FEMS Microbiology Letters 157 (1997) 279^283
Puri¢cation and characterization of an K-L-rhamnosidase from
Aspergillus niger
Paloma Manzanares, Leo H. de Graa¡, Jaap Visser *
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen,
The Netherlands
Received 21 July 1997; revised 1 September 1997; accepted 6 October 1997
Abstract
An enzyme with K-L-rhamnosidase activity was purified by anion exchange chromatography from an Aspergillus niger
commercial preparation. The K-L-rhamnosidase was shown to be N-glycosylated, and had a molecular mass of 85 kD on
sodium dodecylsulfate-polyacrylamide gel electrophoresis of which approximately 12% was contributed by carbohydrate. The
enzyme was optimally active at pH 4.5 and 65³C. When tested towards p-nitrophenyl-K-L-rhamnopyranoside it showed Km and
Vmax values of 2.9 mM and 20.6 U mg31 , respectively whereas it was inhibited competitively by L-rhamnose (Ki 3.5 mM).
Substrate specificity studies showed K-L-rhamnosidase to be active both on K-1,2 and K-1,6 linkages to L-D-glucose. Moreover,
the enzyme was able to release L-rhamnose from geranyl-L-D-rutinoside and 2-phenylethyl-L-D-rutinoside.
Keywords: Aspergillus niger ; K-L-Rhamnosidase; Puri¢cation; Geranyl-L-D-rutinoside; 2-Phenylethyl-L-D-rutinoside
1. Introduction
L-rhamnose has been found as a constituent of
plant pigments and gums, glycolipids and glycosides.
Among glycosides L-rhamnose is known to be
present as an K-L-rhamnopyranosidic residue in naringin and hesperidin, citrus £avonoid compounds,
and in grape terpenyl glycosides, the glycosidic precursors of aromatic terpenols.
Although rhamnosidases are not common enzymes, several industrial applications have been reported. The use of K-L-rhamnosidases for removing
naringin, the main bitter component of several citrus
juices is a common industrial practise [1^4]. Also, the
* Corresponding author. Tel.: +31 (317) 484439; Fax: +31
(317) 484011; E-mail: [email protected]
hydrolysis of hesperidin by K-L-rhamnosidases to release rhamnose and hesperetin glucoside, an important precursor in sweetener production has been described [5]. Recently the interest in K-Lrhamnosidases mainly focused on their action towards terpenyl glycosides such as 6-O-K-L-rhamnopyranosyl-L-D-glucopyranosides (rutinosides) for the
enhancement of aroma in grape juices and derived
beverages [6,7].
Among fungi information about K-L-rhamnosidases is limited to that related to commercial enzyme
preparations derived from Aspergillus niger [4,8^10],
Aspergillus aculeatus [11] and Penicillium decumbens
[7,12,13]. Recently the production and characterization of an Aspergillus terreus K-L-rhamnosidase with
a possible application in the enhancement of wine
£avour has been published [14].
0378-1097 / 97 / $17.00 ß 1997 Published by Elsevier Science B.V. All rights reserved.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 4 8 7 - 4
280
P. Manzanares et al. / FEMS Microbiology Letters 157 (1997) 279^283
Hesperidinase is a fungal enzyme preparation
containing two enzymic activities, an K-L-rhamnosidase that splits hesperidin to rhamnose and the hesperetin glucoside, and a L-glucosidase that subsequently splits the hesperetin glucoside to glucose
and hesperetin. Hesperidinase obtained from A. niger
has been used for immobilization studies and for
measurements of the K-L-rhamnosidase activity on
hesperidin suspensions [8]. However, no separation
of the individual enzymic components was performed. In the present study we report the puri¢cation and physico-chemical characterization of an A.
niger K-L-rhamnosidase activity from a commercial
hesperidinase preparation. Substrate speci¢city studies towards several rhamnoglucosides are also described.
2. Materials and methods
2.1. Enzyme activity assays
K-L-rhamnosidase and L-D-glucosidase activities
were measured using p-nitrophenyl-K-L-rhamnopyranoside (PNP-Rha) and p-nitrophenyl-L-D-glucopyranoside (PNP-Glc) respectively as described previously [15]. One unit of enzyme activity was de¢ned
as the amount of enzyme that released 1 Wmol of pnitrophenol per min at 30³C in McIlvaine bu¡er pH
4.5.
