New assay of α-l-rhamnosidase

Article abstract of DOI:10.1007/s00706-017-2055-0

Abstract: Free rutinose was prepared by enzymatic hydrolysis of rutin using defatted seed meal from tartary buckwheat. This disaccharide was used as substrate in spectrophotometric assay of α-l-rhamnosidase. The assay is based on hydrolysis of rutinose and subsequent determination of released glucose by a standard glucose oxidase assay kit. The method is easy to perform and requires no expensive equipment. The assay was applied in α-l-rhamnosidase estimation in ten commercial enzyme preparations and compared with standard assay on chromogenic substrate.

Full text of DOI:10.1007/s00706-017-2055-0

Monatsh Chem (2018) 149:167–174
DOI 10.1007/s00706-017-2055-0
ORIGINAL PAPER
New assay of α-L-rhamnosidase
Elena Karnišová Potocká1 Mária Mastihubová1 Iveta Čičová2 Vladimír Mastihuba1
·
·
·
Received: 9 June 2017 / Accepted: 4 September 2017 / Published online: 1 December 2017
© Springer-Verlag GmbH Austria 2017
Abstract Free rutinose was prepared by enzymatic
hydrolysis of rutin using defatted seed meal from tartary
buckwheat. This disaccharide was used as substrate in
spectrophotometric assay of α-L-rhamnosidase. The assay
is based on hydrolysis of rutinose and subsequent deter-
mination of released glucose by a standard glucose oxidase
assay kit. The method is easy to perform and requires no
expensive equipment. The assay was applied in α-L-
rhamnosidase estimation in ten commercial enzyme
preparations and compared with standard assay on chro-
mogenic substrate.
Keywords Carbohydrates · Enzymes · Hydrolysis ·
Oxidation · Rhamnosidase
Introduction
α-L-Rhamnosidases (E.C. 3.2.1.40) are glycosidases which
cleave terminal α-L-rhamnose from a great variety of nat-
ural
compounds
such
as
pectins,
O-antigen
polysaccharides, and rhamnolipids [1–3] as well as various
less complex natural glycosides like rutin, quercitrin, nar-
ingin, hesperidin, and saponin rhamnosides [4–7]. This
type of enzymes can be found among bacteria, yeasts,
fungi, plants, and animals, and α-L-rhamnosidase activity
frequently occurs within enzyme complexes together with
the β-D-glucosidase activity, e.g., hesperidinase [6],
naringinase [7], and rhamnodiastase [8]. Rhamnosidases
are frequently used in food industry for debittering of citrus
fruit juices and prevention of their turbidity caused by
precipitation of hesperidin [9, 10] or in aroma release in
beverages [11].
Graphical abstract
4-Nitrophenyl α-L-rhamnopyranoside (1) is commer-
cially available but relatively expensive substrate for α-L-
rhamnosidase assay. Although its preparation has been
reported [12], the compound is not accessible without
experience with techniques of organic synthesis. Moreover,
determination of α-L-rhamnosidase activity exclusively
with chromogenic substrates does not offer the proper
insight into the enzyme specificity, and therefore, it is
necessary to perform other assays to uncover affinity to
given natural substrates, such as molecules harboring
rutinose motif [13].
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-017-2055-0) contains supplementary
material, which is available to authorized users.
´
& Vladimır Mastihuba
vladimir.mastihuba@savba.sk
1
Laboratory of Biocatalysis and Organic Synthesis, Institute of
´
´
Chemistry, Slovak Academy of Sciences, Dubravska cesta 9,
845 38 Bratislava, Slovakia
Naringin can be used as the natural substrate, but dis-
advantage of its use is in the reaction complexity: the
method follows overall disappearance of the substrate
2
National Agricultural and Food Centre, Research Institute of
´
ˇ
Plant Production, Bratislavska cesta 122, 921 68 Piesťany,
Slovakia
123

´
E. K. Potocka et al.
168
naringin and intermediate prunin at the same time and
additional testing is necessary to distinguish between these
two reactions [14]. The reaction can be obviously followed
by HPLC, but this is not supposed to be a fast way. Another
option is to use commercial rhamnose dehydrogenase kits
to follow the released rhamnose photometrically, but this
method is also costly, although it can be universally
modified to virtually any rhamnoside substrate. Therefore,
there still exists a demand for robust, fast, and economic
rhamnosidase assay.
