Invertase from a strain of Rhodotorula glutinis

Article abstract of DOI:10.1016/S0031-9422(02)00336-9

An invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from Rhodotorula glutinis was purified by ammonium sulfate fractionation, gel filtration and anion exchange chromatography. Invertase molecular weight was estimated to be 100 kDa by analytical gel filtration and 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular mass determinations indicated that the native enzyme exists as a homodimer. It is a glycoprotein that contains 19% carbohydrate. The enzyme attacks β-D-fructofuranoside (raffinose, stachyose and sucrose) from the fructose end. It has a K_m of 0.227 M and a V_max of 0.096 μmol/min with sucrose as a substrate. Invertase activity is stable between pH 2.6 and 5.5 for 30 min, maximum activity being observed at pH 4.5. The activation energy was 6520 cal/mol. The enzyme is stable between 20 and 60°C. Mg²⁺ and Ca²⁺ ions stimulated invertase activity 3-fold, while Fe²⁺, K⁺, Co²⁺, Na⁺ and Cu²⁺ increased activity about 2-fold. The transfructosylation reaction could not be observed. This enzyme is of particular interest since it appears to have a high hydrolytic activity in 1 M sucrose solution. This fact would make the enzymatic hydrolysis process economically efficient for syrup production using by-products with high salt and sugar contents such as sugar cane molasses.

Full text of DOI:10.1016/S0031-9422(02)00336-9

Phytochemistry 61 (2002) 605–609
www.elsevier.com/locate/phytochem
Invertase from a strain of Rhodotorula glutinis
Marıa C. Rubio*, Rosa Runco, Antonio R. Navarro
Department of Industrial Microbiology, Institute of Biotechnology, Faculty of Biochemistry, Chemistry and Pharmacy,
´ ´
University of Tucuman, Ayacucho 491-4000, San Miguel de Tucuman, Argentina
Received 5 April 2002;received in revised form 15 July 2002
Abstract
An invertase (b-d-fructofuranoside fructohydrolase, EC 3.2.1.26) from Rhodotorula glutinis was purified by ammonium sulfate
fractionation, gel filtration and anion exchange chromatography. Invertase molecular weight was estimated to be 100 kDa by
analytical gel filtration and 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular mass
determinations indicated that the native enzyme exists as a homodimer. It is a glycoprotein that contains 19% carbohydrate. The
enzyme attacks b-d-fructofuranoside (raffinose, stachyose and sucrose) from the fructose end. It has a Km of 0.227 M and a Vmax of
0.096 mmol/min with sucrose as a substrate. Invertase activity is stable between pH 2.6 and 5.5 for 30 min, maximum activity being
ꢀ
observed at pH 4.5. The activation energy was 6520 cal/mol. The enzyme is stable between 20 and 60 C. Mg2+ and Ca2+ ions
stimulated invertase activity 3-fold, while Fe2+, K+, Co2+, Na+ and Cu2+ increased activity about 2-fold. The transfructosylation
reaction could not be observed. This enzyme is of particular interest since it appears to have a high hydrolytic activity in 1 M
sucrose solution. This fact would make the enzymatic hydrolysis process economically efficient for syrup production using by-pro-
ducts with high salt and sugar contents such as sugar cane molasses.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Rhodotorula glutinis; Hemiascomycetidae;Food industry;Enzymatic characterization;Invertase;
Invert sugars
b-Fructofuranoside fructohydrolase;
1. Introduction
The yeast Rhodotorula produces large amounts of fats
(Prescott and Dunn, 1962;Rose and Harrison, 1970)
and has been used for the production of single-cell pro-
teins from ethanol, acetic acid and acetaldehyde (Hofer
and Misra, 1978;Yech, 1996). Recently, epoxide
hydrolase (EH) has been described by Kronenburg et al.
(1999) in this yeast;however, as far as we know, it has
not been studied as a possible producer of invertase.
It is the aim of this paper to report our investigation
concerning the physical and kinetic properties of an
invertase produced by Rhodotorula glutinis in order to
identify its possible economic advantages in industrial
processes such as syrup production.