2.2. Enzyme puri¢cation
Hesperidinase from A. niger was obtained from
Sigma. Hesperidinase (20 g) was dissolved in a 20
mM piperazine-HCl bu¡er pH 6 (200 ml) and dialysed against the same bu¡er. Column chromatography was carried out on DEAE-Sepharose Fast Flow
(2.5U25 cm) at 4³C. The column was equilibrated
with a 20 mM piperazine-HCl bu¡er pH 6 and elution of the bound proteins occurred by using a NaCl
gradient up to 0.5 M in the same bu¡er. The £ow
rate was 2 ml min31 and 20 ml fractions were collected. Fractions collected were screened for protein
content (A280 ), K-L-rhamnosidase activity and L-Dglucosidase activity.
2.3. Analytical methods
Protein concentrations were measured using the
BCA protein assay reagent (Sigma) using BSA as
standard. The puri¢cation of the K-L-rhamnosidase
was monitored by SDS/PAGE [16]. The proteins
were stained with Coomassie Brilliant Blue R-250.
For the calculation of the protein molecular mass a
10% SDS/polyacrylamide gel was used, calibrated
with protein test mixture 4 (Serva). Deglycosylation
of the enzyme with N-glycanase F followed the instructions of the supplier (Boehringer Mannheim,
Germany). The enzyme was O-deglycosylated by incubation in 0.1 M NaOH for 30 min at 25³C. The pI
of the K-L-rhamnosidase was determined by isoelectric focusing at 4³C in the pH range 3.5^9.5, using
the pI calibration kit from Pharmacia (broad range).
The proteins were stained with Coomassie Brilliant
Blue G-250. For the detection of K-L-rhamnosidase
activity after IEF, the gels were soaked in 50 mM
sodium acetate bu¡er pH 4.5 containing 1 mM 4methylumbelliferyl-K-L-rhamnoside (MU-Rha; Sigma). After 5 min of incubation at 25³C active bands
were visualized under UV light.
2.4. Enzyme characterization
The optimum pH for K-L-rhamnosidase activity
was determined by incubating the enzyme preparation with PNP-Rha in McIlvaine bu¡ers in the pH
range 3 to 8. The pH stability was assessed by preincubating the enzyme in McIlvaine bu¡ers over a
pH range from 3 to 5 at 30³C and measuring activities at 8 and 22 h using the standard protocol. The
optimum temperature was determined at the optimum pH by assaying the enzyme at di¡erent temperatures (25^80³C). The thermal stability of the enzyme was measured by pre-incubating the enzyme
in McIlvaine bu¡er pH 4.5 at di¡erent temperatures
and following the activity with time. Kinetic experiments were carried out at 30³C at the pH optimum.
The Michaelis-Menten constants were determined by
non-linear regression, using PNP-Rha at concentrations ranging from 0.067 to 4.65 mM. Inhibition
studies were performed using L-rhamnose at concentrations ranging from 0 to 20 mM.
P. Manzanares et al. / FEMS Microbiology Letters 157 (1997) 279^283
281
2.5. Substrate speci¢city towards rhamnoglucosides
Substrate speci¢city studies of the K-L-rhamnosidase activity towards the rhamnoglucosides hesperidin, naringin, quercitrin and rutin (Sigma) were performed by high performance anionic exchange
chromatography (HPAEC) using a Dionex system
equipped with a Dionex CarboPac PA-100 and a
pulsed electrochemical detection unit in the pulsed
amperometric detection mode. For the standard assay the rhamnoglucosides were dissolved at 0.25%
(m/v) concentration in 50 mM sodium acetate bu¡er
pH 4.5 and incubated with puri¢ed enzyme, at a ¢nal
concentration in the incubation mixture of 4.5 Wg
ml31 , for up to 6 h at 30³C. L-Rhamnose was determined isocratically using 40 mM NaOH at a £ow
rate of 1 ml min31 . Substrate speci¢city towards
the glycosides geranyl-L-D-rutinoside and 2-phenylethyl-L-D-rutinoside was performed by thin layer
chromatography (TLC) as described previously [7].
