
cone is released. In a second step a nucleophile reacts with the
galactosyl–enzyme complex to form a new galactoside and the free
1
7–19
induction of T-cell apoptosis allowing for immune response eva-
enzyme (Scheme 1).
Some features that should be considered
sion and cell migration which may facilitate metastasis.7
–12
Galec-
in transglycosylation systems are that: (i) the new galactoside can
be substrate of the enzyme and so prone to hydrolysis; (ii) hydro-
lysis of lactose may occur and can be present as a secondary reac-
tins are molecular targets for cancer therapy and therefore galectin
inhibitors are potential antitumor agents.4 Thus, the generation
and availability of novel low molecular weight galactosides en-
ables a more comprehensive examination of the antitumor proper-
ties of these compounds.
–7
1
3,14,16
tion.
These can lead to a decrease in synthesis yields and so
transglycosylation becomes a kinetically controlled process. Nev-
ertheless, if parameters such as donor and acceptor concentration
as well as temperature and reaction time are optimized, successful
results can be obtained using transglycosylation for galactoside
⇑
Corresponding author. Tel.: +598 2 924 18 06; fax: +598 2 924 19 06.
E-mail addresses: dcpg@fq.edu.uy (C. Porciúncula González), eacastilla@gmail.
2
0–22
synthesis.
and structural characterization of a new galactoside, 2-aminoethyl
b- -galactopyranoside.
In this study we propose the enzymatic synthesis
com (A. Castilla), lgarofalo@higiene.edu.uy (L. Garófalo), ssoule@fq.edu.uy (S.
Soule), mgidrv@fq.edu.uy (G. Irazoqui), cgiacomi@fq.edu.uy (C. Giacomini).
D
0
008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.12.009

C. Porciúncula González et al. / Carbohydrate Research 368 (2013) 104–110
105
OH
H
OH
OH
A
H
O
H
HO
O
HO
OH
H
H
OH
OH
H
HO
H
OH
H
H
O
Glucose
H
H
OH
H
OH
Galactose
H
H
H
O
OH
H
H
HO
OH
HO
O
β-Gal
+
H
OH
O
H
H
H
OH
OH
H
OH
OH
H
Lactose
O
2
H N
H
HO
O
B
H
OH
NH2
OH
OH
O
O
H
H
OH
-O
OH
O
H
H
O
1-(2-aminoethyl)-β-D-galactopyranoside
H
H
O
+
R
O
H
HO
OR
H
H
OH
+
HO
H
OH
H
H
-Gal
β
H
O-
H
O
O
-
O
O
H
OH
OH
H
O
H
-O
HO
OH
O
OH
O
H
O
OH
OH
H
OH
OH
R
1
H
H
H
O
H
O
+
O
H
H
H
HO
OR
1
HO
Galactose
H
H
OH
OH
H
H
H
+
O-
O
O-
OH
2
H N
2- Aminoethanol
1
R = Glucose or 2-Aminoethanol; R = H or 2-Aminoethanol
Scheme 1. (A) Transglycosylation mechanism for b-galactosidase using lactose as donor and 2-aminoethanol as acceptor in an aqueous medium. (B) Catalytic mechanism for
retaining glycosidases
2
. Results and discussion
course of the reaction was followed by HPLC. Figure 1 shows a
chromatographic profile of the reaction mixture after 30 min at
50 °C. As expected, four peaks were observed corresponding to
the reaction products and un-reacted substrates. The first three
Synthesis of 2-aminoethyl b-D-galactopyranoside was achieved
by transgalactosylation of lactose catalyzed by Aspergillus oryzae b-
galactosidase (EC 3.2.1.23) using 2-aminoethanol as acceptor. The
peaks were assigned to 2-aminoethanol (t
galactose which co-elute at 7.8 min, and lactose (t
R
4.6 min), glucose and
11.5 min),
R
according to retention times of the corresponding standards. The
fourth peak had a retention time of 17.4 min and was provisionally
assigned to 2-aminoethyl b-D-galactopyranoside. This assignment
was later confirmed by structural analysis of purified material as
described in Section 2.4.