Acid invertase is an important industrial enzyme with
applications in the production of non-crystallizable
invert sugars and soft-centered chocolates (Wiseman,
1979). Invertase hydrolyses sucrose into a mixture of
glucose and fructose under sucrose concentrations lower
than 10% (wt./vol.) and has transfructosylating activity
under sucrose concentrations higher than 10% (wt./
vol.). b-d-fructosyl-transferase (FTase, EC 2.4.1.9)
obtained from plants and fungi (Chen and Liu, 1996)
has a similar activity. Invertase has been widely studied,
especially in the yeasts Saccharomyces cerevisiae and
Schwanniomyces occidentalis (Fontana et al., 1992;
Costaglioli et al., 1997) and in filamentous fungi such as
Aureobasidium sp. ATCC 20524 (Hayashi et al., 1992b)
and Aspergillus niger (Romero-Gomez et al., 2000).
2. Results and discussion
2.1. Invertase production
The biosynthesis of the enzyme was strongly influ-
enced by the sugar used as the main carbon source.
Among the sugars tested (fructose, glucose, lactose,
* Corresponding author. Tel.: +54-381-4247752x261;fax: +54-
381-248169.
E-mail address: biotec@unt.edu.ar (M.C. Rubio).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00336-9

606
M.C. Rubjo et al. / Phytochemistry 61 (2002) 605–609
Table 1
Purification scheme of the invertase from Rhodotorula glutinis. The values given are the means from at least two experiments
Purification steps
Total activity
(mkat)
Total protein
(mg)
Specific activity
(mkat/mg protein)
Purification
factor
Yield (%)
Crude extract
0.32
0.05
0.04
0.03
7.80
0.60
0.20
0.15
0.04
0.08
0.20
0.20
1.00
2.00
5.00
5.00
100
15.6
12.5
9.4
Ammonium sulfate (40–80%)
Sephadex G-150
DEAE-Sephacell
maltose, raffinose and sucrose) only the sucrose-grown
and the raffinose-grown cultures possessed invertase
activity, indicating that the enzyme is substrate induci-
ble. Similar results were obtained with invertases from
S. occidentalis (Costaglioli et al., 1997) and A. niger
(Rubio et al., 1997). Under the conditions described
above, invertase production in sucrose culture reached a
maximum after incubation for 36 h. At this time, the
total specific activity of the invertase was 0.04 mkat/mg
protein. However, enzyme synthesis declined with age,
as did the concentration of free glucose in the medium.
In this respect, invertase synthesis in R. glutinis is simi-
lar to that of S. cerevisiae, where production of the
enzyme is regulated by the concentration of glucose
present in the medium (Fontana et al., 1992).
2.3. Effect of pH and temperature
The optimum pH of the enzyme on the hydrolysis of
sucrose was 4.5. It maintained 48% of its relative activ-
ity after 30 min at pH 3.0 but at pH 8.0, 93% of the
relative activity was lost. Invertase is stable under acidic
conditions between pH 2.6 and 5.5, and maintains 90%
of its residual activity at pH 6.0 for 30 min (Fig. 1).
The activity of the enzyme was determined between 20
ꢀ
and 70 C. The activation energy, calculated from the
Arrhenius plot, was 6520 cal/mol. Invertase from Rho-
ꢀ
dotorula was stable at 60 C while its half-life was 30
min. This stability was higher than that exhibited by the
invertase from Azotobacter chroococcum, whose half-life
ꢀ
at 60 C was 3 min (ꢀDe la Vega et al., 1991). Stability
R. glutinis did not grow on carboxymethylcellulose,
cellobiose or starch. This indicates that it did not form
cellulases, b-glucosidases or amylases.
decreased 33% at 70 C. Results suggested that in these
conditions bacterial contaminations decreased.
2.4. Effect of metal ions on invertase activity
2.2. Purification and characterization of invertase
Table 2 shows that Ca2+ and Mg2+ stimulated
invertase activity about 3-fold while Fe2+, Co2+, Na+,
Cu2+ and K+ increased it about 2-fold as compared to
enzyme activity in the absence of such ions (control).