3. Results and discussion
3.1. Puri¢cation
The dialysed hesperidinase fraction containing K(0.57 U mg31 ) and L-D-glucosidase
activities (0.41 U mg31 ) was applied onto a DEAESepharose Fast Flow column. Ion exchange chromatography separated the two enzyme activities. K-LRhamnosidase eluted as a single peak at 0.1^0.15
M NaCl whereas L-D-glucosidase eluted as two peaks
between 0.17^0.3 M NaCl. The speci¢c activity of
the fractions containing K-L-rhamnosidase activity
was found to be 3.1 U mg31 , with an approximately
5-fold increase in speci¢c activity and a level of recovery of 84%. Speci¢c activities of 2.04 U mg31 (pH
3.5, 57³C) and 31.7 U mg31 (pH 4, 37³C) were reported for the puri¢cation of the K-L-rhamnosidase
component of two di¡erent naringinase preparations
[9,13].
L-rhamnosidase
3.2. Biochemical characterization
The puri¢ed K-L-rhamnosidase gave a single band
on SDS/PAGE with an apparent molecular mass of
85 kDa. The molecular mass after N-deglycosylation
Fig. 1. SDS/PAGE of the puri¢ed K-L-rhamnosidase before and
after N- and O-deglycosylation treatments. Lane 1, 6.9 Wg K-Lrhamnosidase untreated; lane 2, 6.9 Wg N-glycanase treated K-Lrhamnosidase; lane 3, 6.9 Wg O-deglycosylated K-L-rhamnosidase.
The molecular mass values of the markers (M) are shown on the
left.
decreased by about 12% resulting in a value of 75
kDa. Alkali treatment had no in£uence, which indicates that the enzyme is solely N-glycosylated (Fig.
1). Values of 87 kDa and 90 kDa have been described for K-L-rhamnosidases from A. aculeatus
[11] and A. terreus [14], respectively. The 90 kDa
K-L-rhamnosidase from P. decumbens was reported
to contain approximately 50% carbohydrate [13].
K-L-Rhamnosidase showed microheterogeneity
upon isoelectric focusing. Several protein bands
could be seen in the pH range 4.5^5.2, all having
K-L-rhamnosidase activity towards MU-Rha (results
not shown). pI values within the range from 4.6 to 5
have been reported for A. niger [10], A. aculeatus [11]
and A. terreus [14] K-L-rhamnosidase activities. In
the case of Penicillium sp. [7] two K-L-rhamnosidase
isoenzymes (pI 5.7 and 6.2) were resolved by chromatofocusing.
The pH optimum of the puri¢ed K-L-rhamnosidase
was found to be 4.5 in McIlvaine bu¡er. Between pH
3.5 and 5 the enzyme showed 90% of its maximum
activity. K-L-Rhamnosidase activity was stable in the
pH range from 3 to 5 for up to 8 h at 30³C. After 22
h of incubation at 30³C the enzyme retained 60% of
its initial activity over the same pH range. A similar
pH optimum pro¢le has been described for three
282
P. Manzanares et al. / FEMS Microbiology Letters 157 (1997) 279^283
Fig. 2. TLC analysis of the enzymic degradation of glycosides.
Lane 1, K-L-rhamnosidase blank; lane 2, L-rhamnose (Rf 0.53)
and D-glucose (Rf 0.18) standards; lane 3, geranyl-L-D-glucoside
(Rf 0.73); lane 4, geranyl-L-D-rutinoside (Rf 0.55); lane 5, geranyl-L-D-rutinoside after treatment with K-L-rhamnosidase; lane 6,
2-phenylethyl-L-D-glucoside (Rf 0.68); lane 7, 2-phenylethyl-L-Drutinoside (Rf 0.52); lane 8, 2-phenylethyl-L-D-rutinoside after
treatment with K-L-rhamnosidase.
di¡erent A. niger K-L-rhamnosidase preparations
[4,8,9] and for the Penicillium sp. K-L-rhamnosidase
activity [12]. A. aculeatus and A. terreus K-L-rhamnosidases showed a pH optimum in the range from
5.5 to 8 [11,14].
