Bioresource Technology 115 (2012) 222–227

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Preparatory production of quercetin-3-b-D-glucopyranoside using alkali-tolerant

thermostable a-L-rhamnosidase from Aspergillus terreus
Lenka Weignerová a, Petr Marhol a, Daniela Gerstorferová a,b, Vladimír Křen a,⇑
a
Institute of Microbiology, Center for Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ 142 20, Prague 4, Czech Republic
b
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Chemická 5, CZ 160 00, Prague 6, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: Extensive screening for a robust producer of a-L-rhamnosidase activity from well-defined strains of fila-
Available online 11 August 2011 mentous fungi, including multifactorial optimization (inducers, cultivation conditions) was accom-
plished. Enzyme production of the optimal producer Aspergillus terreus (non-toxigenic) was scaled up
Keywords: to 50 L. a-L-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus
Isoquercitrin enabling effective operation at 70 °C and pH 8.0. These conditions allow for a very high substrate (rutin)
Quercetin-3-b-D-glucopyranoside load up to 100–300 g/L, thus enabling very high volumetric productivity of the reaction product querce-
Rutin
tin-3-b-D-glucopyranoside (isoquercitrin). Here, a novel concept of ‘‘immobilised substrate’’ is used. Iso-
a-L-Rhamnosidase
Aspergillus terreus
quercitrin is a highly effective and biocompatible antioxidant with strong anti-inflammatory activities.
Rutin biotransformation was optimized and scaled up to ca 10 kg production and thus the robustness
of the large-scale production was demonstrated. Isoquercitrin can be produced to a very high purity
(98%) in multikilogram amounts, without any quercetin and directly applicable in nutraceuticals.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Flavonoids are a large group of polyphenolic compounds that
occur ubiquitously in vascular plants and therefore are a regular
component of the human diet. Many flavonoids have antioxidant,
antiinflammatory, antiallergenic, antibacterial, and generally
chemoprotectant capabilities (Formica and Regelson, 1995; Rice-
Evans et al., 1996; Hollman and Katan, 1997; Middleton et al.,
2000; Terao et al., 2008). Moreover, a number of epidemiologic
studies indicated the protective effects of flavonoids against car-
diovascular diseases and cancer (Hertog et al., 1995; Hollman
et al., 1996; Knekt et al., 1997; Xing et al., 2001). Quercetin (3, 2-
(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one)
(for all structures and the reaction scheme see Supplementary
data, Fig. 1S) is one of the most abundant flavonoids present in
plants; it occurs in high concentrations in onions, apples, tea, and
many other natural sources. However, the low oral bioavailability
of quercetin, due to its insolubility in water, limits its use as a food
additive or dietary supplement. Recent studies indicate that glyco-
syl conjugation substantially enhanced the bioavailability of quer-
cetin (Lesser et al., 2004). Isoquercitrin (2, quercetin-3-b-D-
glucopyranoside; 2-(3,4-dihydroxyphenyl)-3-b-D-glucopyranosyl-
oxy-5,7-dihydroxy-4H-1-benzopyran-4-one) is the most common
naturally occuring quercetin derivative (denoted also as Bioquerce-
⇑ Corresponding author. Tel.: +420 296442510; fax: +420 296442509.
E-mail address: kren@biomed.cas.cz (V. Křen).
tin) with a higher water solubility than quercetin and better intes-
tinal absorption than rutin (1, 3-[[6-O-(a-L-rhamnopyranosyl)-b-D-
glucopyranosyl]oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-
1-benzopyran-4-one) (Cermak et al., 2003; Shimoi et al., 2003;
Makino et al., 2009; Jung et al., 2010).
Moreover, quercetin – despite the fact that it is one of the best
flavonoid antioxidant – has a rather poor reputation, giving a posi-
tive Ames test. This may be caused by its intercalation into DNA,
causing topoisomerase II inhibition. Quercetin glycosides, which
maintain the antioxidant activity of quercetin, do not interact with
DNA (Webb and Ebeler, 2004). Therefore, the availability of, e.g.
quercetin-3-b-D-glucopyranoside at a good quality (devoid of free
quercetin) and at a reasonable price for application in nutraceuti-
cals is of the utmost importance.
Probably the most feasible procedure for preparing quercetin-3-
b-D-glucopyranoside is selective enzymatic ‘‘trimming’’, e. g. der-
hamnosylation of rutin, which is available for an affordable price
at pharmaceutical quality. Rutin is typically obtained from the Bra-
zilian tree Fava d’anta (Dimorphandra mollis). Enzymatic derham-
nosylation of rutin has, however, numerous technical problems:
(i) the selectivity of enzymatic glycoside hydrolysis thus avoiding
the generation of (highly undesirable) free quercetin; (ii) the gen-
erally low water solubility of all quercetin derivatives, including
glycosides; (iii) the high sensitivity of all quercetin derivatives to
oxidation; (iv) the strict necessity of avoiding the use or presence
of any heavy metals due to their strong complexation by quercetin
(generally, by most flavonoids). The required biotechnological