This effect of metal ions could be important in the use of
raw materials with a high salt content. In contrast, these
The purification of invertase from R. glutinis is shown
in Table 1. The specific activity of the final purified
preparation was 0.20 mkat/mg protein, representing a
total purification factor of 5. The invertase yield
obtained by this purification procedure is low;never-
theless, our objective was to purify and study the
enzyme properties. The elution profiles from DEAE
Sephacell chromatographic column, from which a
homogeneous enzyme was eluted with a linear gradient
of (1 M) NaCl, showed a single peak with a symmetrical
distribution of activity. The molecular weight estimated
by gel filtration was 100 kDa. However, SDS-PAGE of
the enzyme revealed a single protein band, whose esti-
mated molecular weight was 47 kDa. This difference
suggested the presence of subunit structures in the
enzyme, similar to those reported for S. cerevisiae
Y2180 and A. niger invertases (Babczinski, 1980;
Rubio and Maldonado, 1995). The carbohydrate
content of the enzyme was 19% (carbohydrate/pro-
tein, wt./wt.), a value similar to the carbohydrate
content (20%) of Aspergillus japonicus invertase
(Hayashi et al., 1992a) but lower than that of Aur-
eobasidium sp. ATCC 20524 invertases (30–53%)
(Hayashi et al., 1992b).
Fig. 1. Effect of pH on the activity (relative activity *) and stability
(residual activity &) of the invertase from Rhodotorula glutinis. The
values given are means of three experiments done in duplicate.

M.C. Rubjo et al. / Phytochemistry 61 (2002) 605–609
607
Table 2
occidentalis, 0.020 M (Klein et al., 1989) and A. niger,
0.060 M (Rubio and Maldonado, 1995).
Effect of various metal ions on the activity of the invertase from Rho-
dotorula glutinis. The values given are means of three experiments
done in duplicate
The enzyme was able to hydrolyze raffinose, stachyose
and sucrose, but not cellobiose, lactose, melezitose,
maltose, inulin and trehalose. These results suggest that
the enzyme is a b-d-fructofuranosidase fructohydrolase,
able to attack the b-d-fructofuranosides from the fruc-
tose end.
Ions
Relative
activity (%)
Cu2+
Ca2+
Mg2+
K+
Hg2+
Na+
Co2+
Fe2+
200
340
350
100
100
200
170
240
2.6. Effect of sucrose concentration on transfructosylating
activity
1 M sucrose is a syrup with a low free water content.
ꢀ
The reaction catalyzed by invertase at 60 C shows that
Control
100
the enzyme activity is not affected by 1 M sucrose solu-
tion, which implies that its activity does not require
large amounts of free water. It is known that FTases
can act on sucrose by cleaving the b-1,2 linkage and
transferring the fructosyl moiety to a suitable acceptor
molecule such as sucrose. By contrast, FFases com-
monly possess both hydrolytic and transfructosylating
activity in sucrose concentrations higher than 10% (wt./
vol.) and demonstrate the only hydrolytic activity in
sucrose concentrations lower than 10% (wt./vol.). In
order to determine whether the enzyme produced by R.
glutinis possessed high transfructosylating activity, we
examined it with various initial sucrose concentrations
(0.15–1 M) in the reaction mixture. Fig. 3 shows that the
hydrolytic activity of the invertase increases with the
increase in sucrose concentration in the reaction med-
ium. Chromatographs from these samples show no oli-
gosaccharide production, a fact that indicates that the
invertase has no transfructosylating activity in the
assayed concentrations. In contrast, fungal invertase has
more transfructosylating activity with a 1 M sucrose
solution (Hayashi et al., 1993;Chen and Liu, 1996).
metal ions inhibited invertase activity in other micro-
organisms such as A. japonicus (Hayashi et al., 1992a)
and A. chroococcum (De la Vega et al., 1991). Hg2+
does not affect invertase activity, suggesting that tryp-
tophan and cysteine are not essential for its activity, a
fact that would make it different from invertases from
S. cerevisiae and A. niger (Hoshino and Momose, 1966;
Rubio and Maldonado, 1995).