tartary buckwheat were found as a practical material due to
their availability and ease of storage and handling. In
agreement with findings of Morishita et al. [23], the seeds
of common buckwheat Fagopyrum esculentum did not
release any free rutinose from rutin in our hands. On the
other hand, hydrolysis of rutin catalyzed by tartary buck-
wheat ran to the completion, and besides rutinose, only
traces of monosaccharides were detected in the reaction
mixture. Using simple isolation and purification proce-
dures, we obtained rutinose as a pale yellow powder with
purity above 97% in 64% yield. Zhou et al. [19] isolated
rutinose from similar reaction in 92% yield; their product
Rutinose (6-O-α-L-rhamnopyranosyl-D-glucopyranose,
2) is a disaccharide with potential medicinal applications
referred to the L-rhamnose moiety [15] and it may serve
also as a natural substrate for assay of α-L-rhamnosidase.
Its chemical synthesis is known but is difficult to accom-
plish. Contrariwise, fruit processing side products are
inexpensive and easily available sources of rutin (quercetin
3-O-β-rutinoside, 3) and hesperidin which can be hydro-
lyzed to rutinose in quality sufficient for non-medical uses.
Hydrolysis of rutin was earlier performed, for example, by
rhamnodiastase from leaves of Rhamnus californica in
1962 [16].
ˇ
ˇ´
´
had, however, appearance of yellow gum. Simcıkova et al.
[29] prepared rutinose in a reaction catalyzed by recom-
binant fungal rutinosidase in 63% overall yield as a
colorless amorphous solid. These two preparative reactions
were, however, carried out in scale of tens of milligrams of
product. We have run the hydrolysis in multi-gram scale
due to simple and inexpensive isolation techniques which
make the product accessible in any chemical or biological
laboratory in gram quantities.
Rutinosidase (EC 3.2.1.168) is a diglycosidase specific
to hydrolysis of rutinosides liberating the whole disaccha-
ride and the free aglycon. Rutinosidases hydrolyse rutin
and hesperidin; in some cases, they hydrolyze also naringin
or they may possess β-glucosidase activity towards
monoglucosides [17]. Application of these diglycosidases
is not limited to liberation of aroma in food production, but
there is growing importance of their use in the synthesis of
rutinosides [18].
Development of the assay
The principle of this assay is notoriously known from
enzymatic estimations of disaccharides such as maltose,
lactose, or sucrose or from assays of corresponding gly-
cosidases [31–34]. Rutinose is hydrolyzed to rhamnose and
glucose. Portions of reaction mixture are periodically
withdrawn, stopped by mixing with methanol, and glucose
enzymatically released from the disaccharide is estimated
by its oxidation with glucose oxidase. Hydrogen peroxide,
an equimolar by-product of this oxidation, oxidizes a
phenolic substance (3-methylphenol in our case) in the
presence of peroxidase and 4-aminoantipyrine to form
quinone–imine colored product (Scheme 2). BIO-LA-
TEST® Glucose, an enzymatic test for estimations of glu-
cose in medicine, was used as an inexpensive and reliable
ready-to-use kit for glucose determination in the assay.
Several precautions had to be, however, taken into
account during development of the assay including sub-
strate specificity of rhamnosidase (acceptation of rutinose
as substrate of rhamnosidase), substrate specificity of glu-
cose oxidase (potential oxidation of rutinose), interferences
of phenolics in rutinose (rutin and quercetin as potential
impurity) and occurrence of glucose or glucose precursor
in the tested rhamnosidase.
The purpose of this paper is to introduce simple assay of
α-L-rhamnosidase based on hydrolysis of rutinose by
rhamnosidase and to demonstrate preparation of the nec-
essary substrate rutinose through hydrolysis of rutin, using
techniques available in every chemical or biochemical
laboratory.
