Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 α-L-rhamnosidase and their role on hydrolysis of naringin solutions

Article abstract of DOI:10.1271/bbb.66.1442

The kinetics of thermal inactivation of A. terreus α-rhamnosidase was studied using the substrate p-nitrophenyl α-L-rhamnoside between 50°C and 70°C. Up to 60°C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60°C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60°C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.

Full text of DOI:10.1271/bbb.66.1442

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Thermal Inactivation and Product Inhibition of
Aspergillus terreus CECT 2663 α-L-Rhamnosidase and
Their Role on Hydrolysis of Naringin Solutions
Fernando SORIAa & Guillermo ELLENRIEDERa
a Chemical Industry Research Institute (INIQUI), National University of Salta-CONICET
Buenos Aires 177-4400 Salta, Argentina
Published online: 22 May 2014.
To cite this article: Fernando SORIA & Guillermo ELLENRIEDER (2002) Thermal Inactivation and Product Inhibition
of Aspergillus terreus CECT 2663 α-L-Rhamnosidase and Their Role on Hydrolysis of Naringin Solutions, Bioscience,
Biotechnology, and Biochemistry, 66:7, 1442-1449, DOI: 10.1271/bbb.66.1442
To link to this article: http://dx.doi.org/10.1271/bbb.66.1442
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Biosci. Biotechnol. Biochem., 66 (7), 1442–1449, 2002
Thermal Inactivation and Product Inhibition of Aspergillus terreus CECT
2663
a- -Rhamnosidase and Their Role on Hydrolysis
L
of Naringin Solutions
†
Fernando SORIA and Guillermo ELLENRIEDER
Chemical Industry Research Institute (INIQUI), National University of Salta-CONICET,
Buenos Aires 177-4400 Salta, Argentina
Received October 26, 2001; Accepted March 8, 2002
hydrolysis products of natural glycosides6). The
manufacture of the sugar rhamnose from naringin is
an example of the latter application.7) For this proc-
ess it is important that the enzyme be stable at high
temperatures where the solubility of the substrate,
the stability of a supersaturated solution, and the
reaction rate are high.8) Inhibition of the enzyme by
high concentrations of products is also important,
because it limits the rate and yields of hydrolysis
products.
The kinetics of thermal inactivation of A. terreus
rhamnosidase was studied using the substrate p-
nitrophenyl -rhamnoside between 50 C and 70 C.
Up to 60 C the inactivation of the puriˆed enzyme was
completely reversible, but samples of crude or partially
puriˆed enzyme showed partial reversibility. The
presence of the product rhamnose, the substrate narin-
gin, and other additives reduced the reversible inactiva-
tion, maintaining in some cases full enzyme activity at
a-
a-
L
9
9
9
609C. A mechanism for the inactivation process, which
permitted the reproduction of experimental results, was
proposed. The products rhamnose (inhibition constant,
In previous papers the characterization and puriˆ-
cation of an Aspergillus terreus
a- -rhamnosidase
L
2.1 m
M
) and prunin (2.6 m
M
) competitively inhibited the
with high speciˆc activity was described.9–11) In view
of the importance for its applications, we report here
the thermal stability and inhibition by products of
the enzyme.
enzyme reaction. The maximum hydrolysis of super-
saturated naringin solution, without enzyme inactiva-
tion, was observed at 60
reached 99 with 1 naringin solution, although the
hydrolysis degree of naringin was only 40 due to
products inhibition when the initial concentration of
‰avonoid was 10 . The experimental results ˆtted an
9
C. Hydrolysis of naringin
%
%
%
Materials and Methods
%
Materials. Naringin, p-nitrophenyl
a
-
L
-rhamno-
equation based on the integrated Michaelis-Menten's,
including competitive inhibition by products satisfac-
torily.
side (pNPR), and p-nitrophenyl
b
-
D
-glucoside
(pNPG) were from Sigma (St.Louis, Mo, USA).
Glutaraldehyde (50 in water) was from Fluka AG
z
and all other reagents were of analytical grade. Stan-
dards for HPLC of naringin and prunin were from
Extrasynthese (France).
Key words:
a
-
L
-Rhamnosidase; Rhamnose; thermal
inactivation; product inhibition
Prunin (4?,5,7-trihydroxy‰avanone 7-glucoside)
The enzyme
a
-
L
-rhamnosidase (Rase, EC 3.2.1.40)
z
was prepared by hydrolyzing a 10 supersaturated
was ˆrst used in the citrus industry in applications
such as debittering of grapefruit juices by hydrolysis
naringin solution with 7 Uml„1 of an A. niger narin-
ginase (which was a gift from Tanabe, Japan) at
of the bitter component naringin (4
?,5,7-trihydrox-
50
tem was kept at 4
rated by ˆltration, dissolved to 10
hydrolyzed again at 50 C with a new portion of en-
zyme. After a new crystallization at 4 C, the solid
was ˆltered, washed with cold water, and dried at
50 C. The purity of the obtained prunin was 92z,
9
C and pH 4.0 for four hours. After that the sys-
y‰avanone 7-rhamnoglucoside)1–2) and elimination of
hesperidin crystals from orange juices.3) Rase was the
main component of the commercial enzyme complex-
es naringinase and hesperidinase used for these pur-
poses. Later Rase was also used for aroma improve-
ment of wines4–5) and enzymatic preparation of
9
C for 48 h, and the solid was sepa-
z
at 80–90 C, and
9
9
9
9
†
To whom correspondence should be addressed.
Abbreviations: Rase,
a-L-rhamnosidase; Glase, b-D-glucosidase; pNPR, p-nitrophenyl rhamnoside; pNPG, p-nitrophenyl glucoside; [E]0,
initial enzyme concentration; E, active enzyme; I, irreversibly inactivated enzyme; R, reversibly inactivated enzyme; k1, k„1, k2, k3, rate con-
stants of thermal inactivation; f1, k, l1, l
2, constants of integrated equation 3, 4 and 5; K mobs, Michaelis constant in presence of inhibitor;
V mobs, maximum velocity observed in presence of inhibitor; KI, inhibition constant; [P], concentration of product; [S]0, initial concentration
of substrate; [S], concentration of substrate; a, b, constants of equation 8; X, conversion of enzymatic reaction