2
.1. Influence of reaction conditions
As noted above, transglycosylation is a kinetically controlled
process and successful galactoside synthesis depends on several
parameters such as donor and acceptor concentration, tempera-
2
3–25
ture, and reaction time.
described below.
Optimization of these parameters is
2
.1.1. Influence of acceptor concentration
-Aminoethyl b- -galactopyranoside synthesis was assayed at
different concentrations of 2-aminoethanol (0.5, 1.0, 2.0 M) and
.35 M lactose. As shown in Figure 2 maximum synthesis yield
49% corresponding to 0.17 M of the galactoside) was achieved at
0 min with 0.5 M 2-aminoethanol. Further galactoside hydrolysis
until complete disappearance upon 24 h was observed. This dem-
onstrates that 2-aminoethyl b- -galactopyranoside was substrate
2
D
0
(
3
Figure 1. HPLC chromatogram of 2-aminoethyl b-
profile with 0.2 M lactose, 0.5 M 2-aminoethanol, 6 U/mL b-galactosidase after
0 min at 50 °C.
D-galactopyranoside synthesis
3
D

1
06
C. Porciúncula González et al. / Carbohydrate Research 368 (2013) 104–110
Figure 2. Influence of 2-aminoethanol concentration on 2-aminoethyl b-
lactose, 6 U/mL soluble b-galactosidase at 50 °C.
D
-galactopyranoside synthesis yields.
2.0 M,
1.0 M and
0.5 M 2-aminoethanol with 0.35 M
of the enzyme, which turns the reaction time into a key parameter
for the enhancement of the reaction yields. When the concentra-
tion of 2-aminoethanol was increased to 2.0 M, rates of both, syn-
thesis and galactoside hydrolysis were slower (Fig. 2). Under these
conditions maximum synthesis yield was 30% after 2 h, and a de-
crease in lactose conversion rate was also observed. These results
indicated that high 2-aminoethanol concentrations had an overall
inhibitory effect. The lower activities could not be attributed to en-
zyme denaturation since b-galactosidase retained over 80% activity
after 6 h at 50 °C in 2.0 M 2-aminoethanol (data not shown). Inhi-
bition of transglycosylation by high acceptor concentrations was
mechanism predicts binding of the inhibitor both to the free en-
zyme giving an EI complex with a dissociation constant Kic, and
to the galactosyl–enzyme complex giving an E–galactose–I com-
plex with a dissociation constant Kiu. The Kic and Kiu determined
were 0.310 ± 0.040 M and 0.604 ± 0.035 M, respectively. These
inhibition constant values are consistent with the decrease of both
2-aminoethyl b-D-galactopyranoside synthesis and hydrolysis rate
for 2-aminoethanol concentrations higher than 1.0 M previously
observed.
2.3. Effect of enzyme immobilization on transglycosylation
1
8,22
previously reported,
and experiments were done to fully de-
scribe the type of inhibition observed (see Section 2.2).
The advantages of solid-phase enzyme immobilization are
widely acknowledged. Three clear improvements over solution
phase synthesis are that enzyme stability is typically increased,
the enzyme can be easily separated from other reaction compo-
2
.1.2. Influence of donor concentration
Increased lactose concentrations can enhance the amount of
2
7,28
galactoside since it is the limiting reactant, and also because lac-
tose competes with the galactoside for the enzyme, leading to a
reduction in the rate of galactoside hydrolysis. High lactose con-
centrations, on the other hand, increase solution viscosity and
residual lactose can remain under optimal reaction conditions.
The presence of high residual lactose concentrations compromises
galactoside purification from the reaction mix. Thus optimization
of the donor concentration was required.
nents and the immobilized enzyme can be recycled.
In our
study b-galactosidase was immobilized onto glutaraldehyde-aga-
rose. This immobilization strategy involves the reaction of primary
amine groups on the enzyme surface with aldehyde groups on the
support to produce Schiff bases, which are then reduced to alkyl
2
9,30
amine covalent bonds.
