Enzymatic properties and transglycosylation of α-galactosidase from Penicillium oxalicum so

Article abstract of DOI:10.1016/j.foodchem.2010.10.095

Penicillium oxalicum SO α-galactosidase demonstrated weak hydrolysing activity but a high rate of transglycosylation in the reaction with melibiose, where the major product was 6-α-galactosyl melibiose. The transfer ratio was 83.6% and was maintained over

Full text of DOI:10.1016/j.foodchem.2010.10.095

Food Chemistry 126 (2011) 177–182
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Enzymatic properties and transglycosylation of a-galactosidase
from Penicillium oxalicum SO
⇑
Masahiro Kurakake , Youichirou Moriyama, Riku Sunouchi, Shinya Nakatani
Department of Life and Nutritional Science, Fukuyama University, 1-985 Sanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 April 2010
Received in revised form 9 September 2010
Accepted 22 October 2010
Penicillium oxalicum SO
glycosylation in the reaction with melibiose, where the major product was 6-
a
-galactosidase demonstrated weak hydrolysing activity but a high rate of trans-
-galactosyl melibiose. The
a
transfer ratio was 83.6% and was maintained over a long reaction time of 80 h. The molecular weight was
estimated to be 124,000 by SDS–PAGE. The optimal pH was ꢀ3 and a stable pH, with a range of 2.4–9.5,
was found. The optimal temperature was ꢀ60 °C and the activity was stable below 60 °C. With respect to
acceptor specificity, mono-alcohols, sugar alcohols and sugars were poor acceptors, but the di-alcohol
ethylene glycol and the tri-alcohol glycerin were good acceptors. The percentage of transglycosylation
to glycerin increased up to 41.7%, as that to melibiose decreased, with the initial glycerin concentration
Keywords:
a-Galactosidase
Melibiose
Oligosaccharides
Glycerin
of 40%. The production of
enzymatic reaction.
a
-D
-galactosylglycerol was 293 mg for each gram of melibiose used by the
Ó 2010 Elsevier Ltd. All rights reserved.
Transglycosylation Penicillium oxalicum
1. Introduction
Poulos & Beckman, 1978). For example, Fabry’s disease is caused
by a lack of
a-galactosidase in the body, leading to a buildup of
a
-Galactosidase (EC 3.2.1.22) is widely distributed in microor-
ganisms and plants, and its enzymaticproperties have beenreported
(Dey & Campillo, 1984). -Galactosidase hydrolyses the -1,6-link-
trihexosylceramide with terminal a
-galactosyl residue in the
blood vessels, impairing their ability to function. Trihexosylcera-
mide is also a cell-surface ligand of verotoxin that is produced
by Escherichia coli (VTEC). Hence, the physiological function of
a
a
age of galactosyl residues on saccharides, such as melibiose and raf-
finose. The non-reducing end of the galactosyl residue is recognised
and released in the active site of the enzyme. On application of the
enzyme, improvements in the rheological properties of guar gum
(Cyamopsis tetragonolobus), used as a natural food additive, have
the
field.
Transglycosylation has been performed using either melibiose
or pNP- -galactopyranoside as the donor, resulting in the syn-
a-galactosyl residue is a significant target in the medical
a-D
been noted. Guar galactomannan consists of a b-1,4-linked
nose backbone and -1,6-linked -galactose side chains (galactose
38%, mannose 62%). Some -galactosidases are able to partially re-
lease galactosyl residues from guar gum, improving its rheological
properties such that it behaves similar to that of the more expensive
locust bean gum (Ceratonia siliqua) (Bupin, Gidley, & Jeffcoat, 1990;
McCleary, Amado, Waibel, & Neukom, 1981). In the production of
D
-man-
thesis of various oligosaccharides and alkyl galactosides (Hashim-
oto, Katayama, Goto, Okinaga, & Kitahata, 1995; Hinz et al., 2005;
Spangenberg et al., 2000; Van Laere et al., 1999). In particular,
the enzyme from Candida guilliermondrii has demonstrated broad
acceptor specificity to monosaccharides, disaccharides and alco-
hols (Hashimoto et al., 1995). In gene technology, the development
of recombinant enzymes, to improve the transglycosylation activ-
ity, has been studied (Mi et al., 2007).
a
D
a
beet sugar,
a-galactosidase is used to release galactosyl residues
from raffinose, which prevents sucrose crystallisation during the
industrial process (Yamane, 1971).