Thermal Inactivation of A. terreus
a
-Rhamnosidase
1443
measured by HPLC, and it was free from residual
naringin.
cinic acid Na succinate buŠer, pH 5.5, at 80–909C,
W
cooling down to the reaction temperature and imme-
diately adding the enzyme. To register the reaction
course of the hydrolysis of naringin, residual sub-
strate and prunin were measured by high pressure liq-
uid chromatography with a Shimadzu LC-4 A equip-
ment with a C18 column and UV detector (280 nm).
Enzyme preparation and assays. Production of
culture ˆltrates, inactivation of b-glucosidase (Glase)
from the culture ˆltrates at pH 11, and puriˆcation
of Rase from Aspergillus terreus strain CECT 2663,
were done as described previously.10) The enzyme was
also freed from Glase by dye a‹nity chromatography
The carrier was a 32:68 acetonitrile water mixture.
W
Triplicate kinetics experiments are shown in the
ˆgures. Inactivation was tested in at least two repli-
cates of each puriˆcation or culture. The graphics,
non-linear regressions, and statistic calculations were
done using the PRISM software of GraphPad, USA.
using a column of Red HE-3b sepharose according
W
to the following procedure: part of the Glase of the
enzyme complex was retained in a ˆrst step at pH 5.5,
then Rase and the rest of the Glase were retained in a
second passage at pH 4.7, ˆnally Rase was separated
from Glase by eluting the column at pH 5.5 (F. Soria,
Results and discussion
G. Ellenrieder, M. Graselli, A. N. del Can
ã izo, and O.
Cascone, unpublished results).
Kinetics of the thermal inactivation of puriˆed
rhamnosidase
a-
For the crosslinking of the protein, samples of 1 ml
of the puriˆed enzyme solution (10 Uml„1) were incu-
bated with 10–50 ml of 50 glutaraldehyde at room
z
The knowledge of the mechanism of thermal inac-
tivation of enzymes can be of importance in the selec-
tion of the most convenient experimental conditions
and the deˆnition of strategies for thermostabiliza-
tion.14–15) In order to obtain information of utility for
applications of the enzyme, the kinetics of the ther-
mal inactivation of Rase was studied at pH 5.5 be-
temperature from 15 up to 60 min, then the excess of
reagent was removed by Sephadex G-25 gel ˆltration.
The activity of Rase was measured using pNPR as
described by Romero et al.,12) and 0.05
M
succinic
acid sodium succinate pH 5.5 was used as buŠer.
W
Glase activity was estimated with pNPG.13) For both
enzymes, one unit of activity (U) was deˆned as the
9 9
tween 50 C and 70 C. To detect and avoid possible
interferences of contaminant proteins, puriˆed
Rase10) was used, and the results were compared with
that of the crude and partially puriˆed preparations
of the enzyme. The values of residual activity are
plotted as a function of time in Fig. 1(A). It was
observed that the activity was completely recovered
amount of catalyst that liberates 1
nitrophenol per minute.
mmol of p-
Kinetics studies. The kinetics of the thermal inacti-
vation of the Rase were measured as follows: A sam-
ple of 0.8 to 26 units (3.5 to 91
one to three ml of 0.05
cinate buŠer, pH 5.5, was left at the inactivation tem-
perature. Portions of 20 to 100 l were taken out at
m
g protein) of Rase in
when the inactivated enzyme at 50
cubated at 40 C for about ˆve h. The recovery of the
activity was only partial for treatments at 65 C and it
was not observed at 70 C (Fig. 1(A)). From these
9 9
C or 60 C was in-
M
succinic acid sodium suc-
9
W
9
m
9
diŠerent times and the activity measured immedi-
ately. The reversibility of the inactivation was tested
results it could be assumed that at least three diŠerent
states of the enzyme were present during the process:
active or ``native'' enzyme (E), deactivated enzyme
that can be reversibly recovered (R), and irreversibly
inactivated enzyme (I). Assuming that only these
three species were present, their concentrations were
calculated from the data of Fig. 1(A) and plotted as a
by incubating the samples at 409C until no more in-
crease of activity was observed. In order to detect
possible interferences in measurements of residual
activity in the presence of naringin or its hydrolysis
products, blanks, incubated without enzyme under
the same conditions of the activity measurements,
were tested. Enzyme recovery after hydrolysis was
done using Microsep centrifugal concentrators of
Filtron (USA).
The measurement of the inhibition constant of
prunin was done by dissolving it in buŠer at 909C,
then cooling it down to the reaction temperature,
then mixing it with pNPR. After the selected hydrol-
function of time for the inactivation at 659C in
Fig. 1(B). The existence of the enzyme state R is
postulated only taking into account the reversibility
of the thermal inactivation. If E and R diŠer appreci-
ably in size, form, or number of ionic charges ex-
posed to the solvent, their separation by chro-
matography or electrophoresis could be proposed.
However, traditional chromatographic and elec-
trophoretic methods are not fast processes compared
with the rate of transformation of R to E (Table 3),
so this hypothetic separation could be ineŠective. Ex-
ysis time, a sample of 50
ml was added to 1.5 ml of
0.5 NaOH and the absorbance measured at one-
M
min intervals. Blanks without enzyme were run in
parallel in order to correct for the interference pro-
duced by prunin.
periments at pH 4.0 and 60
9C showed that the resid-
ual activity was 20 after 30 min. This indicated that
z
Hydrolysis of supersaturated naringin solutions
the thermal stability of the enzyme was appreciably
lower than that observed at pH 5.5. These values are
were done by dissolving the substrate in 0.05
M
suc-