The performance of soluble versus
-galactopyranoside syn-
immobilized enzyme for 2-aminoethyl b-
D
thesis, with 0.2 M lactose, 0.5 M 2-aminoethanol and 6 U/mL en-
zyme concentration at 50 °C, was analyzed. Both, yield and
synthesis rate, were lower using immobilized enzyme (Fig. 5). It
is noteworthy that in the immobilized enzyme system, galactoside
hydrolysis was also lower and galactoside concentration remained
unchanged after 6 h. These observations could be attributed to re-
stricted diffusion of substrates to the immobilized enzyme.3
Immobilized enzyme could be reused at least nine times as de-
scribed in the experimental Section 4.7. The average yield of galac-
toside product using immobilized enzyme was 0.56 M (27%
synthesis yield). After nine cycles of synthesis the immobilized en-
zyme retained 50% of its initial activity, demonstrating the feasibil-
ity of its use for synthetic purposes.
The effect of different lactose concentrations (0.07, 0.20, and
0
.35 M) for production of 2-aminoethyl b-
was evaluated using 0.5 M 2-aminoethanol. Maximum concentra-
tion of 2-aminoethyl b- -galactopyranoside in the reaction mixture
0.17 M) was achieved at 30 min with 0.35 M lactose (Fig. 3). The
D-galactopyranoside
D
(
1,32
amount of lactose remaining at this maximum level of galactoside
concentration was very high and hindered the purification process.
So, subsequent experiments were done with somewhat lower level
of lactose (0.2 M).
2
.2. Inhibition constants determination
Enzyme inhibition by the acceptor was analyzed using o-Nitro-
phenyl b-
D-galactopyranoside (ONPG) as the galactosyl-donor in
2.4. Structural characterization of 2-aminoethyl
the presence of different concentrations of 2-aminoethanol. Re-
sults were plotted as follows: (i) the reciprocal of the initial veloc-
ity versus 2-aminoethanol concentration and (ii) the ratio between
ONPG concentration and the initial velocity versus 2-aminoethanol
concentration. The profiles shown in Figure 4 were compatible
with those described for mixed inhibition.26 This inhibition
b-
D
-galactopyranoside
The reaction mixture obtained under optimal conditions (0.2 M
lactose, 0.5 M 2-aminoethanol, 6 U/mL soluble enzyme, for 30 min
at 50 °C) was fractionated by gel filtration. Fractions collected
were analyzed by HPLC for carbohydrate composition. Fractions

C. Porciúncula González et al. / Carbohydrate Research 368 (2013) 104–110
107
Figure 3. Effect of lactose concentration on the 2-aminoethyl b-
U/mL soluble b-galactosidase at 50 °C.
D-galactopyranoside synthesis yields, 0.07 M, 0.20 M, and 0.35 M lactose with 0.5 M 2-aminoethanol,
6
Figure 4. Reciprocal plots for 2-aminoethanol inhibition constant determination considering a mixed inhibition model. Inhibition constants were determined using ONPG as
galactose donor (2.5–15 mM) in activity buffer, and 2-aminoethanol (0.10–0.45 M) at 25 °C.
containing pure 2-aminoethyl b-
D
-galactopyranoside (t
R
17.4)
between the galactosyl moiety of the donor molecule and a hydro-
1
4
were pooled, lyophilized, and purity checked by HPLC (Fig. 6).
The purified fraction was subsequently analyzed by thin layer
chromatography (TLC), NMR spectroscopy, and GC–MS
spectrometry.
Based on the enzymatic mechanism reported for b-galactosi-
dases it can be predicted that the new galactoside formed was
xyl group of the acceptor molecule (Scheme 1B).