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.08.029
 L. Weignerová et al. / Bioresource Technology 115 (2012) 222–227
procedure must also use no pathogenic or toxigenic organisms at
any stage of production. The use of only chemicals acceptable for
food and drug use is also a must.
The enzymatic processes published so far (or patented Bucholz
et al., 2002; Chang and Muir, 2004) try to circumvent these prob-
lems. Virtually all a-L-rhamnosidase preparations contain b-gluco-
sidase and sometimes also rutinosidase, which cause the
simultaneous production of unwanted quercetin from the isoqu-
ercitrin produced or directly from rutin, respectively. The purifica-
tion of a-L-rhamnosidase is not technologically feasible due to the
high price of the process; a recombinant enzyme would solve this
problem in an elegant way, however GMO materials are not al-
lowed under some legislations.
Unwanted b-glucosidase could be knocked-out by selective
inhibitors (d-gluconolacton, Bhiri et al., 2008; Yoon et al., 2008;
Nijikken et al., 2007; Seidle et al., 2004) or by some specific phys-
ical conditions based, e.g. on the better thermostability of rham-
nosidase vs. glucosidase.
The issue of the very low water solubility of rutin (0.12 g/L,
20 °C, Chebil et al., 2007) was previously addressed mostly by
the addition of water-miscible cosolvents – typically methanol
(You et al., 2010), acetonitrile, acetone, and tert-amyl alcohol (Che-
bil et al., 2007), which were not only incompatible from a toxicol-
ogy point of view, but often substantially decreased the a-L-
rhamnosidase activity and the solubility only just reached 10 g/L,
thus providing poor volumetric productivity.
The solubility of all flavonoids (typically polyphenols) is
strongly pH-dependent. Polyphenols are weak acids; therefore,
their solubility strongly increases with rising pH and naturally
with higher temperature. High pH is, however, often incompatible
with the activity of enzymes.
In this study, screening for an efficient and robust alkali- and
thermo-stable a-L-rhamnosidase – preferably an extracellular one
– from a known microbial strain that is neither pathogenic nor
toxigenic was performed. Based on the selected enzyme from A.
terreus, we developed a robust enzymatic biotechnological proce-
dure with very high volumetric productivity and simple product
processing, thus producing high-purity isoquercitrin, devoid of
quercetin at the multi-kilogram scale and for an affordable price.
The very good stability of the a-L-rhamnosidase allowed its use
in the form of a crude enzyme without further cost and time con-
suming purification, and also enabling its repeated use, employing
the concept of an ‘‘immobilised substrate’’.
2. Methods
2.1. Materials
p-Nitrophenyl a-L-rhamnopyranoside (pNP-a-L-Rha), HPLC
standards of quercetin and rutin were from Sigma–Aldrich, USA,
p-nitrophenyl b-D-glucopyranoside (pNP-b-D-Glc) was from Senn
Co., CH; rutin was from Alchimica, CZ.
2.2. Fungal strains used
All strains used in the screening were from the Culture Collec-
tion of the Institute of Microbiology, Prague or from the Culture
Collection of Fungi (CCF), Department of Botany, Charles University
in Prague. The production strain Aspergillus terreus CCF 3059 is
deposited in the CCF.
2.3. Enzyme activity assay
a-L-Rhamnosidase and b-D-glucosidase activities were assayed
using pNP-a-L-Rha and pNP-b-D-Glc as substrates, respectively.
223
One unit of enzymatic activity is defined as the amount of enzyme
releasing 1 mol of p-NP per minute in 50 mM citrate–phosphate