1444
F. SORIA and G. ELLENRIEDER
Fig. 1. Thermal Inactivation of Puriˆed
a
-Rhamnosidase at DiŠerent Temperatures.
Initial activity: 5.24 Uml„1; BuŠer: 0.05
M succinic acid Na succinate pH 5.5 A) EŠects of temperature. Residual activity immediately
W
after the inactivating treatment (RA): Full symbols and solid lines. Activity after a recovering treatment at 40
and dotted lines. B) Time course of the concentration of the enzyme species. [E] active enzyme ([E] RA); [R] reversibly deactivated
enzyme ([R] RAR-R); [I] irreversibly inactivated enzyme ([I] [E]0-RAR). Solid lines were obtained by non-linear regression using
equations 3, 4, and 5. Temperature 65 C.
9
C (RAR): Open symbols
＝
＝
＝
9
Fig. 2. EŠects of Enzyme Level and Presence of Substrate on Thermal Inactivation.
A) EŠects of enzyme concentration. Initial activity:
0.8 Uml„1 5.24 Uml„1
(bovine serum albumin). B) Inactivation in presence of 4 naringin. C with 4 naringin; : 70
with 4 naringin; : 65 C without naringin;    65 C without naringin, after incubation at 40 C; : 609C with 4z
21 Uml„1
;