The purified fraction was analyzed by TLC, and revealed a spot
with a lower R (0.06) than hydrolysis products (galactose and glu-
f
cose) or lactose (Table 1). This suggests the presence of a new com-
pound with higher polarity. In addition, the spot corresponding to
the new compound reacted strongly with ninhydrin, confirming
3
3
2
-aminoethyl b-
D
-galactopyranoside. b-galactosidases are retain-
the presence of a nitrogenous compound different from 2-amino-
ethanol which had an R of 0.9.
ing glycosidases that catalyze the formation of O-glycosidic bonds
f

1
08
C. Porciúncula González et al. / Carbohydrate Research 368 (2013) 104–110
Figure 5. Effect of enzyme immobilization on galactoside synthesis yield.
aminoethanol, and 6 U/mL of soluble or immobilized enzyme at 50 °C.
Soluble enzyme;
immobilized enzyme. Synthesis was done using 0.2 M lactose, 0.5 M 2-
GC–MS analysis of pentaacetate pyranose compounds termed ser-
3
4
ies A fragments. In addition, peaks corresponding to the series A
fragments were found at signals m/z: 211 (A2); 169 (A3); and 109
(
A4). Another interesting fragment was m/z 288, which is typically
present in the spectra of b- -galactopyranose acetates due to the
D
proximity of the 4- and 6-acetoxy groups in cis. The presence of
other ions at m/z 432 and m/z 389 indicated the loss of two acetyl
groups, which is consistent with fragmentation at
a-carbonyl
groups.
1
The H NMR spectra of the sample provided information on the
3
5
1
anomeric configuration of the sugar residue. The H NMR spec-
trum of 2-aminoethyl b- -galactopyranoside in D O at 30 °C
showed a signal corresponding to one anomeric proton at d 4.89
J = 8 Hz), indicating the presence of a monosaccharide moiety.
D
2
H
(
The value of the vicinal coupling constant between the anomeric
H1 and H2 indicates the axial configuration generally present in
pyranose structures and supports evidence indicating the presence
3
5
Figure 6. HPLC chromatogram of purified 2-aminoethyl b-
D
-galactopyranoside.
of a b-anomer. The signal at d
of methylene protons coupling with a primary amine group.
These results were consistent with both TLC and GC–MS analysis
H
2.85 ppm suggested the presence
36,37
Table 1
and indicated that a novel, galactoside, 2-aminoethyl b-D-galacto-
pyranoside, was produced by enzymatic synthesis.
f
R values for standards (lactose, glucose, galactose, and 2-aminoethanol) and the new
galactoside (2-aminoethyl b-D-galactopyranoside)
Compound
R
f
3
. Conclusions
Lactose
Glucose
Galactose
0.15
0.29
0.20
0.90
0.06
A new low molecular weight galactoside was synthesized by
transgalactosylation of lactose with 2-aminoethanol as acceptor.
The product was purified and identified as 2-aminoethyl b- -galac-
2
2
-Aminoethanol
-Aminoethyl b-
D
-galactopyranoside
D
topyranoside. Optimal reaction conditions for transgalactosylation
and purification were: 0.2 M lactose, 0.5 M 2-aminoethanol, 6 U/
mL (soluble enzyme) for 30 min at 50 °C. 2-Aminoethanol proved
to be an enzyme inhibitor, fitting a mixed inhibition model with
The TLC was done in Silica-Gel TLC plates, using methanol:chloroform:ace-
tone:ammonium hydroxide (42:17:25:17) as mobile phase and developed with
orsinol and ninhydrin as described in Section 4.9.2.
Kic = 0.310 M and Kiu = 0.604 M.
The purified fraction was peracetylated for analysis by GC–MS
spectrometry.