2.5. Kinetic parameters and specificity for substrate
A Lineweaver–Burk plot of the enzyme affinity for
sucrose gave a straight-line plot (y=3.3118 x+14.763;
R2 is 0.968) from which the Km was calculated as 0.227
M and the Vmax of the enzyme for sucrose was 0.096
mmol/min (Fig. 2). These values were similar to that
obtained with the invertase from A. japonicus (Hayashi
et al., 1992a) and seemed to be larger than those from S.
Fig. 2. Lineweaver–Burk plot of initial velocity against sucrose concentration. The values given are means of three experiments done in duplicate.

608
M.C. Rubjo et al. / Phytochemistry 61 (2002) 605–609
reagent (Somogyi, 1952). One mkat was defined as the
amount of enzyme catalyzing the hydrolysis of 1
mmol of sucrose per second under the above condi-
tions. The soluble protein was measured by the
method of Bradford (1976), with bovine serum albu-
min as a standard. The carbohydrate content of the
enzyme was measured by the phenol-sulfuric acid
method (Dubois et al., 1956).
3.3. Enzyme purification
The yeast was isolated from a 36 h old-sucrose culture
by centrifuging at 15,000 g for 20 min. Intracellular
enzyme was prepared by resuspending the cell in 0.2 M
acetic acid–sodium acetate buffer (pH 4.5) plus 1.4 mM
of 2-mercaptoethanol and disrupting in a ballistic dis-
integrator Braun B (Melsungen A.G.). Disrupted cells
were centrifuged at 15,000 g for 10 min and the super-
natant solution was used as an intracellular enzyme
source (crude extract). Extracellular enzymatic activity
was tested in the culture broth. The crude extract was
first precipitated with solid ammonium sulfate (40%
saturation). The precipitate obtained was collected by
centrifugation at 15,000 g for 20 min, and dissolved in
0.2 M acetic acid–sodium acetate buffer with 1.4 mM of
2-mercaptoethanol. Solid ammonium sulfate was added
to the supernatant solution to achieve 80% saturation.
Precipitated proteins were collected again by cen-
trifugation at 15,000 g for 20 min and dissolved in 1 ml
of the same buffer. The invertase activity was in the
80% fraction, which was dialyzed overnight against the
same acetate-buffer. Purification was effected by gel fil-
tration (Sephadex G-150 column;45 Â2.5 cm) equili-
brated and eluted with 0.2 acetic acid–sodium acetate
buffer (pH 4.5) containing 1.4 mM of 2-mercaptoetha-
nol. The active fractions (11–17) were pooled and
applied to a DEAE-Sephacell column (11Â1.5 cm), and
then eluted with a linear gradient from 0 to 1 M NaCl
solution in the same buffer. Fractions (13–16) contain-
ing invertase activity were pooled. The purified extract
was dialyzed against 0.2 M acetic acid–sodium acetate
buffer plus 1.4 mM of 2-mercaptoethanol and stored at
Fig. 3. Effect of the sucrose concentration on the hydrolytic activity of
the invertase from Rhodotorula glutinis (R2, 0.954). The values given
are means of three experiments done in duplicate.
In brief, the invertase isolated from R. glutinis is a b-
fructofuranosidase (invertase, EC 3.2.1.26). Interesting
ꢀ
properties were found such as its stability at 60 C, its
activation by ions (Ca2+, Mg2+, K+, Co2+, Fe2+
,
Na+ and Cu2+) and its lack of transfructosylating
activity on a 1 M sucrose solution, Therefore, invertase
from R. glutinis may have a potential for industrial
application in syrup production because of its strong
hydrolytic activity in a 1 M sucrose solution, in con-
strast to other fungi.
3. Experimental
3.1. Enzyme production
R. glutinis strain CIM 1001 was obtained from our
own laboratory culture collection. The strain was grown
in liquid Czapek medium. The pH was adjusted to 4.5
with 0.1 N HCl. Erlenmeyer flasks (250 ml) containing
50 ml of medium were inoculated with 2.6Â106 cells/ml.