3.3 Uml„1+2 mg ml„1 BSA
C without naringin; : 65
naringin.
#

z
9
;

;

: 70
9
z
9
#

9C
z
9
9
near of that published by Gallego et al.,11) which were
done at pH 4.0.
naringin was also measured using membrane-cen-
trifuge concentrators, which separate the enzyme
from the main part of low molecular weight substrate
and products. These results of both methods were
similar. The thermal inactivation of puriˆed Rase in
The eŠect of enzyme concentration on inactivation
9 9 9
at 60 C and 70 C is shown in Fig. 2(A). At 70 C
only a small decrease was observed with this inacti-
vated enzyme, although the enzyme level was in-
the presence of 4
z
initial naringin is plotted at diŠer-
creased from 0.8 Uml„1 to 26 Uml„1 (3.5 to 91.3
m
g
ent temperatures as a function of time in Fig. 2(B).
These curves are of importance because they
represent the operational stability of the enzyme dur-
ing the process of production of rhamnose and pru-
nin. It can be seen that the reversible deactivation at
protein ml„1). Even the addition of two mg ml„1 of
bovine serum albumin (BSA) had no appreciable
in‰uence on the inactivation. A similar eŠect was ob-
9
served at 60 C. This behaviour indicates that neither
aggregation nor dissociation are important steps in
the inactivation mechanism. This result is diŠerent
60
(which was not restored by incubation at 40
observed at 65 C, it was appreciably smaller than
9
C was practically eliminated. A loss of activity
9
C) was
from that found for the
a
-rhamnosidase of Penicilli-
9
16)
um decumbens
,
which is stabilized at high enzyme
that of the reversible inactivation of the enzyme
alone, but approximately similar to its corresponding
irreversible process. At 709C, the inactivation was
concentration and also by inert proteins.
The activity of the Rase was measured in the
presence of naringin and prunin. These ‰avonoids
can be transformed to chalcones in the basic medium
used for p-nitrophenol measurement in the test of
activity,12) being a possible source of interference due
to the production of color at 400 nm.17) However,
after dilution, this interference was only of the order
near that of the enzyme alone. These results suggest
that the protective eŠect of this substrate is due to an
inhibition of the reversible inactivation, but the
irreversible process is not aŠected by its presence.
Nearly the same amount of [I] was found in inactiva-
tions of the free enzyme as in presence of naringin.
Other additives, like rhamnose, and polyalcohols
such as sorbitol and glycerol, also reduced the rever-
of 5
z
of the p-nitrophenol absorbance and was
easily corrected by subtracting the value of the con-
trol. Activity after the hydrolysis in presence of
9
sible thermal inactivation at 60 C (Table 1). The