A guar gum-hydrolysing strain, Penicillium oxalicum SO, was
separated from soil using a medium plate with 0.5% guar gum
and 1.5% agar. This strain produced mainly b-mannanase with high
hydrolysing activity that rapidly hydrolysed both guar gum and lo-
cust bean gum (Kurakake, Sumida, Masuda, Oonishi, & Komaki,
Some
a-galactosidases have a transglycosylation action, hence
syntheses of new products, with
a-1,6 linkages, have been stud-
ied. These products are expected to have medical applications
since the galactosyl residues of biomolecules often have impor-
tant roles in organisms (Arya et al., 1999; Coetzee et al., 1996;
2006). It was found that
a-galactosidases of the minor product
had low hydrolysing activity, but showed high transglycosylation
in the reaction with melibiose. In the present study, we investi-
gated the enzymatic properties of
tor specificity, in the transglycosylation.
a-galactosidase, such as accep-
⇑
Corresponding author. Tel.: +81 084 936 2111; fax: +81 084 936 2023.
E-mail address: kurakake@fubac.fukuyama-u.ac.jp (M. Kurakake).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.10.095

178
M. Kurakake et al. / Food Chemistry 126 (2011) 177–182
tions of 8% melibiose, 8% acceptor and 0.032 U/ml enzyme, and
2. Materials and methods
incubated at pH 5 and 40 °C for 24 h. The reaction mixture was
deactivated by boiling and the resulting sugars were analysed as
described above. The acceptors used were alcohols (methanol,
ethanol, 1-propanol, 2-propanol, n-butanol, 3-methyl-1-butanol,
ethylene glycol (1,2-ethanediol), and glycerin), sugar alcohols
(erythritol, xylitol, myo-inositol, sorbitol, mannitol, and lactitol),
2.1. Materials
Guar gum and locust bean gum were purchased from Sigma–
Aldrich (St. Louis, MO, USA). The sugar compositions were 38%
galactose/62% mannose and 23% galactose/77% mannose, respec-
tively. Melibiose, raffinose and stachyose were purchased from
Wako Pure Chemical Industries Limited (Osaka, Japan). All other
chemicals were of reagent grade.
sugars
-mannose,
palatinose, trehalose, isomalto-oligosaccharides, and
trin) and other agents (hesperidin, -ascorbic acid, -tyrosine, and
-serine). The transglycosylation to the acceptor was evaluated
by the peak area of the transferred product shown on the HPLC
chromatogram, whereby ++++ denoted >150,000 ( Vs), +++ de-
(L
-arabinose,
D
-xylose,
D
-ribose,
-sorbose, N-acetylglucosamine,
-cyclodex-
D-glucose, D-galactose,
D
D-tagatose,
D
-fructose,
L
a
L
L
L
2.2. Production and purification of enzymes
l
P. oxalicum SO was cultivated in liquid medium containing 1%
guar gum, 0.5% yeast extract and 0.2% Na2HPO412H2O (100 ml)
in a 500 ml flask at 150 rpm and 30 °C for 4 days. The spores of
the SO strain were inoculated. The inoculum was grown in a plate
medium (0.5% guar gum and 1.5% agar). The supernatant of the cul-
ture broth, after centrifugation at 1500g for 10 min, was used to
determine enzyme activity. The sediment strain was washed with
distilled water, after centrifugation, and homogenised. Enzyme
activity of the strain suspension was determined as either intracel-
lular or binding enzyme.
The culture filtrate (360 ml) was desalted by saturation in 80%
ammonium sulphate. After filtration, the precipitate was dissolved in
100 mM sodium acetate buffer (pH 5). Cold acetone was added to the
enzyme solution (3 ꢁ volume). After incubation at ꢂ20 °C for 30 min,
precipitated proteins were separated by centrifugation at 1500g for
10 min. The precipitate was dissolved in 100 mM sodium acetate buf-
noted 120,000–150,000, ++ denoted 50,000–120,000, + denoted
<50,000, and – denoted no peak.
