Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β- glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside

Article abstract of DOI:10.1016/j.carres.2011.11.008

Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-β-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na]⁺ ions correlated with the theoretical monoisotopic mass (calcd [M+Na]⁺: 507.44 m/z; obs. [M+Na]⁺: 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-β-glucosidases by zymographic analysis.

Full text of DOI:10.1016/j.carres.2011.11.008

Carbohydrate Research 347 (2012) 69–75
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Transglycosylation specificity of Acremonium sp. -rhamnosyl-b-glucosidase
a
and its application to the synthesis of the new fluorogenic substrate
4-methylumbelliferyl-rutinoside
Laura S. Mazzaferro a, Lucrecia Piñuel a, Rosa Erra-Balsells b, Silvana L. Giudicessi b, Javier D. Breccia a,
⇑
a INCITAP-CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Departamento de Química, Facultad de Ciencias Exactas y Naturales,
Universidad Nacional de La Pampa (UNLPam), Av. Uruguay 151, 6300 Santa Rosa, La Pampa, Argentina
b CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, 3[er] P., Ciudad Universitaria,
1428 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Transglycosylation potential of the fungal diglycosidase
a-rhamnosyl-b-glucosidase was explored. The
Received 13 September 2011
Received in revised form 8 November 2011
Accepted 11 November 2011
Available online 19 November 2011
biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This
feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-ruti-
noside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbellifer-
one, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated
reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound
was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the
[M+Na]+ ions correlated with the theoretical monoisotopic mass (calcd [M+Na]+: 507.44 m/z; obs.
[M+Na]+: 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside
in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to
explore in nature the occurrence of endo-b-glucosidases by zymographic analysis.
Keywords:
Diglycosidase
Hesperidin
Zymogram
Acremonium sp. DSM 24697
Ó 2011 Published by Elsevier Ltd.
1. Introduction
features which determine solubility, stability in the body, and also
transport pathways.4,5
The study of the degradation of natural polysaccharides or gly-
coconjugates by an enzyme consortium—and even the purified
proteins—is a complex task. Endo-glycosidases usually present an
extended binding site cleft composed of several subsites for mono-
mer units. As a consequence, substrate specificity depends on the
recognition of more than one glycosidic moiety, sometimes an
aglycone moiety, and the linkage(s) between them.1 Both polymers
and commercial substrates are utilized to study substrate specific-
ity of these enzymes.2 The techniques that measure release of a
chromophoric aglycone upon enzymatic hydrolysis of a glycocon-
jugate allow the easy monitoring of the reaction course by contin-
uous UV–vis or fluorescence spectroscopy.3 However, commercial
substrates are not always available and it is particularly true in
the case of the screening of enzymatic hydrolysis of some sugar
moieties found in secondary metabolites of plants. These com-
pounds are nowadays gaining attention because of their biological
activities; and their absorption and metabolism are different
depending on the sugar moieties due to differences in structural
The disaccharidic moiety rutinose (6-O-
D-glucopyranoside) is an important constituent of flavonoids,
terpenoids, anthocyanins, and other secondary metabolites of
plants. The most commonly reported deglycosylation mechanism
a-L-rhamnopyranosyl-
b-
involves an
3.2.1.21) that act in a sequential mode9 (Table 1). We recently re-
ported the diglycosidase activity -rhamnosyl-b-glucosidase (EC
3.2.1.168) that acts on hesperidin and other bioactive flavonoids,
a-rhamnosidase (EC 3.2.1.40) and a b-glucosidase (EC
a
splitting off a rutinose residue (6-O-a-L-rhamnopyranosyl-b-D-
glucopyranoside) in one step (Table 1).10 It is not clear, however,
which of the mechanisms is the dominant one in nature because
of the lack of tools for the screening of a-rhamnosyl-b-glucosidase
activity. Comparable problems are faced when searching for other
diglycosidases (for a review, see Ref. 11). Yamamoto et al.12 de-
signed an ingenious plate screening system for b-primeverosidases
consisting of the use of the chromogenic substrates 4-nitrophenyl-
b-primeveroside
(6-O-b-D-xylopyranosyl-b-D-glucopyranoside)
(medium P) and 4-nitrophenyl-b-xylopyranoside (medium X).