buffer at pH 6.0 and 35 °C. After incubation of the reaction mixture
at 35 °C for 10 min, the liberated p-NP was determined spectro-
photometrically at 420 nm under alkaline conditions (50 L of the
reaction mixture was added to 1 mL of 0.1 M Na2CO3); p-NP (0–
2 mM) was used for calibration.
2.4. Preparation of the crude enzyme
2.4.1. Cultivation conditions
The culture was maintained on the following slants [g/L]: agar-
agar, 20; bacto-peptone, 5; and malt extract 35. The cultivation med-
ium contained [g/L]: 15.0, KH2PO4; 4.0, NH4Cl; 0.5, KCl; 5.0, yeast ex-
tract; 1.0, Casamino acids (Oxoid, UK). The medium was
supplemented with the specific inducer (L-rhamnose; 0–70.0 g/L
or rutin; 5 g/L or quercetin; 5 g/L) and 1 mL/L of trace-element Vish-
niac solution (Vishniac and Santer, 1975), pH adjusted to 4.0 or 6.0
and after sterilization, MgSO4.7H2O sterile solution (10% w/v) was
added. Conical flasks (500 mL) with 100 mL of medium were inocu-
lated with 0.5 mL of a spore suspension in 0.1% (v/v) Tween 80 solu-
tion and cultivated on a rotary shaker at 200 rpm and 28 °C or 26 °C.
Cultures were sampled (filtration) over the given periods and as-
sayed for extracellular a-L-rhamnosidase activity.
2.4.2. Large scale fermentation
Batch fermentations were carried out in a 10 L bioreactor (Bio-
engineering, CH) with a working volume of 8.5 L and in a 75 L bio-
reactor (Bioengineering, CH) with a working volume of 50 L. The
reactors were equipped with pH, dissolved oxygen and tempera-
ture probes (all Ingold, CH). The medium (see Section 2.4.1, indu-
cer: L-rhamnose 10 g/L, pH 6.0) was sterilized in the fermentor
and inoculated aseptically with 10% (v/v) of seed culture (prepared
as described in Section 2.4.1. using a three-day cultivation in
flasks). The medium was stirred (3 bottom radial stirrers, 4 baffles,
400 rpm) and the fermentation continued at 28 °C for 3–10 days.
2.5. Bioconversion of rutin
2.5.1. Analytical scale
After the cultivation (Section 2.4.1) the mycelium was sepa-
rated by filtration (Filter Discs, grade 1392, P-Lab, CZ). The crude
enzyme solution (50 mL) was supplemented with ethanol to final
concentrations of 0%, 10% or 20% and the pH was adjusted to 6.5,
7.0, 7.5, 8.0 or 8.5. The solution was heat-treated at 70 °C for
30 min. The rutin was then suspended (concentration 20, 50, 70,
100, 150, 200, 250 or 300 g/L) in the enzyme solution. The reaction
mixture was stirred at 70 °C and the rutin conversion was moni-
tored by HPLC.
2.5.2. Pilot plant scale
After the fermentation (Section 2.4.2) the mycelium was re-
moved by filtration. The medium filtrate (45 L) was heat-treated
at 70 °C for 30 min and the pH was adjusted to 8.0 by KOH. The
medium has good buffering capacity, probably due to remaining
unconsumed phosphate. Rutin (6.75 kg, 150 g/L) was suspended
in the buffered enzyme solution; the reaction mixture was stirred
(3 bottom axial stirrers, 4 baffles, 600 rpm) at 70 °C and the conver-
sion was monitored by HPLC. After the substrate was completely
consumed (24 h), the reaction was stopped by heating to 95 °C
(for 20 min). After cooling down to 30 °C (ca 2 h) the lower phase
containing the bulk of the sedimented product (isoquercitrin)
was removed by careful decantation to avoid tedious filtration. Iso-
quercitrin was washed by decantation with warm water (3  10 L,
30 °C) to remove all released L-rhamnose as well as further impu-
rities (salts). The thick yellow suspension was then spray-dried
224 L. Weignerová et al. / Bioresource Technology 115 (2012) 222–227