Results and discussion
Preparation of rutinose
Rutinose is available on the market in prices which are
unacceptable for routine enzyme assays. It was, therefore,
prepared in our laboratory by hydrolysis of rutin (3) cat-
alyzed by rutinose in seed meal from tartary buckwheat
(Fagopyrum tataricum, Scheme 1). Such hydrolysis toge-
ther with transrutinosylations was reported by several
authors [19–23], but it has never been used for gram scale
preparation of rutinose.
Two enzymes were preliminary tested in the proposed
assay—prokaryotic α-L-rhamnosidase from Megazyme and
Viscozyme L, a glycanase from Aspergillus aculeatus
which was ultrafiltered and lyophilized before testing to
remove sugars, salts, and preservers from its original
preparation. The results were compared with standard
Although rutinosidase was found in several sources of
plant [24–26] or microbial origin [17, 27–30], seeds of
123

New assay of α-L-rhamnosidase
169
Scheme 1
Scheme 2
rhamnosidase assay using p-nitrophenyl α-L-rhamnopyra-
noside (Table 1). Activity assays were performed at pH 6.0,
according to recommendations from producer of the
prokaryotic α-L-rhamnosidase. This latter enzyme has sig-
nificantly higher affinity towards the synthetic substrate.
On the contrary, Viscozyme L shows opposite substrate
preference, although the difference is less dramatic (almost
five time higher activity on rutinose). According to the
literature, differences in specificity towards natural and
synthetic substrates are rather frequent. As an example,
Grandits et al. [13] reported that the difference in speci-
ficity towards natural and synthetic substrate enabled
resolution of two distinct α-L-rhamnosidases from the same
source.
Table 1 α-L-Rhamnosidase activity towards substrates 1 and 2 at pH
6.0
Enzyme
Activity
1
2
Prokaryotic rhamnosidase
Viscozyme L (lyoph.)
260.3 35.7a
14.3 1.0b
1.2 0.3a
67.1 2.1b
a
Activity in U cm−3
b
Activity in U g−1
123

´
E. K. Potocka et al.
170
Optimization of the assay
Ultrafiltered and lyophilized preparation of Viscozyme L
was selected for further optimization of the assay as an
inexpensive enzyme model with good response to rutinose.
Viscozyme L is a fungal enzyme and fungal glycosidases
have typically pH optima within the interval 4.5–5.5.
Profiles of optimal pH of rhamnosidase activity in Vis-
cozyme L on both substrates similarly peaked at pH 5,
although the profile of rutinose hydrolysis had sharper
decline of activity outside this pH value (Fig. 1). Obvi-
ously, pH 5.0 was used in further α-L-rhamnosidase assays.
Examination of the rate of hydrolysis of rutinose as a
function of its initial concentration revealed that the
velocity reached its plateau at 139.3 mM (Fig. 2). The
initial concentration of rutinose 139.3 mM (corresponding
to 50 mg cm−3 substrate solution before mixing with the
enzyme) was then standardly used in assays of
rhamnosidase.
The relation between enzyme concentration and kinetics
of rutinose hydrolysis was tested in interval of Viscozyme
concentrations 0.23–3.64 mg cm−3. Enzyme loads above
0.9 mg cm−3 (corresponding to rhamnosidase activity
0.1 U cm−3) markedly shortened the interval of linear
course of rutinose hydrolysis (Fig. 3), and therefore, it is
preferable to stop the reaction after 5 min. Using 5 min
hydrolysis, the relation between velocity of hydrolysis and
enzyme load was linear in the tested interval of Viscozyme
concentrations (Fig. 4).