Thermal Inactivation of A. terreus
a
-Rhamnosidase
1445
stabilization eŠect caused by the bifunctional reagent
glutaraldehyde is also shown in Table 1. It reduced
the reversible deactivation without any eŠect on the
irreversible one. The crosslinking produced by this
substance prevents normal unfolding of the en-
zyme,18) however in this case the eŠect is somewhat
lower than that of the additives.
Thermal inactivation of crude and partially puri-
ˆed -rhamnosidase
The residual activity after treatments at 60
a
9
C of
several crude and partially puriˆed samples are
shown in Table 2. It can be observed that the inacti-
vation of Rase in crude culture ˆltrates depends on
the carbon source used in its production. Cultiva-
tions using rhamnose as the carbon source showed an
inactivation of the same order as that of the puriˆed
enzyme. Gallego-Custodio et al.9) found comparable
results. However, when naringin was used as the car-
bon source, the enzyme was more stable, and was
9
scarcely inactivated at 60 C. This diŠerence could be
Table 1. EŠects of Additives and Cross-linking on the Thermal
attributed to the rest of naringin in the culture
ˆltrates. However, one of these samples maintained
its stability after it was treated at pH 11 for selective
Stability of Puriˆed
a-Rhamnosidase
Additive
Residual activity (
Time (h)
z)
T
or
Treatment
(
9
C)
inactivation of
b-glucosidase, precipitated with
treatment
None
1
2
3
(NH4)2SO4, redissolved and then dialyzed. These
steps certainly removed the rest of the naringin. This
diŠerence in thermal stability was caused perhaps by
diŠerent glycosylation of the enzymes during the
60
60
65
65
Inactivationa 70
I+Recoveryb 99
Inactivation
±
±
±
±
1
2
.3
3
65
100
56
±
±
±
±
0
1
4
63
99
54
±
±
±
±
2
4
1
.3
67
I+Recovery 91
84
.5 74
production step.19) The recovery of activity at 40
9C
Rhamnose
60
Inactivation
99
87
±
±
4
98
±
3
96
±
5
2
of the non-puriˆed Rases produced with rhamnose as
carbon source, was however only partial. In samples
taken during the diŠerent steps of the puriˆcation, it
was only after the last gel ˆltration chromatography
(5 g„1
)
Sorbitol
(0.1
60
65
65
Inactivation
Inactivation
I+Recovery
5
95
75
76
±
±
±
3
2
3
93
±
M
)
that the inactivation at 609C became fully reversible.
In spite of the partial irreversibility of the inactiva-
tion, the presence of naringin prevented the loss of
Glycerol
(25
60
Inactivation
90
90
±
5
95
±
4
88
±
1
z
)
activity of the crude enzyme at 60
9
C. Probably the
GA cross-
linking
60
60
65
65
Inactivation
±
±
±
±
5
3
1
1
89
87
59
58
±
±
±
±
.4 82
±
±
±
±
1
3
.3
1
transformation of the state R to I at 60
9
C was
I+Recovery 89
Inactivation 77
I+Recovery 77
1
0
2
82
45
44
favored by impurities, but naringin inhibited the
transformation of E to R. Due to this eŠect of the
substrate, crude preparations of Rase could be nearly
±
Values are the mean of two experiments standard deviation.
Residual activity immediately after incubation.
as eŠective for the hydrolysis at 60
enzyme.
9
C as the puriˆed
a
b
Inactivation follow by incubation at 40
GA: glutaraldehyde.
9C, 10 hours.
Table 2. Thermal Inactivation at 60
9C of
a
-Rhamnosidase Preparations of DiŠerent Purity
Residual activity (
2
z
) Time (h)
Sample
Treatment
1
3
4
Inactivationa
66
77
4
67
80
3
2
61
75
3
59
70
2
2
±
±
±
±
±
±
1. Culture ˆltrate carbon source: rhamnose
I+Recoveryb
±
3
±
1
±
3
±
±
2
±
2. Culture ˆltrate carbon source: naringin
Inactivation
98
100
3
98
104
5
3. Partially puriˆed sample carbon source: naringinc
Inactivation
I+Recovery
96
99
1
3
95
95
1
1
90
93
1
3
96
100
0
4
±
±
±
±
±
±
±
±
4. Partially puriˆed sample carbon source: rhamnosed
5. Puriˆed enzyme carbon source: rhamnose
Inactivation
I+Recovery
70
70
2
0
63
2
62
2
62
1
±
±
±
±
68 .3
±
±
65 .4
±
±
±
±
±
±
±
61 .4
±
4
Inactivation
I+Recovery
70 .3
65
100
0
1
63
108
2
2
±
2
99
99
Values are the mean of two experiments
±
standard deviation.
C.
C, 10 hours.
Puriˆcation includes: treatment pH 11, (NH4)2SO4 precipitation, and dialysis.
a
Residual activity immediately after incubation at 60
Incubation at 60 C follow by incubation at 40
9 9
9
b
c
d
Puriˆcation includes: (NH4)2SO4 precipitation, dialysis, and DEAE sepharose cromatography.