Activity (U/ml)
4

0
3 5 7 9 11
Cultivation (days)
Fig. 1. Induction of a-L-rhamnosidase activity in A. terreus culture with L-rhamnose (different concentrations).  0 g/L, d 5 g/L, j 10 g/L, N15 g/L, e 25 g/L, h 35 g/L, s 50 g/L,
x 70 g/L.

(Anhydro Compact, Wotol, FR, capacity 20 L/h, input/output tem-

perature 230/85 °C) and isoquercitrin was obtained as a soft yellow
fluffy powder in a very high purity (98%). The structure of isoqu- A 120 350
ercitrin was confirmed by 1H and 13C NMR and MS conforming to
the published data (Bouktaib et al., 2002). 300

Rhamnosidase/Glucosidase
100

2.6. Analytical HPLC

250
Activity (%)

80
A very quick and simple chromatographic method to monitor
rutin biotransformation had to be developed. This method using 200
a modern silica-based monolithic column enables the separation 60
of all analytes within 6.5 min. HPLC assays were performed using 150
a Shimadzu Prominence system (Kyoto, JP) consisting of a DGU-
20A mobile phase degasser, two LC-20AD solvent delivery units, 40
a SIL-20AC cooling autosampler, CTO-10AS column oven and 100
SPD-M20A diode array detector with standard analytical cell vol-
20
ume (10 lL). Chromatographic data was collected and processed 50
using Shimadzu LC solution software (Kyoto, JP) at a rate of
40 Hz and a detector time constant of 0.025 s. A Chromolith Perfor-
0 0
mance RP-18e 100  3 mm monolithic column equipped with a
20 30 40 50 60 70 80 90
guard column (5  4.6 mm) (all Merck, DE) was used with binary
gradient elution, acetonitrile/water/trifluoroacetic acid (5/95/0.1, Temperature (°C)
v/v/v, phase A) and acetonitrile/water/trifluoroacetic acid (80/20/
0.1, v/v/v, phase B); 0–4 min 7–30% B, 4–5 min 30% B, 5 – 6 min B 120 100
30–7% B. The mobile phase was filtered prior to use through a
0.45 lm nylon membrane filter (Whatman, USA) and used at flow 100 Rhamnosidase/Glucosidase
rate of 1.5 mL/min. The column oven temperature was set to 25 °C 80
and the samples were kept at 20 °C in the sample manager. The
PDA data were acquired in the 190–500 nm range and the 80
Activity (%)

360 nm signal was used for quantification. 60

60
2.7. Spectral characterization
40
40
NMR spectra were recorded on a Bruker Avance III-400 MHz
spectrometer (400.13 MHz for 1H, 100.61 MHz for 13C) in CD3OD. 20
Mass spectra were obtained using an ultraFLEX III spectrometer 20
(MALDI TOF, Bruker-Daltonics, DE). A 10 mg/mL solution of 2,5-
dihydrobenzoic acid (DBH) in 50% acetonitrile/0.3% acetic acid 0 0
was used as the MALDI matrix.
3 4 5 6 7 8 9
pH
3. Results and discussion
Fig. 2. A: Effect of temperature on activity of a-L-rhamnosidase (d),b-D-glucosidase
3.1. Screening for a-L-rhamnosidase activity (j) and a-L-rhamnosidase/b-D-glucosidase ratio (D) at pH 7.0. B: Effect of pH on the
activity of a-L-rhamnosidase (d), b-D-glucosidase (j) and a-L-rhamnosidase/b-D-
glucosidase ratio, the measurements at pH 4.0–6.5 were carried out in 50 mM
The screening of a large library of filamentous fungi (40 strains) citrate–phosphate buffer and the measurements at pH 6.5–9.0 were carried out in
was performed under various cultivation conditions using L-rham- 50 mM borate buffer.
 L. Weignerová et al. / Bioresource Technology 115 (2012) 222–227 225