Fig. 1 Activity of α-L-rhamnosidase in Viscozyme L as a function of
pH determined with p-nitrophenyl α-L-rhamnoside (a) and rutinose
(b). Reaction mixture A (1 cm3, 40 °C) comprised 0.25 mg of
lyophilized Viscozyme in 4.75 mM solution of the pNP α-L-
rhamnopyranoside in 0.95 M acetate buffer, pH 5.0. The reaction
mixture B (1.1 cm3, 40 °C) comprised 2 mg of lyophilized Viscozyme
and 50 mg rutinose in 0.09 M acetate (closed circle) or 0.09 M
phosphate (open circle) buffer
Interferences and precautions
To develop a reliable rhamnosidase assay, several issues
had to be resolved to eliminate interferences and error
readings coming from the fact that the method is based on
estimation of glucose. The response to glucose may vary
depending on the storage of the working solution. Standard
of glucose must be, therefore, estimated together with
every assay. The enzymatic reaction in the withdrawn
sample must be immediately stopped to correctly estimate
the amount of produced glucose within time intervals.
Mixing the sample with three volumes of methanol and
immediate vortexing inhibited the tested enzymes. This
was proven by stability of final absorbance reads after
mixing with glucose oxidase solution. No inhibition effect
of methanol in the sample on glucose oxidase test was
observed. Obviously, methanol contents in the glucose
standard and in real samples were kept identical.
Glucose oxidase is known for negligible oxidation of
several other sugars than glucose [35]. Moreover, the
working solution of rutinose may contain traces of glucose
either as impurity from its synthesis or from spontaneous
hydrolysis of the disaccharide if low pH is necessary for
Fig. 2 Effect of rutinose concentration on velocity of its hydrolysis
by Viscozyme L. Incubations were carried at 40 °C in 0.1 M acetate
buffer pH 5.0 containing 27.9–278.6 mM rutinose and 1.82 mg cm−3
lyophilized Viscozyme
123

New assay of α-L-rhamnosidase
171
Fig. 5 Response of glucose oxidase test to 139.3 mM rutinose in
varying pH (expressed as concentration of glucose)
Fig. 3 Time course of the hydrolysis of rutinose expressed as
concentration of released glucose. Incubations were carried at 40 °C
in 0.1 M acetate buffer pH 5.0 containing 139.3 mM rutinose and
lyophilized Viscozyme in amounts: 0.23 mg cm−3 (open diamond),
0.45 mg cm−3 (closed circle), 0.91 mg cm−3 (closed triangle),
1.82 mg cm−3 (closed square), and 3.64 mg cm−3 (closed diamond)
be estimated directly, the bulk extenders may stepwise
hydrolyze after dissolving in assay mixture if a glycosidase
with respective activity is present. For example, commer-
´
cial pectinase Lallzyme Cuvee Blanc comprises low
rhamnosidase activity according to the assay with pNP
rhamnoside. When the enzyme was dissolved in buffer
with no rutinose added, gradual release of glucose occurred
(Fig. 6). On contrary, enzyme inactivated after 10 min
comprised only starting glucose level which did not rise up
during the further incubation. This is a proof that the
enzyme preparation itself can be source of gradual glucose
release, thus imitating rutinose hydrolysis. Such situation
may lead either to false positive results in rhamnosidase
screenings or to high background glucose hampering esti-
mation of low level of rhamnosidase. It is, therefore,
necessary to provide enzyme blanks (without rutinose)
within the assay.
Fig. 4 Velocity of rutinose hydrolysis after 5 min as a function of
load of Viscozyme L. Incubations were carried at 40 °C in 0.1 M
acetate buffer pH 5.0 containing 139.3 mM rutinose
rhamnosidase assay. Responses corresponding to the level
of glucose 0.26–0.28 mM over the pH range 3.5–7.0 were
observed for samples containing 139.3 mM rutinose
(Fig. 5). The stabilized absorbance reading did not rise up
in the time interval between 5 and 60 min, i.e., rutinose
itself is not substrate of glucose oxidase. The mentioned
negligible responses (below 0.2%) come probably from
traces of glucose present as impurity in the substrate. In
case of such low background response of pure substrate,
the blank of rutinose may be omitted.