1446
F. SORIA and G. ELLENRIEDER
Table 3. Kinetic Constants of the Thermal Inactivation of Puriˆed a-Rhamnosidase
Constant Temp.(
9
C)
Value
Evaluation procedure
k1
k1
k„1
k„1
50
60
50
60
0.32
0.44
1.7
±
±
±
±
.04
Data ˆt by non-linear regression using the equation of a reversible ˆrst order process.
.06
0.3
0.1
0.7
k2
65
70
0.099
1.4
±
±
.002
0.5
Fit of the data of Fig. 1(B) by non-linear regression using equation 5.
k2
65
70
0.09
1.2
±
±
0.01
0.6
Fit of the data of inactivation in the presence of naringin (Fig. 2(B)) by non-linear regression
using the equation of a ˆrst order decay.
f1
l1
l2
k
65
65
65
65
0.31
2.3
0.084
0.40
±
±
±
±
0.03
0.5
0.009
0.02
Data ˆt by non-linear regression using equations 3 and 4.
＝
k3 (as it was observed at 70
Mechanism of thermal inactivation
coe‹cient of step (3), k3
?
Assuming that only the enzyme species E, R, and I
participate in the inactivation process of the puriˆed
enzyme, the following mechanism is proposed:
9
C) k2[R]+k3[E] k3([E]+[R]). This last equation
＝
＝
indicates that k2 k3, a relation that permits us to
simplify the diŠerential equations corresponding to
the proposed inactivation mechanism. Taking into
account that k2 k3, from the reactions scheme, the
following system of independent ˆrst-order diŠeren-
tial equations can be easily derived:
k1
＝
1)
E
R
k„1
k2
2)
3)
R
E
I
I
k3
[E ]
d
d

＝
„(k1
+
k
2)[
E
]+k„1
[
]
R
]
(1)
(2)
dt
The step 3 was introduced considering that narin-
gin reduced the concentration [R] without modiˆca-
tion of the rate of production of I, so, the species I
could be produced directly from E. The ˆrst two
steps resemble the classical mechanism of inactiva-
tion including reversible unfolding followed by an ir-
reversible inactivation,15) however R is not an inter-
mediate between E and I, and actual unfolded states
between E or R and I could be present. In a lapse of 4
hours the irreversible inactivation became apprecia-
[
R]
＝
k1 ]„(k„1
[
E
+
k
2)[
R
dt
the resolution of this system leads to:
l
1t
l
2t
[
E
]
＝
[
E
]0( f1e－ +(1„f1
)
e－
)
(3)
(4)
(5)
l
2t
l1t)
＝
]
[
R
[
E
]0(1„f1)k( „
e－ e－
]0(1„e„
[E )
k2t
＝
]
[
I
where f1,
the kinetics constants k1, k„1, and k2. The curves of
Fig. 1(B) at 65 C and 70 C were ˆtted by non-linear
k, l1 and l2 are constants that depend on
ble only at 659C (Fig. 1(A)), but at lower tempera-
tures the process was reversible and only the ˆrst two
steps could be observed. Therefore the kinetic con-
9
9
regression using these eq. (3) to (5). In the three cases
the run test of the residues indicated that the devia-
tion from the models were not signiˆcant. The kinetic
constants are shown in Table 3.
stants k1 and k„1 were evaluated at 50
using the rate equation of a reversible ˆrst order
process, (Table 3). At 65 C and at 70 C all steps
9C and at 609C
9
9
must be taken into account. The results obtained in
the presence of substrate can be summarized accord-
ing to the following: if during inactivation of the en-
In the presence of naringin, step 1 is presumably
inhibited, with R being absent in the system, equa-
tion 5 is yet valid and [E] can be evaluated consider-
zyme alone [E]0
＝
[E]+[R]+[I] and in the presence
ing that: [E]0
＝
[E]+[I]. The decay of E became a
＝
of substrate [E
?]
[E?
]+[R
[E ]0 and it was observed that
0 (no reversible inactivation in the presence of
substrate). As the rate of production of I was in-
?]+[I?], experimentally
0
ˆrst-order process with a rate constant k2.
This is a simpliˆed mechanism for the inactivation
of puriˆed Rase, and probably additional steps could
be included, however the derived equations ˆtted the
experimental results.
it was taken that [E]0
＝
?
＝
[R?]
dependent of the presence of substrate, d[I] dt
＝
W
＝
d[I
?
] dt and [I] [I
?
]. As a consequence of these equ-
W
ations [E
?]
＝
[E]+[R], and considering the steps (2)
Inhibition by products
＝
and (3) of the proposed mechanism d[I] dt
W
Measurements of the enzyme activity done in the
presence of rhamnose or prunin showed that these
products inhibited the hydrolysis. For each level of
＝
＝
＝
k?3([E]+[R]), as-
k2[R]+k3[E] d[I
?] dt k3
?
[E
?
]
W
suming that the substrate does not modify the rate