nose and different L-rhamnose-containing flavonoid glycosides as three-day cultivations were sufficient for the preparatory experi-
specific inducers (Monti et al., 2004) with the aim of finding a tech- ments. Operational costs of the pilot plant fermentor (75 L) are
nologically suitable producer of a-L-rhamnosidase. The level of very high and the production in three days was sufficient to per-
undesired b-D-glucosidase activity was also a decisive parameter. form a batch reaction. Naturally, the scale-up to larger volumes
The preparation obtained from an A. terreus CCF 3059 strain under will be strongly dependent on specific local technological condi-
L-rhamnose induction displayed a maximum a-L-rhamnosidase/b- tions but this also demonstrates that the process has still further
D-glucosidase activity ratio (20:1, cultivation at pH 6.0 and potential to improve.
28 °C).a-L-Rhamnosidase production by A. terreus was tested at
various L-rhamnose concentrations (0–70 g/L, Fig. 1). During the 3.3. Influence of pH and temperature on the activity and stability of a-
cultivation, the extracellular activities of a-L-rhamnosidase and L-rhamnosidase and b-D-glucosidase
b-D-glucosidase were monitored. The addition of L-rhamnose did
not influence the production of b-D-glucosidase. The highest a-L- The presence of b-D-glucosidase activity is highly undesirable for
rhamnosidase production was in the medium with 15 g/L L-rham- the selective rutin biotransformation (Fig. 1S, Supplementary data)
nose (5.5 U/mL), which accounted for an 11-fold increase com- and its removal or inactivation is a fundamental requirement. D-Glu-
pared to the medium without L-rhamnose. Considering the price- cose was tested as a selective b-D-glucosidase inhibitor; however, it
performance criterion (L-rhamnose price vs. production increase) also inhibited a-L-rhamnosidase activity and cannot be used (Fig. 6S
(Fig. 1), 10 g/L of L-rhamnose as the inducer was used in the prepa- in Supplementary data). Co-solvents, such as ethanol and glycerol
ratory experiments, as the increase from 10 to 15 g/L only produces did not improve the process mainly due to rather strong inhibition
a negligible improvement. Rapid decline of the enzyme activity in of a-L-rhamnosidase (Figs. 3S, 4S, 5S Supplementary data).
the case of higher rhamnose concentration (25 and 35 g/L) can be The pH and temperature optima and stabilities of both a-L-
explained by simultaneous pH decrease (<pH 2, data not shown), rhamnosidase and b-D-glucosidase were measured (Fig. 2). a-L-
which is caused by deregulation of the citric cycle with high carbo- Rhamnosidase from A. terreus compared to b-D-glucosidase is a
hydrate concentration analogously as, e.g. in the citric or itaconic strongly alkali- and thermo-tolerant enzyme. a-L-Rhamnosidase
acid production by aspergilli (Mitsuyasu et al., 2009). has an optimum at 70 °C while b-D-glucosidase quickly lost its
activity above 55 °C. The pH profile of both enzymes somewhat
3.2. Large scale a-L-rhamnosidase production correlated but the a-L-rhamnosidase exhibited a higher activity
at pH levels above 8. Moreover, a-L-rhamnosidase displayed far
The enzyme cultivation was scaled up using 10 g/L of L-rham- greater temperature stability than b-D-glucosidase (data not
nose as an optimal inducer concentration. Batch fermentations shown). High temperature (70 °C, 30 min) treatment completely
were tested in a 10 L bioreactor and then in a 75 L bioreactor deactivated b-D-glucosidase, whereas in contrast the activity of
(see Supplementary data, Figs. 2S-A,B). The extracellular produc- a-L-rhamnosidase slightly increased. a-L-Rhamnosidase is inhib-
tion of both a-L-rhamnosidase and b-D-glucosidase did not change ited by rhamnose as a feedback inhibitor (50 mM L-rhamnose,
in both cases and were comparable to the production at the sha- 30% activity; 150 mM L-rhamnose, 10% activity) (Soria and Ellen-
ken-flask scale. Considering the price-performance criterion rieder, 2002). To summarize, the crude heat-treated enzyme (culti-
(a-L-rhamnosidase activity vs. bioreactor operational costs) vation medium alone) was a suitable biocatalyst for the efficient

A 175 100 B 175 100 C 175 100

Relative concentration (%)

150
150
Relative concentration (%)

150

Relative concentration (%)

75
Concentration (g/L)

Concentration (g/L)

75
Concentration (g/L)

125 75 125
125
100 100 100
50 50 50
75 75 75

50 50 50
25 25 25
25 25 25

0 0 0 0 0 0
0 5 10 0 5 10 0 5 10 15 20 25
Conversion (hours) Conversion (hours) Conversion (hours)

D 350 100

300
Relative concentration (%)

75
Concentration (g/L)

250

200
50
150

100
25
50

0 0
0 10 20 30 40 50
Conversion (hours)