The microorganisms that were more deeply colored in medium P
than medium X were selected as promising candidates. In this
work we studied the Acremonium sp.
transglycosylation ability using different acceptors bearing OH
a-rhamnosyl-b-glucosidase
⇑
Corresponding author. Tel.: +54 2954 436787x26; fax: +54 2954 432535.
E-mail address: javierbreccia@exactas.unlpam.edu.ar (J.D. Breccia).
0008-6215/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.carres.2011.11.008

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L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
Table 1
Enzymatic activities acting on hesperidin, and substrates used for their detection
groups. Such ability was exploited for the one step synthesis of 4-
methylumbelliferyl-rutinoside using kinetic control of the
reaction. This compound can be used as a fluorogenic substrate
2.3. Enzyme reactions
For quantification of
a
-rhamnosyl-b-glucosidase activity, each
L of substrate (0.11% w/v hesperi-
din in 50 mM sodium citrate buffer pH 5.0) and 50 L of suitably
diluted enzyme solution. The reaction was performed for 30 min
for screening of
graphic staining.
a-rhamnosyl-b-glucosidase activity by zymo-
reaction (1 mL) contained 450 l
l
at 60 °C and stopped by adding 500 lL of 3,5-dinitrosalicylic acid
2. Experimental
(DNS).13 The tubes were placed in a boiling water bath for
10 min and cooled before measuring the absorbance at 540 nm.
2.1. Chemicals and stock solutions
One unit of
the amount of enzyme required to release 1
a
-rhamnosyl-b-glucosidase activity was defined as
mol of rutinose per
l
4-Nitrophenol, 4-methylumbelliferone (4-MU), 4-methylum-
belliferyl-b-
trihydroxy-4’-methoxyflavanone) and hesperidin (hesperetin
7-O-(6-O- -rhamnopyranosyl-b- -glucopyranoside) were pur-
D
-glucopyranoside (4-MU-Glc), hesperetin (30,5,7-
min. For transglycosylation activity, 10%v/v alcohols or 1.8 mM 4-
methylumbelliferone was added to the reaction mixture and incu-
bated at 30 °C with 0.0023 U/mL of
a-rhamnosyl-b-glucosidase
a
-L
D
activity for 1 h (otherwise indicated in text). LogP of the alcohols
where obtained from PhysProp (http://www.syrres.com/what-
we-do/databaseforms.aspx?id=386).
chased from Sigma Chemical (St. Louis). HPLC grade methanol
LiChrosolvÒ was obtained from Merck (Darmstadt). The other
chemicals were from standard sources.
The immobilized enzyme on chitosan composites was per-
formed as previously described.14 The products of enzymatic reac-
tion were analyzed by thin layer chromatography (Silica Gel 60 W)
using ethyl-acetate/2-propanol/water (3:2:2) as mobile phase and
stained with anthrone reagent. Total activity (hydrolysis + trans-
glycosylation) was quantified by measuring hesperetin released
at 320 nm.15 Spectra of analytes were obtained using a USB4000
UV–vis spectrophotometer (Ocean Optics).
To prepare stock solutions for enzymatic reactions, hesperidin
and 4-methylumbelliferone were solubilized in dimethylsulfoxide
(180 mM) and suspended in water. To adjust pH 100 mM sodium
citrate (pH 5) and sodium phosphate (pH 8) buffers were used.
Standards for HPLC were prepared in dimethylsulfoxide
(180 mM) and suitably diluted in the mobile phase before
injection.
2.2. Enzyme source
2.4. Analytical assays
Acremonium sp. DSM 24697 was cultured using hesperidin as
Reaction substrates and products were analyzed by high perfor-
mance liquid chromatography (HPLC) using a KONIK-500-A series
carbon source for induction of
a-rhamnosyl-b-glucosidase and
the enzyme was purified as described previously.10

L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
71
Table 2
Transglycosylation activity of
acceptors
HPLC system attached to a KONIK Uvis 200 detector. The column
was a reversed-phase LiChroCARTÒ 125-4 (12.5 cm length, 4 mm
internal diameter) LiChrospherÒ
m, RP 18 (pore size 100 Å).