4. Experimental
4.1. Materials
The following m/z ions were detected in the fraction containing
the peracetylated 2-aminoethyl b-D-galactopyranoside: 475, 432,
3
89, 331, 288, 211, 169, and 109. As expected the molecular ion
+
M (m/z 475) corresponding to the highly substituted 2-aminoethyl
b- -galactopyranoside, was of very low intensity. Other peaks in
the higher mass range were due to the loss of acetyl groups and
were m/z 432 (M-CH CO), m/z 389 (M-2 CH CO), and m/z 331
M-OC N(CH CO) ). The presence of the ion at m/z 331 (A1)
likely represents one of the fragments usually obtained by
o-Nitrophenyl b-
D
-galactopyranoside (ONPG), lactose, galactose,
D
2-aminoethanol, and b-galactosidase (b-D-galactoside galactohy-
drolase; EC 3.2.1.23) from A. oryzae, were purchased from Sigma–
Aldrich (St. Louis, MO). Chloroform, orcinol and ninhydrin were
from Merck (USA). TLC plates were from Machery Nagel (Duren,
Germany). All other chemicals used were of analytical grade.
3
3
(
2
H
4
3
2

C. Porciúncula González et al. / Carbohydrate Research 368 (2013) 104–110
109
4
.2. Enzymatic activity
Aliquots of 100 L of diluted A. oryzae b-galactosidase solution
4.7. Re-use of immobilized enzyme
l
Ninety mg of immobilized b-galactosidase were incubated with
8.5 mL 0.5 M 2-aminoethanol, 0.2 M lactose at 50 °C for 3 h, under
continuous stirring. The enzyme derivative was separated from the
reaction mixture by filtration, washed with activity buffer, and
stored at 4 °C until the next use. The filtrates obtained were ana-
lyzed for carbohydrate as described below in Section 4.9.
were added to 2.0 mL of 25 mM ONPG in 50 mM sodium acetate
buffer at pH 5.5 (activity buffer), at room temperature. The rate
of formation of free o-nitrophenol (ONP) was recorded spectropho-
tometrically at 405 nm using a 1 cm path length. One enzyme unit
(
U) was defined as the amount of enzyme hydrolyzing 1
lmol of
substrate per minute in the above defined conditions. The extinc-
2
À1
À1
tion coefficient used for ONP at pH 5.5 was 7.5 Â 10 M cm
.
4.8. Galactoside-purification
Enzyme activity was expressed as U/mL.
Immobilized enzyme activity was measured under identical
conditions by incubating appropriate aliquots of a gel suspension
with the substrate solution described above. Enzyme activity was
expressed as U/g suctioned dried gel.
2-Aminoethyl b-D-galactopyranoside was purified using size
exclusion chromatography in a 1.6 cm id  70 cm Sephadex G10
column and eluted with deionized water at a flow rate of
À1
0.06 mL/min (1.8 cm h linear flow rate).
4
.3. Protein assay
4.9. Carbohydrate analysis
Protein was determined by the bicinchoninic acid (BCA)
assay.
4.9.1. HPLC analysis
3
1,38
Galactoside synthesis was monitored by HPLC, Waters High-
Performance Liquid Chromatography system, using a Shodex
Asahiapak NH2P50 4E, 4.6 mm i.d. Â 250 mm length column
equipped with refractive index detector. Compounds were sepa-
rated under isocratic conditions with acetonitrile/water (75:25 v/
v) at a flow rate of 1 mL/min at 25 °C. Previously purified and char-
4
4
.4. Enzyme immobilization
.4.1. Synthesis of glutaraldehyde-agarose
Glutaraldehyde-agarose containing 90
of suction-dried gel was prepared as described previously by
l
mol glutaraldehyde/g
acterized 2-aminoethyl b-D-galactopyranoside was used as
standard.
2
9
Guisan et al.