Fig. 3. Bioconversion of rutin, a-L-rhamnosidase 2.4 U/mL, V = 20 mL, initial concentration: (A) 70 g/L, (B): 100 g/L, (C): 150 g/L, (D): 300 g/L. Rutin [g/L] , conversion[%] e;
isoquercitrin [g/L] j, [%] h; quercetin [g/L] d, [%]). s.
226 L. Weignerová et al. / Bioresource Technology 115 (2012) 222–227

and selective biotransformation of rutin. This technological ‘‘trick’’
(see also Schepers et al., 2006; Mueller et al., 2006; Gödde et al.,
2005; Wolterink-van Loo et al., 2005) circumvented tedious en-
zyme purification that moreover results in considerable activity
losses and it was substantially more economical and fully suitable
for the large-scale production of isoquercitrin.
3.4. Biotransformation of rutin
The following procedures were optimized, mainly taking into
account the economics of the process, its scalability and the purity
of the final product. Therefore, some parameters employed are at
the sub-optimal level taking into account the price-performance
criterion.
The cultivation medium (typically 3–4 days old, a-L-rhamnosi-
dase activity 1.5–2.5 U/mL) was separated from the mycelium of
A. terreus by filtration. Extending the cultivation would yield
somehow higher activity of 3–4 U/mL (see Supplementary data
Fig. 2S), but this would also considerably increase the operational
costs.
The pH level of the crude enzyme was adjusted to pH 8.0, heat-
treated (70 °C, 30 min) and then rutin was directly suspended with
stirring into the enzyme solution, the pH and temperature were
kept constant. Under relatively high substrate loads (rutin) of
70–150 g/L, the bioconversion was successfully completed within
24 h (Fig. 3A–C). Further increase in rutin concentration was lim-
ited due to the feedback inhibition of a-L-rhamnosidase with liber-
ated L-rhamnose (Fig. 3D) and, therefore, the reaction was
practically stopped. The L-rhamnose released could in principle
be removed during the bioconversion process, e.g. by dialysis.
Although the high pH and temperature improves rutin solubil-
ity, the majority of both the product and the starting material re-
mains in the solid phase, nevertheless, the reaction proceeds very
well. This is an ‘‘immobilised substrate’’ concept that also enables
the eventual reuse of the enzyme solution after the reaction is
completed by simple filtration.