The elution consisted of an isocratic flow of 40%v/v methanol and
60%v/v water at a flow rate of 1.0 mL/min at 40 °C. Peak areas and
extinction coefficient were calculated from chromatograms of
authentic standards detected at 285 nm. For 4-methylumbellife-
a
-rhamnosyl-b-glucosidase using different alcohols as
Transglycosylation activity (ꢀ10ꢁ3 U/mL)
5
l
Acceptor
Methanol
Ethanol
Propanol
Isopropanol
Butanol
Isobutanol
tert-Butanol
Amyl-alcohol
Benzyl-alcohol
4.6 1.7
5.6 0.6
4.0 1.3
4.1 0.4
2.9 2.0
2.7 0.4
ND
ryl-b-D-rutinoside quantification, 4-methylumbelliferyl-b-D-gluco-
pyranoside was used as standard. Triplicate samples were used.
Samples were deproteinized according to Contin et al.16
<0.5
<0.5
2.5. Production and purification of 4-methylumbelliferyl-b-D-
rutinoside
ND. Not detected.
For production of 4-methylumbelliferyl-b-D-rutinoside, a 60 mL-
sterically hindered alcohols.19 Recently, Zhow et al.20 described
the syntheses of R-b-rutinosides by means of a crude rutin-degrad-
ing solution prepared from the seeds of Fagopyrum tataricum.
working volume reactor was used under low stirring at 30 °C. The
reaction mixture contained 1.8 mM hesperidin and 1.8 mM 4-
methylumbelliferone as sugar donor and acceptor, respectively,
and 2%v/v DMSO in 50 mM sodium citrate buffer pH 5.0. The immo-
bilized catalyst was added at a final concentration of 0.0023 U/mL
(2 mL chitosan composites beads). The reaction mixture (60 mL)
was filtered with Double Rings filter paper No. 103. A solvent
extraction was performed twice by adding 0.5 volume of ethyl-ace-
tate. Aqueous phase was freeze-dried and suspended in ethyl-ace-
tate/2-propanol/water (3:2:2). Separation of the products was
performed by column chromatography using Silica Gel 60 W
(10 cm length ꢀ 1 cm internal diameter) as the stationary phase
and ethyl-acetate/2-propanol/water (3:2:2) as the mobile phase.
a-Rhamnosyl-b-glucosidase is able to use hesperidin instead of
rutin as a rutinose donor due to its specificity for 7-O-flavonoid-
rutinosides. Advantageously, 7-O-flavonoid-rutinosides are the
major flavonoids in some citrus industry by-products, constituting
an inexpensive raw material.21
An inverse relationship was found between the transglycosyla-
tion activity for primary and secondary aliphatic alcohols and the
alcohols logP (Fig. 1). In all cases, activity was enhanced (2–4-fold)
in comparison with the hydrolysis, achieving the highest yields
with methanol and ethanol as acceptors. Since hesperidin solubil-
ity is low in water, and increases in presence of water-soluble sol-
vents, addition of short-chain alcohols is expected to enhance the
whole activity (transglycosylation + hydrolysis) of the enzyme.
Retaining glycoside hydrolases usually catalyze the glycosyla-
tion of short alkyl alcohols while examples of aromatic alcohols
being used as acceptors are less common. Since the alcohols act
as substrates, the affinity interaction with the surrounding envi-
ronment of the active site is likely to be different depending on
the alcohol. Studies performed with crystallized glycoside hydro-
lases have demonstrated the importance of hydrophobic residues
for the aglycone binding pocket.22,23 This could explain the fact
that the enzyme preferably transfers the sugar moiety to –OH
acceptors other than water, even when the water concentration
is up to 50 times higher. Another factor to be considered is the pro-
tein stability in aqueous-alcoholic systems. Because alcohols desta-
bilize the protein by lowering the dielectric constant of the
medium, the product yield will depend on the equilibrium of the
variables mentioned above.