Thermal Inactivation of A. terreus
a
-Rhamnosidase
1447
Fig. 3. Inhibition of
A) Rhamnose; buŠer: 0.05
C.
a
-Rhamnosidase by Products. ``Observed'' Michaelis Parameters as a Function of Inhibitor Concentration.
M
Na succinate pH 5.5. Temperature: 50
9
C. B) Prunin; buŠer: 0.05
M
Na succinate pH 5.5. Temperature:
60
9
Fig 4. Hydrolysis of Supersaturated Naringin Solutions.
A) EŠects of temperature and enzyme level on yield after 4 hours of hydrolysis. : 4


naringin, 609C; : 10z naringin, 7 U ml B)
W
z
EŠects of initial substrate concentration at the necessary time to reach diŠerent conversions. Symbols: experimental points; lines: theo-
retical curves obtained with eq. 8 and 9.
inhibitor the plott of initial rate versus pNPR concen-
tration was ``Michaelian'' as demonstrated by run
tests. The values of the ``observed'' Michaelis con-
stant (K mobs and V mobs) were calculated by non-linear
regressions and plotted as a function of inhibitor
concentration in Figs. 3(A) and 3(B), respectively.
The linear dependence of K mobs indicated that both
inhibitors acted competitively.20) According to the
equations for enzyme inhibition,21) the values of V mobs
must be constant for competitive inhibition. They
showed a very low decrease, which may be due to the
Hydrolysis of supersaturated naringin solutions
Using enzymes to produce rhamnose from narin-
gin, the low solubility of this ‰avonoid produces
some problems associated with the manipulation of
suspensions. They can be avoided by hydrolyzing a
supersaturated solution. As the solubility of naringin
increases abruptly above 70
tions can be prepared at 80
fast cooling to 50 C–60 C form homogeneous super-
9
C, concentrated solu-
9
C–90 C, which after a
9
9
9
saturated solutions. The supersaturated solutions are
stable during several hours, and the enzymatic reac-
tion can be run in this time interval. This procedure
increases the reaction rate and yield.8) Special atten-
tion was given to the process taking place within the
ˆrst 4–6 hours of reaction because in this time the su-
persaturated naringin solutions were stable and the
conversions were appreciable. Using puriˆed Asper-
experimental deviation. From the equation: K mobs
Km Km KI ] the inhibition constants were calculat-
ed, being 1.65 m for rhamnose at 50 C and
2.15 m for prunin at 60 C.
Other -rhamnosidases also show competitive
inhibition by rhamnose, with Ki ranging from 0.3 to
＝
+
[I
W
M
9
M
9
a
12,22–23)
4.2 m
M
.
Glucose inhibits the enzyme in a less
gillus terreus a-rhamnosidase as the catalyst, the
proportion, it was found that in a concentration of
hydrolysis yield depended on the enzyme activity and
temperature, as shown in Fig. 4(A). It can be seen
that the optimum temperature for the hydrolysis was
100 m
M
z
the inhibition is about 20 , similar to that
reported by other authors.9) The enzyme from A.
niger, is more inhibited by glucose, this eŠect being
609C, which was higher than that of the enzymes
5,24)
about 50
z
for a concentration of 29
z
.
from P. decumbens and A. niger previously as-
sayed.8) The results on the kinetics of thermal inacti-
vation are in accordance with this behaviour and in-