Fig. 4. Schematic diagram of production process: (i) Fermenter was inoculated aseptically with 10% (v/v) of inoculum. (ii) Culture was grown at 28 °C for 5 days. (iii) Medium
filtrate was heat-treated at 70 °C for 30 min and pH was adjusted to 8.0. (iv) Rutin (6.75 kg, 150 g/L) was suspended in crude enzyme. (v) Reaction was stirred and monitored
by HPLC. After the substrate was consumed (24 h), the reaction was stopped by heating to 95 °C (for 20 min). (vi) Isoquercitrin was washed by decantation with warm water
(3  10 L, 30 °C) to remove all released L-rhamnose. (vii) Thick yellow suspension was spray-dried. (viii) Enzyme solution containing isoquercitrin (<1.0 g/L, RT) and free L-
rhamnose (>150 mM, inhibitor of a-L-rhamnosidase). (ix) After L-rhamnose removal, enzyme solution could be used for rutin bioconversion. (x) After salt and nutrient
supplementation, solution could be used as a further cultivation medium. (xi) L-Rhamnose source.
 L. Weignerová et al. / Bioresource Technology 115 (2012) 222–227
After the reaction was completed, the suspension was cooled to
15 °C and pH adjusted to 6, thus promoting complete precipitation
and decantation of the product. The liquid phase after filtration still
contained active a-L-rhamnosidase (having an activity only in 10%
lower than the initial activity) and the liberated L-rhamnose could
be reused as: (i) starting material for the cultivation medium of A.
terreus to save on rather expensive L-rhamnose (after salt and
nutrient supplementation), (ii) for a second rutin bioconversion
(after the removal of inhibitory L-rhamnose (e.g. dialysis or ultrafil-
tration) the enzyme solution could be reused, (iii) as a source of L-
rhamnose as a commodity chemical (Fig. 4).
After decantation, the crude isoquercitrin was rinsed three
times with water and finalized by spray-drying, yielding isoquerci-
trin in a ca 92% yield as a fine yellow powder with a 98% purity
(HPLC, see Fig. 7S – Supplementary data).
4. Conclusion
Isoquercitrin is an antioxidant and chemoprotective nutraceuti-
cal with better (solubility, bioavailability) properties than rutin.
Wider use, e.g. in animal experiments, has been hindered by its
high price (isoquercitrin > 90%, 50 mg 59.70 EUR, Sigma 2011). This
procedure is based on ‘‘green chemistry’’ principles without any
toxic additives. This original, efficient and fully scalable and
waste-free bioproduction of isoquercitrin enables the production
of this natural product by biocompatible enzymatic process from
another natural product (rutin) available in bulk quantities. Pilot
plant processing and the finalization make the product suitable
for either use in nutraceuticals or as a high purity fine chemical.
Acknowledgements
Financial support from EU FP7 project NOVOSIDES FP7-KBBE-
2010-4-265854, Czech Science Foundation Grant P301/11/0767,
and by research concept of the Institute of Microbiology
AV0Z50200510 is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2011.08.029.
References
Bhiri, F., Chaabouni, S.E., Limam, F., Ghrir, R., Marzouki, N., 2008. Purification and
biochemical characterization of extracellular b-glucosidases from the
hypercellulolytic Pol6 mutant of Penicillium occitanis. Appl. Biochem.
Biotechnol. 149, 169–182.
Bouktaib, M., Atmani, A., Rolando, Ch., 2002. Regio- and stereoselective synthesis of
the major metabolite of quercetin, quercetin-3-O-b-D-glucuronide. Tetrahedron
Lett. 43, 6263–6266.
Bucholz, H., Koppe, T., Schleehahn, M., 2002. Method for enzymatic splitting of
rutinosides. US Patent No. 6, 420, 142 B1, 16 November.
Cermak, R., Landgraf, S., Wolffram, S., 2003. The bioavailability of quercetin in pigs
depends on the glycoside moiety and on dietary factors. J. Nutr. 133, 2802–
2807.
Chang, P.R., Muir, A., 2004. Extraction, purification and conversion of flavonoids
from plant biomass. Patent Application WO 2004/027074 A2.
Chebil, L., Humeau, C., Anthoni, J., Dehez, F., Engasser, J.M., Ghoul, M., 2007.
Solubility of flavonoids in organic solvents. J. Chem. Eng. Data 52, 1552–1556.
Formica, J.V., Regelson, W.J., 1995. Review of the biology of quercetin and related
bioflavonoids. Food Chem. Toxicol. 33, 1061–1080.
227
Gödde, C., Sahm, K., Brouns, S.J.J., Kluskens, L.D., Oost, J., Vos, W.M., Antranikian, G.,
2005. Cloning and expression of islandisin, a new thermostable subtilisin from
Fervidobacterium islandicum, in Escherichia coli. Appl. Environ. Microb. 71, 3951–
3958.
Hertog, M.G.L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F.,