2.6. MALDI-TOF/TOF MS analysis
Samples of substrates and products of the enzymatic reaction
were analyzed by ultraviolet (UV)–MALDI-TOF/TOF MS analyses
performed on the Bruker Ultraflex Daltonics mass spectrometer
in positive and negative ion modes. For matrix preparation, nor-
harmane, 2,5-dihydrobenzoic acid, 20,40,60-trihydroxyacetophe-
none and
a-cyano-4-hydroxycinnamic acid triethylamine solu-
tions (2 mg/mL) were prepared by dissolving selected compound
in acetonitrile/water (1:1 v/v) solution. Carbon nanotubes pre-
pared as described by Nonami et al.17 were also utilized as matrix.
Dry droplet sample preparation method was used according to
Gholipour et al.18 Desorption/ionization was obtained by using a
355-nm solid laser. The accelerating potential was 20 kV. Spectra
were obtained and analyzed with the programs FlexControl and
FlexAnalysis, respectively.
3. Results and discussion
3.1. Acceptor specificity of
a-rhamnosyl-b-glucosidase
The acceptor specificity of the fungal diglycosidase
a-rhamno-
syl-b-glucosidase was investigated using different acceptors bear-
ing hydroxyl groups, and hesperidin as rutinose donor. Primary
and secondary alcohols were shown to be glycosylated by the en-
zyme, and hydrolytic activity was suppressed with the exception of
amyl- and benzyl-alcohols. In the last case, the reaction mixture
formed a two phase-system because of the low water solubility
of the alcohol. For the reason that hydrolytic activity was sup-
pressed, released hesperetin was stoichiometrically equivalent to
the amount of synthesized alkyl-rutinosides and it was used for
estimation of the transglycosylation yield (Table 2). The short-
chain alcohols (C1–C3) were found to give the highest yields of
alkyl-rutinosides. On the other hand, tert-butanol was not glycos-
ylated. This result is in agreement with the behavior of most
retaining glycoside hydrolases, which are reported to be inert to
Figure 1. Transglycosylation activity of
acceptor logP.
a-rhamnosyl-b-glucosidase as a function of

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L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
3.2. Synthesis of 4-methylumbelliferyl-rutinoside
-Rhamnosyl-b-glucosidase was incubated with the aromatic
alcohol 1.8 mM 4-methylumbelliferone (4-MU) as acceptor and
1.8 mM hesperidin as rutinose donor. Thin layer chromatography
(TLC)oftheenzymaticreactionmixturegavea weakfluorescentspot
(Rf = 0.72), similar to that of 4-MU-glucoside (4-MU-Glc, Rf = 0.80).
The spot was found to be a glycoconjugate that, taking into account
the enzyme mechanism,10 strongly suggested the synthesis of
4-MU-rutinoside (Fig. 2). The yield of transglycosylation was 28%
after the first hour of reaction at 30 °C (Fig. 3). Subsequently, the
amount of transglycosylation product remained constant for at least
3 h, while the hydrolysis was shown to proceed up to completion.
This behavior is in agreement with that observed for other glycosi-
dase-catalyzed synthesis. Transglycosylation rate is usually higher
than free sugar formation rate during the first stage of the reaction
and, later on, transglycosylation product concentration reaches a
plateau or diminishes because it acts as a substrate of the
a
a
4-MU
4-MU-Glc
4-MU-Rutinoside
Rha
Glc
Mal
SS 4MU-Glc RP
SS 4MU-Glc RP
b
Figure 2. (a) Rutinose transglycosylation from hesperidin to 4-methylumbelliferone performed by
a
-rhamnosyl-b-glucosidase. SS sugar standards (Glc glucose, Rha
rhamnose, Mal maltose), 4-MU-Glc 4-methylumbelliferyl-b-
rhamnosyl-b-glucosidase.
D-glucopyranoside, RP reaction products. (b) Hydrolysis and transglycosylation reactions catalyzed by a-

L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
73
2500
2000
1500
1000
500
1000
800
600
400
200
0
0
10
20
30
40
50
0
DMSO (%v/v)
0
1
2
3
Figure 4. Effect of DMSO addition on 4-MU-rutinoside yield after (N) 1 h and (d)
3 h.
Time (h)
Figure 3. Time course of enzymatic reaction measured by production of (N)
hesperetin and (d) 4-MU-rutinoside.