1448
F. SORIA and G. ELLENRIEDER
Table 4. Hydrolysis of 10
z
Naringin Solutions by Crude and
dicated that during the hydrolysis the enzyme was not
inactivated. Considering the dependence of yield on
Partially Puriˆed
a-Rhamnosidase
enzyme activity the value of 7 U ml was selected for
W
conversion
4 (h)
prunin
enzyme
(gl„1
)
subsequent runs because the rate of increase of yield
over this value became lower and did not justify the
spending for the expensive enzyme. The in‰uence of
the initial naringin concentration over the necessary
time to reach diŠerent hydrolysis conversions is
shown in Fig. 4(B). It can be seen that, in a lapse of
±
±
±
±
±
±
±
1. culture ˆltrate
0.39
0.41
0.44
2
1
2
9
30
32
33
.4
1
2
2. culture ˆltrate treated at pH 11a
3. dye a‹nity puriˆed enzymeb
4. puriˆed enzyme
±
0.46 .4
1
Values are the mean of two experiments
±
standard deviation.
a
This treatment inactivates
This enzyme preparation (
glucosidase.
b
a
-glucosidase.
seven hours, only the 1
z
naringin solution was total-
b
-rhamnosidase 7 Uml„1) was free from
b-
ly hydrolyzed, and when the initial substrate concen-
tration was increased, the conversion became lower.
It was shown previously, using a commercial enzyme,
that this limitation of the hydrolysis is caused by inhi-
bition by the product rhamnose,8) the concentration
of rhamnose and prunin after a ˆxed time increased
when the initial naringin was increased and conse-
quently the yield decreased. To ˆt the experimental
data of yields as a function of time, the Michaelis-
Menten equation with competitive inhibition by the
product was chosen as a model. It can be easily
demonstrated that replacing the concentration of in-
of 10
z naringin at 609C was done using the follow-
ing enzyme preparations: 1) a crude A. terreus cul-
ture ˆltrate (containing Glase), 2) a culture ˆltrate
after Glase inactivation at pH 11,10) and 3) Rase puri-
ˆed by dye a‹nity chromatography. In case 1), the
aglycone naringenin was formed as a consequence of
the partial hydrolysis of prunin. It can be seen in
Table 4 than the level of prunin in the hydrolysis with
the puriˆed enzyme was near to that of the cases 2)
and 3). The reduction of concentration of naringin
was, however, comparable for the 4 preparations.
The eŠects of inhibition by products depends on
the source of enzyme, the A. niger Rase8) showed a
smaller inhibition than that of A. terreus. In a proc-
ess for rhamnose and prunin production by enzymat-
ic hydrolysis of naringin, a low inhibition is desirable
in order to obtain products in concentrated solutions
and reduce the cost of water elimination in the
recovering step. Although rhamnose and prunin are
expensive chemicals and the relative incidence of cost
of this step on the ˆnal product price is not very high,
it is necessary to take into account these aspects.
＝
hibitor [I] by that of product [P] [S]0„[S] in that
equation, the following expression is obtained:
d
[
S
]
KIVm
[S]
＝
„
(6)
dt
Km(
KI+[
S
]0)+(KI„Km)[S]
Km and Vm are the Michaelis constants and KI the
product inhibition constant. Inverting this equation
to dt d[S], it can be integrated as usual, yielding t as
W
function of the conversion X
Km
(
K
I+[
S
]0)
[
S
]0(KI
„Km)
＝
t
„
ln(1„
aX„b ln(1„
]0„[
X
)+
X
(7)
(8)
VmKI
VmKI
t
＝
X
)
It can be concluded that Aspergillus terreus
a-
[
S
S
]
＝
X
with
rhamnosidase is a very convenient catalyst for several
applications because of its high thermal stability (es-
pecially in the presence of substrate). Its high speciˆc
activity and good production level are also advan-
tageous characteristics. These properties of A. ter-
reus Rase are also convenient for the rhamnose and
prunin production from naringin. Although inhibi-
tion by products limits the rhamnose concentration
to be obtained with maximum yield in only one step,
alternatives could be considered on the production
scale to overcome this fact. Recycling of the enzyme
and non-hydrolyzed naringin and the two-step proce-
dure must be analyzed. Furthermore the concentra-
tion of rhamnose solutions could have not a great rel-
ative incidence in the cost of the total process due to
the high price of this sugar.
[S]
0
It can be easily demonstrated that an equation
similar to eq. 8 is valid also for an enzyme inhibited
competitively by two products. Equation 8 was used
to ˆt the experimental data of Fig. 4(B) and the
values of the constants ``a'' and ``b'' were obtained
by non-linear regression. The ˆt was good, and run
tests indicated that the deviations from the model
were not signiˆcant. The dependence of ``a'' and
``b'' on [S]0 slightly deviated from the linearity, and
could be better reproduced by the equations
＝
a
2.33„4.77[
S
]0+0.167[
S
]20;
2
0
＝
b
„2.73+4.88[ ]0„0.179[
S
S
]
(9)
where [S]0 is expressed as a
z
. Using equations 8 and
9, the solid lines of Fig. 4(B) were drawn, and it can
be seen that the experimental results ˆtted well. These
equations can also be used for predictions of hydrol-
ysis conversions for other substrate concentrations.
In order to compare the puriˆed enzyme with other
cheaper preparations of the same source, hydrolysis
Acknowledgments
The authors would like to thank Universidad
Nacional de Salta, CONICET, and Agencia Nacional
de Promocioá n Cientiˆca y Tecnol. of Argentina for

Thermal Inactivation of A. terreus
a
-Rhamnosidase
1449
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the ˆnancial support that made this work possible.
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