2.6. HPLC analysis of sugars
The sugars formed by the enzymatic reaction were analysed by
HPLC under the following conditions (Kurakake et al., 2006): (1)
column, 250 ꢁ 7 mm i.d. GL-C610 (Hitachi Kasei Limited, Tokyo,
Japan); mobile phase, water; column temperature, 60 °C; flow rate,
1.0 ml/min; and detector, L-3300 differential refractive index mon-
itor (Hitachi High-Technologies Limited, Tokyo, Japan); or (2) col-
umn, 250 ꢁ 4.6 mm i.d. NH2P-50 (Asahi Kasei Co. Limited, Tokyo,
Japan); mobile phase, water:acetonitrile = 25:75; column tempera-
ture, 25 °C; and detector, RI 8020 differential refractive index
monitor (Tosoh Limited, Tokyo. Japan).
fer (pH 5). The crude a-galactosidase was purified by the following
chromatography step. The crude enzyme solution (0.5 ml) was sub-
jected to gel filtration on preparative high-performance liquid chroma-
tography (HPLC), with a 60 ꢁ 2.15 cm i.d. TSKgel G3000SW column
(Tosoh Limited, Tokyo, Japan.), pre-equilibrated with 50 mM phos-
phate buffer (pH 6.8) containing 0.3 M NaCl. Proteins were eluted at
a flow rate of 1.6 ml/min and fractions were collected at 1 min inter-
vals. Proteins were detected by their absorbance at 280 nm in a UV
detector (SPD-7A, Shimadzu Co. Limited, Kyoto, Japan). Each purifica-
tion step was carried out at 4 °C.
2.7. Identification of transfer product
Transfer products were separated by HPLC (column,
250 ꢁ 10 mm i.d. NH2P-50; mobile phase, water:acetoni-
trile = 20:80 or 25:75) and identified by 13C NMR analysis. 13C
NMR spectra were taken on a JEOL JMN-LA 500 (500 MHz for 1H
and 125 MHz for 13C) spectrometer (JEOL Limited, Tokyo, Japan).
Each sample (1%) was dissolved in deuterium oxide and the chem-
ical shifts were calibrated using the signal acetone as an internal
standard.
2.3. Determination of enzyme activity
3. Results and discussion
p-Nitrophenyl
a-D-galactopyranoside (1 mM) was incubated
with an enzyme sample in 0.1 M acetate buffer (pH 5) at 40 °C
for 10 min (working volume of 1 ml). The reaction was stopped
by adding 0.5 ml of 1 M Na2CO3 and the absorbance of the released
p-nitrophenol at 400 nm determined. One unit was defined as the
3.1. Purification and properties of SO a-galactosidase
When used in a liquid culture in a 500-ml flask for 4 days, P.
oxalicum SO produced a maximum of 0.038 U/ml -galactosidase.
a
amount of enzyme that could produce 1 lmol p-nitrophenol/min.
The enzyme was either intracellular or was bound to the cell wall
at an early period, but was released from the cell during culture;
40% of the enzyme was released in 4 days (0.015 U/ml; superna-
tant). When pNP-galactopyranoside was used as the substrate,
2.4. Enzymatic reaction for melibiose
One gram of melibiose was dissolved with 0.1 M acetate buffer
(pH 5) and an enzyme solution (2 ml) added. The mixture (8.3%
melibiose and 0.027 U/ml enzyme) was incubated at pH 5 and
40 °C for 24–80 h. The aliquot was sampled at the given time and
the enzyme deactivated by heating in boiling water for 5 min. After
centrifugation at 1500g for 10 min, the supernatant was filtered
the activity of
a-galactosidase was very low. When melibiose
was used as the substrate; however, the enzyme demonstrated
high transglycosylation.