Another precaution must be taken when commercial
enzymes comprising oligosaccharides as bulk extenders
such as lactose, starch, etc. are to be assayed. In such case,
the enzyme blank must be processed by exactly the same
method as the sample, including the stopping of the reac-
tion after identical time interval. Although free glucose can
Fig. 6 Development of glucose in solution of non-denatured (closed
´
square) and methanol-denatured (closed circle) Lallzyme Cuvee
Blanc (2 mg cm−3) in 0.1 M acetate buffer pH 5.0
123

´
E. K. Potocka et al.
172
α-L-Rhamnosidase assays in real samples
Experimental
Preparations of ten commercial enzymes of microbial ori-
gin were assayed for α-L-rhamnosidase activity both on
rutinose and on 4-nitrophenyl α-L-rhamnopyranoside (pNP
Rhap). Obviously, nominal values of rhamnosidase activity
measured on the two substrates were not identical. Samples
possessing moderate levels of rhamnosidase on the syn-
thetic substrate displayed three-to-four time higher nominal
activity in assays on rutinose (Table 2). The assay on the
synthetic substrate was, however, more sensitive in tests of
samples comprising low rhamnosidase and high glucose
background. The method had detected rhamnosidase
activity under 5 U g−1 of the enzyme, while assays of the
same samples on rutinose displayed zero or low response.
Some exception in reaction behavior was observed for
rhamnosidase in α-galactosidase DS from Amano. Its
response to rutinose was more than 18 times higher over
the response to pNP Rhap suggesting strong preference of
this enzyme to non-glycosylated rhamnosaccharides.
Catalysts and chemicals
Seeds of common buckwheat (Fagopyrum esculentum)
were purchased from a local supplier. Seeds of Fagopyrum
tataricum var. Madawaska were from the seed bank of the
National Agricultural and Food Centre, Research Institute
ˇ
ˇ
of Plant Production Piestany. High-purity recombinant α-L-
rhamnosidase (prokaryote, EC 3.2.1.40) was purchased
from Megazyme International (Ireland). Rapidase AR-
2000 was kind gift from Patrice Pellerin (DSM, USA),
´
Lallzyme BETA and Lallzyme Cuvee Blanc were pur-
chased from Lallemand (Canada). Lypolyve AN was from
Lyven (France), Enzeco Microbial Lipase was a gift from
Enzyme Development Corporation (USA) and Rohament
was a gift from Barentz Slovakia (local supplier of AB
Enzymes GmbH, Germany). Crude enzyme preparations
Viscozyme L and Novozym 188 were a kind gift from
´
´ˇ
Marian Illas (Biotech s.r.o., Slovakia) and were ultrafil-
tered and lyophilized prior use. BIO-LA-TEST Glucose®
kit is product of Erba Lachema (Brno, Czech Republic).
Celite® 545 was purchased from Alfa Aesar GmbH Co KG
(Germany). Powdered activated charcoal was purchased
from Mikrochem s.r.o. (Slovakia). TLC standards: anhy-
drous D-glucose was purchased from Mikrochem s.r.o.
(Slovakia), L-rhamnose 99%, rutin hydrate ≥94% (HPLC),
and quercetin hydrate ≥95% (HPLC) were purchased from
Sigma-Aldrich (USA). All other commercial reagents and
solvents used in experiments were purchased locally in
satisfactory purity (min. 95%).
Conclusion
We report a new photometric assay of α-L-rhamnosidase
based on hydrolysis of rutinose. Although the assay has a
limited application in screenings among commercial
enzymes with high content of glucose or glucose precursor,
it may find use in routine assays of rhamnosidases with
proven specificity towards rutinose provided that a set of
blanks is prepared together with the enzyme sample. The
substrate is easy to prepare in any chemical or biochemical
laboratory in gram quantities by simple, inexpensive
procedures.
Analytical methods and other apparatus
Spectrophotometric determination of liberated glucose was
performed on UV–Vis Spectrophotometer UV-1800 (Shi-
madzu, Kyoto, Japan). 1H NMR (600 MHz) and 13C NMR
Table 2 α-L-Rhamnosidase
activity towards substrates 1 and
2 at pH 6.0
Enzyme
Activity/U g−1
1
2
Viscozyme L (lyoph.)