The enhancement of transglycosylation activity by employment
of organic solvents (2–20%v/v) might be caused by suppression of
hydrolytic activity, increase of substrates solubility, or a combina-
enzyme.24,25 The increment of the reaction temperature up to the
near-optimal for hydrolysis (60 °C) was shown to diminish the yield
of4-MU-rutinoside, probablyby favoringthehydrolysisof thetrans-
glycosylation product. The highest yield was obtained for the accep-
tor donor ratio in the range 0.8–1:1 (data not shown).
Several authors reported the synthesis of 4-nitrophenyl- and 4-
methylumbelliferyl-dissacharides and oligosaccharides26–28 using
glycosidases that catalyze the transfer of one sugar unit from a
polysaccharide to the corresponding monoglycoside. In that way,
Kadi and Crouzet28 synthesized phenyl-b-primeveroside) by
means of Trichoderma longibrachiatum xylanase, which was capa-
ble to transfer a xylose moiety from xylan to phenyl-glucoside.
However, synthesis of rutinosides starting from 4-MU-Glc would
tion of both factors.32 During MUR synthesis,
a
-rhamnosyl-b-glu-
cosidase was shown to produce both transglycosylation product
and free sugar even when high DMSO concentration (30–40%v/v
)
was used. Even though, there was a trend to increase the ratio be-
tween the transglycosylation product and free sugar with increas-
ing DMSO concentration in the range 2–20%v/v (data not shown).
To probe that the major effect of DMSO was due to the solubiliza-
tion of the substrates with a consequent increment in the overall
activity of the enzyme, hydrolytic activity was tested with
1.8 mM hesperidin as a substrate in presence of DMSO at different
concentrations (Table 3). Hydrolytic activity was not modified by
addition of 2%v/v DMSO and was significantly diminished when
DMSO concentration was increased up to 10%v/v. The fact that an
increment in the solubility of hesperidin by DMSO addition did
not raise the reaction rate is explained by its water solubility
only be possible by reverse hydrolysis since
(3.2.1.40) reported up to now are inverting enzymes, and so cannot
catalyze transglycosylation.29 Remarkably, use of
-rhamnosyl-b-
a-L-rhamnosidases
a
glucosidase represents a shortcut for the one step synthesis of
the fluorogenic substrate 4-methylumbelliferyl-rutinoside.
Table 3
Effect of DMSO addition on hesperidin hydrolysis
3.3. Water-soluble organic solvents
Enzyme activity (30 °C, U/mL)
Yield (18 h-incubation, %)
26
43 22
52 15
Control
2%v/v DMSO
10%v/v DMSO
0.401 0.005
0.399 0.005
0.318 0.003
4
The inclusion of an organic co-solvent in the reaction medium
has frequently been used to increase the synthetic efficiency of gly-
cosidase-catalyzed processes; the two most commonly employed
organic co-solvents in this respect being dimethylsulfoxide
(DMSO) and acetone.30 Acetone addition (50%v/v) to
a-rhamno-
syl-b-glucosidase was shown to promote the protein aggregation,
with a concomitant lost in the enzyme activity. When DMSO and
dimethylformamide (10%v/v) were included in the transglycosyla-
tion reaction mixture (0.5 h-reaction) the first did not increase
the product yield (Fig. 4), while the latter was deleterious for the
enzyme.
The solvent DMSO was added to the reaction mixture in concen-
trations in the range of 2%v/v (the minimum required for substrates
stock preparation) to 50%v/v. The addition of 6–10%v/v DMSO did
not change the 4-MU-rutinoside yield in comparison with 2%v/v
DMSO, for 1 h-reaction (Fig. 4). However, it effectively increased
the yield after 3-h reaction. The conversion percentages dimin-
ished at higher DMSO concentrations, and no product was formed
above 30%v/v likely due to the destabilization of the biocatalyst. The
increasing concentration of organic co-solvent usually promotes
protein aggregation decreasing the enzymatic activity. Thus, the
achievement of high activity caused by higher substrate solubility
is counterbalanced by the instability of the protein structure.31
400
300
200
100
0
0
2
4
6
8
10
Time (h)
Figure 5. Time course of transglycosylation reaction in a bench-scale reactor. One-
hundred percent activity corresponded to 0.01 U/mL.