A crude enzyme solution was prepared by desalting in a satu-
rated 80% ammonium sulphate solution, followed by precipitation
in cold acetone and gel filtration on preparative HPLC with a TSKgel
G3000SW column (data not shown). Eluted proteins were detected
by their absorbance at 280 nm. The first small peak corresponded
using a 0.22-lm membrane filter and analysed by HPLC. The trans-
fer ratio [(Glc ꢂ Gal)/Glc ꢁ 100(%)] was calculated from the con-
centrations of galactose (Gal) and glucose (Glc).
to
a-galactosidase activity. On SDS–PAGE, only one band was de-
tected and the molecular weight was estimated to be 124,000
2.5. Acceptor specificity
(Fig. 1). Fig. 2 shows the effects of pH and temperature on the
activity of purified a-galactosidase. The optimal pH, at 40 °C, was
Twenty percent of melibiose (40
ll), 20% of acceptor (40
ll) and
found to be approximately 3 and the enzyme was stable over a
0.16 U/ml of enzyme (20 l) were mixed, giving final concentra-
l
wide pH range (2.4–9.5), as shown in Fig. 2A. The optimal

M. Kurakake et al. / Food Chemistry 126 (2011) 177–182
179
Table 1
Substrate specificity of SO
a-galactosidase.
Substrate
Relative activity (%)
116,000
66,000
Methyl-
pNP-
pNP-b-
a
D
-
D
-galactopyranoside
-galactopyranoside
-galactopyranoside
0
18
0
100
0
a
-
D
Melibiose
Lactose
Raffinose
105
112
0
Stachyose
Guar gum
Locust bean gum
45,000
29,000
0
ucts. Nuclear magnetic resonance analysis (NMR) showed that
the trisaccharide resulted from the formation of an -1,6 linkage
a
between the galactosyl residue and the C6 position of the galacto-
syl residue of the melibiose molecule. This self-transglycosylation
Fig. 1. Analysis of SO a-galactosidase on SDS–PAGE.
to melibiose has also been shown by the
a-galactosidases of
C. guilliermondrii, Bifidobacterium adolescentis, and Lactobacillus
reuteri (Hashimoto et al., 1995; Tzortzis, Jay, Baillon, Gibson, &
Rastall, 2003; Van Laere et al., 1999). As the reaction progressed,
a small amount of tetrasaccharide was formed by transglycosyla-
tion to the trisaccharide.
Table 2 shows the sugar composition over a long reaction time
(33, 56, and 80 h). In the 80-h reaction, the transglycosylation
reached an equilibrium with an oligosaccharide composition of
46.8% (34.4% trisaccharide and 12.4% tetrasaccharide). The small
decrease in the transfer ratio, in comparison with that at 33 h, is
attributable to the hydrolysis of the tetrasaccharide to galactose
and trisaccharide. The transfer ratio was maintained at a high level
temperature was approximately 60 °C at pH 5, and the activity
was stable at temperatures of up to 60 °C (Fig. 2B). There are
few studies of
a-galactosidase from Penicillium. The molecular
weight and enzymatic properties of Penicillium purpurogenum
and simplicissimum enzymes were reported to be different from
those of P. oxalicum SO enzyme (Luonteri, Alatalo, Siika-Aho,
Penttila, & Tenkanen, 1998; Shibuya et al., 1995).
Substrate specificity is shown in Table 1, with the enzymatic
activity presented as a value relative to melibiose. The enzymes
ability to recognise an
hydrolysis of both pNP-b-
activity towards melibiose was much higher than that with pNP-
-galactopyranoside, and the relative activity increased with
a-1,6 linkage was shown by the lack of
D
-galactopyranoside and lactose. Enzyme
of 83.6% at 80 h. As has been reported for
a-galactosidases from
a-
other origins, the hydrolysis reaction of oligosaccharides proceeds
gradually, therefore, a long reaction time will not necessarily in-
crease the transfer ratio (Hashimoto et al., 1995; Van Laere et al.,
D
molecular size (raffinose and stachyose). This specificity differs
from that of the enzymes from Bacillus stearothermophilus, Tricho-
derma reesei, and Aspergillus fumigatus, which have high activity
1999). SO
a-galactosidase has the unique characteristic of low
hydrolysing activity.
to pNP-
Kulminskaya, Savel’ev, Shishlyannikov,
Spangenberg et al., 2000). The SO enzyme has no activity with
the -1,6 galactosyl side chains in the galactomannan of either
guar gum or locust bean gum.