Rapidase AR 2000
Enzeco lipase
25.1 0.1
7.1 0.8
9.9 0.3
14.6 1.0
12.1 0.4
2.5 0.0
6.3 0.2
0.9 0.1
1.6 0.0
4.5 0.4
81.3 4.8
29.6 3.4
34.4 3.4
50.4 8.2
33.9 2.7
3.9 1.4
119.2 1.4
n.d.
Novozym 188 (lyoph.)
Amano lipase A
Lallzyme BETA
α-Galactosidase DS
Ultrazym 100
´
Lallzyme Cuvee Blanc
n.d.
Lipolyve AN
n.d.
123

New assay of α-L-rhamnosidase
173
(151 MHz) spectra were recorded with a Varian 600 NMR
spectrometer. Spectra were taken with about 100 mM
solution in D2O.
subsequently eluted with gradient of ethanol in water
(800 cm3 of 2%, 800 cm3 of 4% and 800 cm3 of 8% ethanol
in water). Fractions containing pure disaccharide were
combined, concentrated on rotavap, and lyophilized over-
night to give 1.7 g (64%) of pale yellow powder with purity
above 97% (HPLC). We have prepared, overally, four
different batches in scales from 1 to 5 g and the chemical
yields of pure rutinose were always above 60%. Rf = 0.15
(glucose = 0.24, rhamnose = 0.46) (chloroform/methanol/
water, 12:7:1, v/v/v); [α]2D0 = −6.0 (c = 1.0, H2O, 24 h
equil.) [Ref. [36] [α]2D0 = −10 (c = 1.0, EtOH), Ref. [19]
[α]2D5 = −0.5 (c = 0.00834, H2O)]. The product decom-
posed at 182–189 °C [Ref. [37] 185–189 °C
(decomposition)]. Structure of the product was verified by
1H and 13C NMR (see supplementary file) and chemical
shifts of atoms agree with data published previously by
Roslund et al. [38]. The lyophilized powder was then used
for activity assay.
Incubations in small scale were performed in Eppendorf
tubes on Thermomixer comfort (Eppendorf AG, Hamburg,
Germany). Incubations in preparative scale were performed
in Thermobox KS 4000 ic control from IKA (Staufen,
Germany). Ultrafiltration was performed on filter VIVA-
FLOW 200 (10,000 MWCO PES) purchased from
¨
Sartorius Stedim Biotech (GmbH, Gottingen, Germany)
with peristaltic pump Masterflex L/S (Cole-Parmer Inter-
national, Vernon Hills, IL, USA). Lyophilization of the
ultrafiltered enzymes was performed on freeze dry system
LABCONCO Freezone 18 overnight. Optical rotations
were measured with a Perkin-Elmer 241 polarimeter at 20 °
C.
TLC alumina plates Silica gel 60 F254 from Merck
KGaA (Darmstadt, Germany) were used for following the
process of rutin hydrolysis by TLC. Plates were eluted by
chloroform/methanol/water (12/7/1, v/v/v). The spots were
detected by charring the plates with 5% (v/v) ethanolic
solution of H2SO4 and heating at ca. 200 °C.
α-L-Rhamnosidase assays
High-performance liquid chromatography was per-
formed on an Agilent 1200 Series apparatus consisting of
quarternary pump, RI and UV–Vis detectors, column
thermostat, and a Rheodyne injector with a 20 mm3 loop.
Purity of rutinose was estimated on Polymer IEX H+ col-
umn (250 9 8 mm) from Watrex eluted with 9 mM H2SO4
(1 cm3 min−1) at 35 °C with detection by RI detector.