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L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
(0.324 mM),33 which is high enough in comparison with hespere-
tin concentration reached in the reaction mixture (0.12 mM after
30 min reaction). Consistently, hydrolysis yield was increased
upon addition of DMSO in the range studied, with hesperetin final
concentrations in the range 0.47–0.95 mM after 18 h-reaction. This
result suggested that when the reaction proceeds, hesperidin solu-
bilization rate becomes limiting for the enzymatic reaction.
acceptor:donor ratio was 1:1. The enzyme a-rhamnosyl-b-glucosi-
dase was immobilized in chitosan composites beads in order to
separate and re-use the biocatalyst. Since agitation increases the
solubilization rates of the substrates, DMSO concentration was
set at suboptimal concentration (2%v/v). The kinetics of the trans-
glycosylation reaction was monitored during 10 h (Fig. 5). The 4-
methylumbelliferyl-rutinoside concentration was shown to reach
a maximum after 4 h. It remained near a constant value of ca.
3.4. Enzymatic reactor for 4-methylumbelliferyl-rutinoside
production
300
l
M up to 7 h-reaction-representing 16% conversion of the
acceptor-, and later on it slightly decreased to ca. 250
lM after
10 h. Reaction was kinetically controlled by separation of the cata-
lyst, and the reaction mixture was filtered to remove suspended
solids.
The production of 4-methylumbelliferyl-rutinoside was per-
formed in a bench-scale agitated reactor (60 mL) making use of
the performed optimization: temperature was set at 30 °C and
3.5. Isolation and identification of 4-methylumbelliferyl-
rutinoside
Liquid–liquid extraction was applied for isolation of the synthe-
sized compound: the filtrate was extracted twice with ethyl-ace-
tate to separate the aglycones. The freeze-dried aqueous phase
underwent preparative chromatography for separation of the sugar
products (Fig. 6). The transglycosylation product was subjected to
MALDI-TOF/TOF. Data recorded for the (M+Na)+ ions indicate that
the observed mass correlates with the calculated monoisotopic
mass of the expected product, 4-MU rutinoside (Fig. 7, calcd
[M+Na]+: 507.44 m/z; obs. [M+Na]+: 507.465 m/z). UV–vis spec-
trum (pH 8) was recorded for the obtained compound, which
showed a peak at 318 nm, being similar to the maximum wave-
length for the commercial substrate 4-MU-Glc (320 nm). When
the product was incubated with the enzyme the typical peak of
4-MU was observed (365 nm) due to the hydrolysis of the newly
synthesized artificial substrate.
Yamamoto et al.12 reported the screening of b-primeverosidase-
producing microbial strains by combining the artificial substrates
4-nitrophenyl-b-xylopyranoside and their own synthesized 4-
nitrophenyl-b-primeveroside. Among commercial substrates, 4-
nitrophenol is a good leaving group for glycosidases and it is
mostly employed for activity detection in liquid mixtures of en-
zymes. On the contrary, 4-methylumbelliferone is the aglycone of
choice for zymographic analysis. While the last gives focused UV-
fluorescent bands on analytical polyacrylamide gel electrophoresis
or electrofocusing, the first diffuses through out the gel and activity
Figure 6. Schematic diagram of the enzymatic synthesis and purification of 4-MU-
rutinoside.
Figure 7. MALDI-TOF/TOF spectrum of reaction product obtained using nor-harmane matrix, positive ion mode, 44% laser.

L. S. Mazzaferro et al. / Carbohydrate Research 347 (2012) 69–75
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bands cannot be clearly recognized. The fluorogenic substrate 4-
methylumbelliferyl-rutinoside is a new contribution for the study
of diglycosidases since it allows the detection of enzymes specific
for rutinose in one step by zymographic analysis.
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This work was supported by Consejo Nacional de Investigaci-
ones Científicas y Técnicas, Universidad Nacional de La Pampa,
Universidad de Buenos Aires and Agencia Nacional de Promoción
Científica y Técnica of Argentina.
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