a
-
D
-galactopyranoside (Puchart & Biely, 2005; Shabalin,
The relationship between transglycosylation and the melibiose
concentration was investigated. Fig. 4 shows the transglycosyla-
tion reaction where the melibiose concentration ranged from
0.4% to 8%. The concentration of glucose released increased with
substrate concentration. The concentration of galactose released,
however, was relatively constant. The transfer ratio showed that
transglycosylation was efficiently enhanced at >1% melibiose. In
general, transglycosylation by glycosidase (hydrolase) can be car-
ried out with a high substrate concentration, for example a concen-
tration of 10%. The SO enzyme also has high transferring activity at
relatively low substrate concentrations. It would be expected,
&
Neustroev, 2002;
a
3.2. Transgalactosylation of SO a-galactosidase
Fig. 3 shows the chromatogram of the reaction mixture in 8.3%
melibiose and 0.027 U/ml -galactosidase. In a 24-h reaction, glu-
a
cose and galactose were released from melibiose; however, the
galactose peak was very small. Many galactosyl residues were
transferred to melibiose, forming trisaccharides as transfer prod-
therefore, that a high production of
would occur during transglycosylation.
a-1,6-linked oligosaccharides
100
100
A
B
80
60
40
20
0
80
60
40
20
0
2
4
6
8
10
0
20
40
60
80
Temperature (
)
pH
Fig. 2. Effects of pH and temperature on SO
a-galactosidase activity. A and B shows effects of pH and temperature, respectively. Symbols; A: optimal pH (d), stable pH (s),
and B: optimal temperature (N), stable temperature (4).

180
M. Kurakake et al. / Food Chemistry 126 (2011) 177–182
0 h
24 h
Melibiose
Melibiose
Trisaccharide
Glucose
Tetrasaccharide
Galactose
0
5
10
15
20
0
5
10
15
20
Retention time min
Retention time min
Fig. 3. HPLC charts of reaction mixture in transglycosylation by SO
a-galactosidase. Melibiose (8.3%) and
a
-galactosidase (0.027 U/ml) were incubated at pH 5 and 40 °C for
24 h.
Table 2
Transgalactosylation with melibiose by SO
a-galactosidase.
Time (h)
Sugar composition (%)
Transfer ratio (%)
Hydrolysis ratio (%)
Glucose
Galactose
Melibiose
Trisaccharide
Tetrasaccharide
33
56
80
17.2
20.5
22.9
2.3
3.3
3.7
37.3
29.5
26.6
36.4
36.5
34.4
6.8
10.2
12.4
86.5
83.8
83.6
13.5
16.2
16.4
Melibiose (8.3%) and
a-galactosidase (0.027 U/ml) were incubated at pH 5 and 40 °C.
3.3. Acceptor specificity in the transgalactosylation of SO
galactosidase
a-
20
15
10
5
A
The acceptor specificity of the transglycosylation reaction of SO
-galactosidase was investigated using melibiose as a substrate
a
(Table 3). No transgalactosylation was observed when mono-alco-
hols were used as acceptors. However, di- and tri-alcohols, such as
ethylene glycol and glycerin, demonstrated good transgalactosyla-
tion activity. Enzymes from Aspergillus niger, Candida guilliermondii,
and Taramyces flavus efficiently transferred galactosyl residues to
mono-alcohols, producing alkyl galactopyranosides (Hashimoto
et al., 1995; Simerska et al., 2006; Tzortzis et al., 2003). The high
degree of transglycosylation to both the di-alcohol ethylene glycol
and the tri-alcohol glycerin is a unique characteristic of SO
0
0
2
4
6
8
Initial concentration of melibiose (%)
a
-galactosidase.
100
80
60
40
20
0
For sugars and sugar alcohols, acceptor specificity was very low.