Assay with rutinose: rutinose (50 mg in 1 cm3 0.1 M
acetate buffer pH 5.0 unless otherwise stated) was pre-
heated at 40 °C and mixed with 100 mm3 of α-L-rham-
nosidase solution. After 5 min, predefined volume of
reaction mixture was withdrawn and mixed with three
volumes of methanol and immediately vortexed to stop the
reaction. The methanolic solution (100 mm3) was then
added to 1 cm3 of pre-tempered working solution of the
glucose oxidase kit. The mixture was incubated for 15 min
at 37 °C along with the standard comprising 100 mm3
glucose (0.55 mM in methanolic buffer) and 1 cm3 working
solution. The absorbance at 498 nm was read not later than
after 40 min. Solution comprising corresponding concen-
tration of rutinose without enzyme and a solution with
corresponding amount of enzyme without rutinose were
used as blanks. All blanks were processed the same way as
the hydrolytic reaction, including time intervals and mixing
with methanol.
Preparation of rutinose
Seeds of tartary buckwheat were homogenized in coffee
grinder. Scaly parts were then separated on 1 mm screen
and the meal was extracted by ethyl acetate in Soxhlet
extractor for 48 h to remove fat and soluble substances.
Dried crude flour was then homogenized by mortar and
pestle and sieved through 0.3 mm screen. This material was
then stored in dark and dry conditions for further use.
Free rutinose was prepared by hydrolysis of rutin. As an
example, 5 g rutin (8.2 mM) was dissolved in 1 dm3 of
distilled water and 2.5 g of powdered tartary buckwheat
meal was added into the reaction. Hydrolysis ran for 3 h at
40 °C and 150 rpm. Solids were then separated from the
reaction mixture by vacuum filtration and the filtrate was
concentrated on rotary vacuum evaporator to volume about
20–30 cm3. The concentrate was applied on Biotage SNAP
cartridge filled with slurry of 50 g mixture of activated
charcoal and Celite® 545 (1:1, w/w) pre-equilibrated with
10 column volumes of distilled water. Monosaccharides
were eluted from the column with 1.2 dm3 of water.
Fractions of about 150 cm3 were continuously collected,
evaporated, and analyzed by TLC. Column was then
Concentration of glucose was calculated according to
the following equation:
As À Aeb À Arb
cglc ¼ cst Â
;
ð1Þ
Ast
where cglc stands for concentration of glucose in the sample
and cst for concentration of glucose in the standard. As, Aeb,
Arb, and Ast stand for absorbances of sample, enzyme blank,
rutinose blank, and standard glucose solution, respectively.
One unit of enzyme activity was defined as the amount
of enzyme catalyzing releasing 1 µmol of glucose min−1
under the described conditions. Within optimization
experiments, conditions of assay were varied according to
123

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E. K. Potocka et al.
174
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rutinose concentration, buffer type and pH, enzyme load,
and time of hydrolysis.
Assay with pNP α-L-rhamnopyranoside: an amount of
50 mm3 of diluted enzyme was mixed with 0.95 cm3 of
5 mM solution of the pNP α-L-rhamnopyranoside in 0.1 M
acetate buffer, pH 5.0. Reaction mixture was incubated for
5 min at 1000 rpm and 40 °C. The enzymatic reaction was
terminated by the addition of six volumes of saturated
solution of borax. For determination of spontaneous
hydrolysis of glycosides, 50 mm3 of buffer was added
instead of the enzyme sample and used as blank value. The
formation of p-nitrophenol was measured at 410 nm and its
respective amount was calculated from the calibration
curve. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the formation of 1 µmol of p-
nitrophenol per minute under the described conditions.
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Acknowledgements This work was supported by the Slovak
Research and Development Agency under the contract no. APVV-
0846-12, the Scientific Grant Agency of the Ministry of Education of
Slovak Republic, and Slovak Academy of Sciences (VEGA) under
the project no. 2/0157/16 and also the Research and Development
Operational Programmes funded by the ERDF (“Centre of Excellence
on Green Chemistry Methods and Processes”, CEGreenI, contract no.
26240120001, as well as the “Amplification of the Centre of Excel-
lence on Green Chemistry Methods and Processes”, CEGreenII,
contract no. 26240120025).
28. Mazzaferro LS, Pin˜uel L, Minig M, Breccia JD (2010) Arch
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