Giampaoli, S., Jansen, A., Menotti, A., 1995. Flavonoid intake and long-term risk
of coronary heart disease and cancer in the seven countries study. Arch. Intern.
Med. 155, 381–386.
Hollman, P.C.H., Hertog, M.G.L., Katan, M.B., 1996. Role of dietary flavonoids in
protection against cancer and coronary heart disease. Biochem. Soc. Trans. 24,
785–789.
Hollman, P.C.H., Katan, M.B., 1997. Absorption, metabolism and health effects of
dietary flavonoids in man. Biomed. Pharmacother. 51, 305–310.
Jung, S.H., Kim, B.J., Lee, E.H., Osborne, N.N., 2010. Isoquercitrin is the most effective
antioxidant in the plant Thuja orientalis and able to counteract oxidative-
induced damage to a transformed cell line (RGC-5 cells). Neurochem. Int. 57,
713–721.
Knekt, P., Jarvinen, R., Seppanen, R., Hellovaara, M., Teppo, L., Pukkala, E., Aromaa, A.,
1997. Dietary flavonoids and the risk of lung cancer and other malignant
neoplasms. Am. J. Epidem. 146, 223–230.
Lesser, S., Cermak, R., Wolffram, S., 2004. Bioavailability of quercetin in pigs is
influenced by the dietary fat content. J. Nutr. 134, 1508–1511.
Makino, T., Shimizu, R., Kanemaru, M., Suzuki, Y., Moriwaki, M., Mizukami, H., 2009.
Enzymatically modified isoquercitrin, a-oligoglucosyl quercetin 3-O-glucoside,
is absorbed more easily than other quercetin glycoside or aglycone after oral
administration in rats. Biol. Pharm. Bull. 32, 2034–2040.
Middleton Jr., E., Kandaswami, C., Theoharides, T.C., 2000. The effect of plant
flavonoids on mammalian cells: implications for inflammation, heart disease,
and cancer. Pharmacol. Rev. 52, 673–751.
Mitsuyasu, O., Dwiarti, L., Shin, K., Park, E.Y., 2009. Biotechnological production of
itaconic acid and its biosynthesis in Aspergillus terreus. Appl. Microbiol.
Biotechnol. 84, 597–606.
Monti, D., Pišvejcová, A., Křen, V., Lama, M., Riva, S., 2004. Generation of an a-L-
rhamnosidase library and its application for the selective derhamnosylation of
natural products. Biotechnol. Bioeng. 87, 763–771.
Mueller, P., Egorova, K., Vorgias, C.E., Boutou, E., Trauthwein, H., Verseck, S.,
Antranikian, G., 2006. Cloning, overexpression, and characterization of a
thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus
abyssi. Protein Expr. Purif. 47, 672–681.
Nijikken, Y., Tsukada, T., Igarashi, K., Samejima, M., Wakagi, T., Shoun, H., Fushinobu,
S., 2007. Crystal structure of intracellular family 1 b-glucosidase BGL1A from
the basidiomycete Phanerochaete chrysosporium. FEBS Lett. 581, 1514–1520.
Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure-antioxidant activity
relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933–
956.
Schepers, B., Thiemann, V., Antranikian, G., 2006. Characterization of a novel
glucoamylase from the thermoacidophilic archaeon Picrophilus torridus
heterologously expressed in E. Coli. Eng. Life Sci. 6, 311–317.
Seidle, H.F., Marten, I., Shoseyov, O., Huber, R.E., 2004. Physical and kinetic
properties of the family 3 b-glucosidase from Aspergillus niger which is
important for cellulose breakdown. Protein J. 23, 11–23.
Shimoi, K., Yoshiyumi, K., Kido, T., Usui, Y., Yumoto, T., 2003. Absorption and urinary
excretion of quercetin, rutin, and alphaG-rutin, a water soluble flavonoid, in
rats. J. Agric. Food Chem. 51, 2785–2789.
Soria, F., Ellenrieder, G., 2002. Thermal inactivation and product inhibition of
Aspergillus terreus CECT2663 a-L-rhamnosidase and their role on hydrolysis.
Biosci. Biotechnol. Biochem. 66, 1442–1449.
Terao, J., Kawai, Y., Murota, K., 2008. Vegetable flavonoids and cardiovascular
disease. Asia Pac. J. Clin. Nutr. 17, 291–293.
Vishniac, V., Santer, A., 1975. The Thiobacilli. Bacteriol. Rev. 21, 195–213.
Webb, M.R., Ebeler, S.E., 2004. Comparative analysis of topoisomerase IB inhibition
and DNA intercalation. Biochem. J. 384, 527–541.
Wolterink-van Loo, S., Levisson, M., Cabrieres, M.C., Franssen, C.R., Oost, J., 2005.
Characterization of a thermostable dihydrodipicolinate synthase from
Thermoanaerobacter tengcongensis. Extremophiles 12, 461–469.
Xing, N., Chen, Y., Mitchell, S.H., Young, C.Y.F., 2001. Quercetin inhibits the
expression and function of the androgen receptor in LNCaP prostate cancer
cells. Carcinogenesis 22, 409–414.
Yoon, J.J., Kim, K.Y., Cha, C.J., 2008. Purification and characterization of thermostable
b-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on
microcrystalline cellulose. J. Microbiol. 46, 51–55.
You, H.J., Ahn, H.J., Ji, G.E., 2010. Transformation of rutin to antiproliferative
quercetin-3-glucoside by Aspergillus niger. Agric. Food Chem. 58, 10886–10892.