B
The transfer product peaks for xylitol, mannose, and tagatose were
clearly detectable; however, the peak size was very small. Man-
nose and tagatose are structurally similar; the only difference
being the position of the hydroxymethylene group (C6 for man-
nose and C1 for tagatose). As is the case in ethylene glycol (C2)
and glycerin (C3), the sugar alcohols have a hydroxyl group bound
to each carbon atom. The low acceptor specificity can therefore be
attributed to the increase in molecular size (>C3), since the small-
est sugar alcohol, erythritol (C4), did not demonstrate any acceptor
activity. This research shows that the acceptor specificity of SO
0
2
4
6
8
10
Initial concentration of melibiose (%)
a-galactosidase, from P. oxalicum, is in contrast to that of a-galac-
Fig. 4. Effect of melibiose concentration on transglycosylation reaction of SO
-galactosidase Melibiose (0–8%) and -galactosidase (0.027 U/ml) were incubated
at pH 5 and 40 °C for 24 h. Symbols; A: glucose (d), galactose (s), and B: transfer
ratio (N).
tosidase extracted from other enzymes. For example, it has been
reported that B. adolescentis and A. fumigatus enzymes have accep-
tor specificity to oligosaccharides such as malto-oligosaccharides
a
a

M. Kurakake et al. / Food Chemistry 126 (2011) 177–182
181
Table 3
Glycerin
Melibiose
Acceptor specificity of transgalactosylation by SO a-galactosidase.
Acceptors
Transfer
product
Acceptors
Transfer
product
Alcohols
Methanol
Sugars
Glucose
TP
–
–
–
–
–
–
+
L
-Arabinose
Galactose
Ethanol
–
D
D
D
D
D
-Xylose
Trisaccharide
1-Propanol
2-Propanol
n-Butanol
–
-Ribose
–
-Glucose
-Galactose
-Mannose
–
20
Retention time (min)
10
3-Methyl-1-
butanol
Ethylene glycol
++
++++
++++
++
–
D
-Tagatose
-Fructose
Glycerin
D
Fig. 6. HPLC analysis with GL C610 column of reaction mixture in transglycosy-
lation to glycerin. Melibiose (8%), glycerin (40%) and -galactosidase (0.027 U/ml)
Sugar alcohols
–
L
-Sorbose
a
Erythritol
Xylitol
myo-Inositol
Solbitol
+
++
–
N-Acetylglucosamine
Palatinose
Trehalose
–
–
–
–
were incubated at pH 5 and 40 °C for 24 h. TP: transfer product.
–
Isomalto-
oligosaccharides
glycerin from 0% to 40%, the transglycosylation to glycerin in-
creased linearly as the rate of self-transglycosylation of melibiose
decreased. Fig. 6 shows the chromatogram of the reaction mixture
in 40% glycerin. The transfer product produced a high peak be-
tween those of melibiose and glucose, whereby the transfer ratio
to glycerin was 41.7%.
Mannitol
Lactitol
+
–
a
-Cyclodextrin
–
Others
Hesperidin
+
–
L
L
L
-Ascrorubic acid
-Tyrosine
–
–
-Serine
In the 13C NMR spectrum of the transfer product, signals of
a-
Melibiose (8%), acceptor (8%) and
pH 5 and 40 °C for 24 h.
a-galactosidase (0.032 U/ml) were incubated at
galactopyranoside were detected (C1 99.8, 99.4, 99.1, C2 69.5, C3
70.4, C4 70.2, C5 72.0, and C6 62.2 ppm) and the C1 and C2 signals
of glycerin (C1 63.5, C2 73.0, and C3 63.5 ppm) were shifted to 72.1
and 79.7 ppm, respectively. Therefore, the transfer product, formed
(Hinz et al., 2005; Puchart & Biely, 2005). Furthermore, a-galacto-
by the interaction of the OS enzyme, was identified with 1-O-a-D-
sidase from C. guilliermondii has broad specificity and efficiently
transfers galactosyl residues to monosaccharides such as glucose
and galactose (Hashimoto et al., 1995). Other substances, such as
amino acids and L-ascorbic acid, were also incubated with SO a-
galactosidase from P. oxalicum, but were not found to act as
galactosylglycerol and 2-O-
a-
D-galactosylglycerol. Although both
b-D-galactosylglycerol and
a-
D-glucosylglycerol are well known
transfer products of transglycosylation (Irazoqui et al., 2009; Nak-
ano, Takenishi, & Watanabe, 1988; Takenaka & Uchiyama, 2000),
very little is known of the product
a-D-galactosylglycerol. This
acceptors.
product may prove to be of great interest for the synthesis of gly-
colipids that have physiological activity.