The optimum temperature for the puri¢ed K-Lrhamnosidase at pH 4.5 was found to be 65³C. At
30³C the activity was approximately 20%. The stability of the enzyme was measured at 30, 40, 50 and
65³C. The enzyme was stable up to 40³C for 4 h
whereas at 50³C the enzyme retained still 85% of
its original activity. At its optimum temperature
the enzyme retained 60% of its initial activity after
1 h of incubation. For K-L-rhamnosidases from various Aspergilli slightly lower temperature optima
have been reported ranging from 50 to 60³C
[4,9,11,14].
3.3. Kinetic parameters
The Michaelis constant Km and the Vmax value of
K-L-rhamnosidase were found to be 2.9 mM and 20.6
U mg31 , respectively (pH 4.5, 30³C). When using
PNP-Rha as substrate similar Km values, 2.32 mM
[10] and 2.65 mM [9] have been described for A.
niger K-L-rhamnosidases, whereas a lower Km value,
1.52 mM, was found for the Penicillium sp. K-Lrhamnosidase activity [12]. With respect to Vmax values, 13 U mg31 (pH 5, 40³C) and 10.7 U mg31 (pH
3.5, 57³C), were found for A. aculeatus [11] and Penicillium sp. [12] K-L-rhamnosidase activities, respectively. The A. niger enzyme evidently is the most
active one.
End-product inhibition by L-rhamnose was also
studied. The reaction was competitively inhibited
by L-rhamnose, with a Ki value of 3.5 mM. Inhibition studies have only been described for the K-Lrhamnosidase activity from Penicillium sp [12].
Rhamnose was found to be a competitive inhibitor
of the K-L-rhamnosidase activity with Ki values of
1.2 mM and 4.27 mM when using PNP-Rha or naringin as substrates, respectively.
3.4. Substrate speci¢city
K-L-Rhamnosidase was active towards naringin,
hesperidin and rutin, but it was not able to release
L-rhamnose from quercitrin (Table 1). In naringin
the L-rhamnose residue is K-1,2 linked to the L-Dglucoside whereas in rutin and hesperidin it is K-1,6
linked. Thus, the enzyme seems to hydrolyse both K1,2 and K-1,6 linkages to L-D-glucosides. In quercitrin, the rhamnosyl residue is linked directly to the
aglycon and this might be the reason why the enzyme is not active. A. niger K-L-rhamnosidase activities showing di¡erent substrate speci¢cities have
been reported. For instance, K-L-rhamnosidase activities from di¡erent A. niger preparations were active
towards naringin and rutin but not against hesperidin [9], or only active against naringin [4] and hesTable 1
Substrate speci¢city of
Substratea
A. niger K-L-rhamnosidase
Rhamnose releasedb (nmol)
Hesperidin
2.4
Naringin
33.0
Rutin
8.2
Quercitrin
n.d.c
a Substrate concentration: 0.25% (m/v).
b Incubation time: 90 min.
c n.d.: not detected.
P. Manzanares et al. / FEMS Microbiology Letters 157 (1997) 279^283
peridin [8]. The K-L-rhamnosidase activity from A.
aculeatus has been described as active towards naringin and hesperidin [13].
The enzyme puri¢ed by us was also active towards
geranyl-L-D-rutinoside and 2-phenylethyl-L-D-rutinoside, natural rhamnoglucosides found in grape juice
and wine [7,17]. K-L-Rhamnosidase cleaved the
rhamnose-glucose linkage (L-rhamnose K-1,6 linked
to D-glucose) of geranyl- and 2-phenylethyl-L-D-rutinosides, as can be seen in Fig. 2 by the release of Lrhamnose (RF 0.53) and the corresponding glucosides, geranyl-L-D-glucoside (RF 0.73) and 2-phenylethyl-L-D-glucoside (RF 0.68). Only the K-L-rhamnosidase activity from P. decumbens has been described
as active towards grape monoterpenyl disaccharide
glycosides [9].
The thermostability, kinetic properties and the
broad substrate speci¢city displayed by the A. niger
K-L-rhamnosidase described here may allow its application in biotechnological processes such as debittering of citrus juices. Its action towards grape glycosides suggests its possible application for aroma
enhancement during winemaking processes.
Acknowledgments
This work has been supported by the EC project
AIR3-CT94-2193. P.M. was the recipient of a Formacioèn Personal Investigador fellowship from the
Spanish Government. We thank Dr. Gunata,
INRA Montpellier, for the TLC analysis.
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