4
.4.2. Immobilization of b-galactosidase onto glutaraldehyde-
agarose
Two grams of suction dried glutaraldehyde-agarose was incu-
4.9.2. TLC analysis
TLC was performed on Silica Gel TLC plates. Samples and stan-
dards (glucose, galactose, lactose, and 2-aminoethanol) were spot-
ted onto the TLC plates and developed to 10 cm in an
11.0 cm  11.0 cm  6.2 cm chamber (saturation time 30 min),
bated with 20.6 mL of A. oryzae b-glactosidase solution (147 UE/
mL; 2.8 mg protein/mL) in 0.1 M sodium phosphate buffer at pH
7
.0 (immobilization buffer). The suspension was gently stirred
3 2 4
using MeOH:CHCl :(CH3) CO:NH OH (42:17:25:17) as mobile
overnight at room temperature, washed by sintered glass filtration
with the immobilization buffer, and then equilibrated in 40 mM
sodium carbonate buffer, pH 10.0. The immobilized enzyme was
suspended in 20 mL of 1 mg/mL sodium borohydride solution in
phase. The TLC plates were air-dried, sprayed with orcinol 0.2%
(w/v) in EtOH:H SO (90:10), then heated 2 min at 110 °C, and
2
4
3
9
sprayed with ninhydrin spray reagent.
4
0 mM sodium carbonate buffer pH 10.0, and the mixture was
4.9.3. Acetylation analysis
gently stirred for 30 min at room temperature, washed with activ-
ity buffer, and stored at 4 °C.
Purified sample material was acetylated by addition of 0.2 mL
methylimidazole, followed by 2 mL acetic anhydride and mixed
for 10 min at room temperature. Five milliliter water was added
to decompose the excess of acetic anhydride. After cooling, 1 mL
AcOEt was added and the mixture was agitated on a vortex mixer.
After separation of phases, the upper phase was removed with a
4
.5. Enzymatic synthesis of 2-aminoethyl b-D-galactopyranoside
Ten milliliter reaction mixtures containing lactose (0.07–
.35 M) and 2-aminoethanol (0.5–2.0 M) in activity buffer were
0
2 4
Pasteur pipette, dried with Na SO , and stored in a 1 mL septum-
3
9
supplemented with soluble or immobilized A. oryzae b-galactosi-
dase to a final concentration of 6 U/mL. The reaction mixtures were
incubated at 50 °C and gently stirred. Aliquots of the mixtures were
withdrawn at regular intervals and the reaction was stopped by
incubation at 100 °C for 5 min for the soluble enzyme or by filtra-
tion for the immobilized enzyme. The samples were analyzed for
carbohydrates as described below in Section 4.9. Synthesis yield
was defined as the amount of galactoside obtained expressed as
a percentage of the initial amount of lactose.
cap vial at À20 °C until analysis.
4.9.4. GC–MS analysis
Peracetylated derivatives of purified samples were analyzed
using an HP 5890 Series II GC with an MD 5971 quadrupole mass
detector, EI mode at 70 eV, He carrier (Hewlett-Packard, Palo Alto,
CA, USA). The column was a HP 5 (crosslinking 5% PH Me siloxane),
30 m  0.25 mm i.d., 0.25 mm film thickness. Injector and detector
temperatures were 300 °C and oven temperature was maintained
at 150 °C for 5 min, then increased to 250 °C at 5°/min (held
2 min) then increased to 350 °C at 5°/min and held for 2 min. Injec-
tions were made in the split mode, with a split flow of 40 mL/min.
Chromatographic peaks were analyzed using HPChem Station soft-
ware (Hewlett-Packard, Palo Alto, CA).
4
.6. Inhibition constant determinations
Inhibition constants were determined using ONPG as galactose
donor (2.5–15.0 mM) and 2-aminoethanol (100–450 mM) in activ-
ity buffer at 25 °C. The rate of ONP formation was determined as
previously described in Section 4.2. Inhibition constants were cal-
4.9.5. NMR experiments
1
culated by plotting 1/v versus 2-aminoethanol concentration (Kic
and the ratio of ONPG concentration and initial velocity versus 2-
aminoethanol concentration (Kiu as described by Cornish-
)
The H spectrum was obtained at 400 MHz, at 30 °C on a Brüker
2
Avance DPX 400 NMR instrument with D O as solvent. Chemical
shifts were reported in ppm, using TMS (d 0.000) as internal
H
reference.
)
2
6
Bowden.

1
10
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2
2
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Ciencias Básicas’ (PEDECIBA) and Project I+D 408 funded by ‘Com-
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