SO
a-galactosidase demonstrated high acceptor specificity to
ethylene glycol and glycerin. The conditions for the transglycosyla-
tion reaction to glycerin were investigated as it is expected that
this reaction would lead to the synthesis of a new glycolipid, as a
result of esterification between the transfer product (
sylglycerol) and fatty acids.
Fig. 5 shows the relationship between the degree of transglyco-
sylation to glycerin and the initial concentration of glycerin.
Although the absolute transfer ratio from melibiose was reduced
from 91% to 79%, with an increase in the initial concentration of
The production of
a-D-galactosylglycerol, in the reaction with
40% glycerin, was 293 mg in 24 h per 1 g melibiose used by the
enzymatic reaction. The rate of production could be improved by
altering the reaction time, the enzyme activity, and the concentra-
tion of glycerin. The synthesis of sugar lipids can be carried out
readily, by the reverse reaction with fatty acids using lipase.
a-D-galacto-
In conclusion,
transferred galactosyl residues to the melibiose substrate, despite
a low hydrolysing activity of pNP- -galactopyranoside. With re-
spect to its acceptor specificity, SO -galactosidase showed little
a-galactosidase from P. oxalicum SO efficiently
a
a
transglycosylation to mono-alcohols, sugar alcohols, and sugars;
however, the di-alcohol ethylene glycol and the tri-alcohol glycerin
were shown to be good acceptors for the transglycosylation
reaction.
100
80
60
40
20
0
References
Arya, P., Kutterer, K. M. K., Qin, H., Roby, J., Barnes, M. L., Lin, S., et al. (1999).
a-
Galactose based neoglycopeptides. Inhibition of verotoxin binding to
globotriosylceramide. Bioorganic and Medicinal Chemistry, 7, 2823–2833.
Bupin, P. V., Gidley, M. J., Jeffcoat, R., & Underwood, D. R. (1990). Development of a
biotechnological process for the modification of galactomannan polymers with
plant a-galactosidase. Carbohydrate Polymers, 12, 155–168.
Coetzee, T., Fujita, N., Dupree, J., Shi, R., Blight, A., Suzuki, K., et al. (1996).
Myelination in the absence of galactocerebroside and sulfatide: Normal
structure with abnormal function and regional instability. Cell, 86(2), 209–219.
0
10
20
30
40
Initial concentration of glycerin (%)
Dey, P. M., & Campillo, E. D. (1984).
Related Areas of Molecular Biology, 56, 141–249.
Hashimoto, H., Katayama, C., Goto, M., Okinaga, T.,
a-Galactosidase. Advances in Enzymology and
Fig. 5. Relationship between transglycosylation to glycerin and the initial concen-
tration of glycerin. Melibiose (8%), glycerin (0–40%) and -galactosidase (0.032
a
& Kitahata, S. (1995).
U/ml) were incubated at pH 5 and 40 °C for 24 h. Symbols; transfer ratio (4) and
Transgalactosylation catalyzed by
H-404. Bioscience, Biotechnology, and Biochemistry, 59(4), 619–623.
a-galactosidase from Candida guilliermondii
transglycosylation to melibiose (d) and to glycerin (s).

182
M. Kurakake et al. / Food Chemistry 126 (2011) 177–182
Hinz, S. W. A., Doeswijk-Voragen, C. H. L., Schipperus, R., Broek, L. A. M., Vincken, J.
P., & Voragen, A. G. J. (2005). Increasing the transglycosylation activity of
Puchart, V.,
&
Biely, P. (2005). Glycosylation of internal sugar residues of
-galactosidase from Aspergillus fumigatus.
a-
oligosaccharides catalyzed by a
galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed
mutagenesis. Biotechnology and Bioengineering, 93(1), 122–131.
Irazoqui, G., Giacomini, C., Batista-Viera, F., Brena, B. M., Cardelle-Cobas, A., Corzo,
N., et al. (2009). Characterization of galactosyl derivatives obtained by
transgalactosylation of lactose and different polyols using immobilized b-
galactosidase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry,
57(23), 11302–11307.
Biochimica et Biophysica Acta, 1726(2), 206–216.
Shabalin, K. A., Kulminskaya, A. A., Savel’ev, A. N., Shishlyannikov, S. M.,
&
Neustroev, K. N. (2002). Enzymatic properties of
Trichoderma reesei in the hydrolysis of galactooligosaccharides. Enzyme and
Microbial Technology, 30(2), 231–239.
a-galactosidase from
Shibuya, H., Kobayashi, H., Park, G. G., Komatsu, Y., Sato, T., Kaneko, R., et al. (1995).
Purification and some properties of
a-galactosidase from Penicillium
Kurakake, M., Sumida, T., Masuda, D., Oonishi, S., & Komaki, T. (2006). Production of
galacto-manno-oligo-saccharides from guar gum by b-mannanase from
Penicillium oxalicum SO. Journal of Agricultural and Food Chemistry, 54,
7885–7889.
purpurogenum. Bioscience, Biotechnology, and Biochemistry, 59(12), 2333–2335.
Simerska, P., Kuzma, M., Monti, D., Riva, S., Mackova, M., & Kren, V. (2006). Unique
transglycosylation potential of extracellular a-D-galactosidase from Talaromyces
flavus. Journal of Molecular Catalysis B, Enzymatic, 39(1–4), 128–134.
Luonteri, E., Alatalo, E., Siika-Aho, M., Penttila, M., & Tenkanen, M. (1998).
a-
Spangenberg, P., Andre, C., Dion, M., Rabiller, C., Mattes & Faculte, R. (2000).
Galactosidases of Penicillium simplicissimum: Production, purification and
characterization of the gene encoding AGLI. Biotechnology and Applied
Biochemistry, 28(2), 179–188.
Comparative study of new
Carbohydrate Research, 329(1), 65–73.
Takenaka, F., Uchiyama, H. (2000). Synthesis of
a-galactosidases in transglycosylation reactions.
&
a-D-glucosylglycerol by a-
McCleary, B. V., Amado, R., Waibel, R., & Neukom, H. (1981). Effect of galactose
content on the solution and interaction properties of guar and carob
galactomannans. Carbohydrate Research, 92, 269–285.
glucosidase and some of its characteristics. Bioscience, Biotechnology, and
Biochemistry, 64(9), 1821–1826.
Tzortzis, G., Jay, A. J., Baillon, M. L. A., Gibson, G. R., & Rastall, R. A. (2003). Synthesis
Mi, S., Meng, K., Wang, Y., Bai, Y., Yuan, T., Luo, H., et al. (2007). Molecular cloning
of a-galactooligosaccharides with a-galactosidase from Lactobacillus reuteri of
and characterization of a novel
CGMCC 1669 and expression in Pichia pastoris. Enzyme and Microbial Technology,
40(5), 1373–1380.
a
-galactosidase gene from Penicillium sp. F63
canine origin. Applied Microbiology and Biotechnology, 63(3), 286–292.
Van Laere, K. M. J., Hartemink, R., Beldman, G., Pitson, S., Dijkema, C., Schols, H.
A., et al. (1999). Transglycosidase activity of Bifidobacterium adolescentis DSM
Nakano, H., Takenishi, S., & Watanabe, Y. (1988). Formation of transfer products
from soybean arabinogalactan and glycerol by galactanase from Penicillium
citrinum. Agricultural and Biological Chemistry, 52(8), 1913–1921.
Poulos, A., & Beckman, K. (1978). Trihexosylceramide alpha-galactosidase of human
leucocytes. Clinica Chimica Acta, 89(1), 35–45.
20083
681–688.
Yamane, T. (1971). Decomposition of raffinose by
a-galactosidase. Applied Microbiology and Biotechnology, 52(5),
a-galactosidase. An enzymatic
reaction applied in the factory-process in Japanese beet sugar factories. Sucre
Belge/Sugar Industry Abstracts, 90, 345–348.