ꢀ
4 C.
ꢀ
The culture was incubated for 36 h at 30 C on a reci-
procating shaker (250 rpm). The carbon sources tested
were carboxymethylcellulose, cellobiose, fructose, glu-
cose, lactose, maltose, raffinose, starch and sucrose at
0.5% (wt./vol.).
3.4. Estimation of molecular weight
The molecular weight (Mr) was estimated, using a
45Â2.5 cm column of Sephadex G-150, equilibrated and
eluted with 0.2 M acetate buffer (pH 4.5) plus 1.4 mM
of 2-mercaptoethanol. The standard proteins were
bovine serum albumin (67,000), ovoalbumin (45,000),
alkaline phosphatase (100,000) and catalase (250,000).
SDS-PAGE was performed in 9% acrylamide gels,
which were calibrated using a proteins standard kit
(36,000–205,000) from Sigma. For detecting proteins,
gels were stained with an alkaline silver nitrate solution.
3.2. Enzyme assays
The reaction mixture is 0.25 ml of 0.40 M sucrose solu-
tion, 0.20 ml of 0.20 M acetic acid–sodium acetate buffer
(pH 4.5) and 0.05 ml of enzyme solution. Enzymatic
ꢀ
activity was determined at 35 C and pH 4.5, measuring
the reducing sugars released with the Somogyi–Nelson

M.C. Rubjo et al. / Phytochemistry 61 (2002) 605–609
609
3.5. Effect of pH and temperature on enzyme activity
and stability
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Secretion of invertase from Schwanniomyces occidentalis. Bio-
technol. Lett. 19, 623–627.
Invertase activity was examined in a pH range of 2.6–
8.0. Activity value was obtained by adding to the reac-
tion mixture 0.2 glycine-HCl buffer (pH 2–3), 0.2 M
acetate buffer (pH 4–5) or 0.2 M phosphate buffer (pH
6–8). The temperature was tested in the range of 20–
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gillus japonicus. Enzyme Microb. Technol. 18, 153–160.
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properties of an extracellular invertase from Azotobacter chroo-
coccum. Enzyme Microbiol. Technol. 13, 267–271.
ꢀ
70 C at pH 4.5. In order to investigate the stability of
the enzyme at various pHs, the enzyme solutions were
pre-incubated for 30 min at each pH. In the heat stabi-
lity testing of the enzyme, enzyme solutions (pH 4.5)
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Colorimetric methods for determination of sugar and related sub-
stances. Anal. Chem. 28, 350–356.
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tinuous alcoholic fermentation of sucrose using flocculating yeast.
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Hayashi, S., Matsuzaki, K., Takasaki, Y., Ueno, H., Imada, K.,
1992a. Purification and properties of b-fructofuranosidase from
Aspergillus japonicus. World J. Microbiol. Biotechnol. 8, 276–
279.
ꢀ
were preincubated at various temperatures (25–80 C)
for 30 min. The residual activity of the treated enzyme was
assayed under the standard assay conditions. The acti-
vation energy was calculated from the Arrhenius plot.
3.6. Effect of metal ions on invertase activity
Hayashi, S., Nonoguchi, M., Shimokawa, Y., Takasaki, Y., Imada,
K., 1992b. Effect of deglycosylation on the properties of b-fructo-
furanoside P-1 from Aureobasidium sp. ATCC 20524. J. Ind.
Microbiol. 9, 251–255.
The following ions: Ca2+, Mg2+, K+, Cu2+, Hg2+
,
Co2+, Na+ and Fe2+ (all as chlorides) were added to
the reaction mixture at a 5 mM concentration (pH 4.5
with 1 N HCl).
Hayashi, S., Matsuzaki, Y., Inomata, Y., Takasaki, Y., Imada, K.,
1993. Properties of Aspergillus japonicus b-fructofuranosidase
immobilized on porous silica. World J. Microbiol. Biotechnol. 9,
216–220.
3.7. Measurement of Km and Vmax
Hofer, M., Misra, P., 1978. Evidence for a proton/symport sugar in
the yeast Rhodotorula gracilis (glutinis). Biochem, J. 172, 15–22.
Hoshino, J., Momose, A., 1966. Comparative studies on the two forms
of yeast invertase. J. Gen. Appl. Microbiol. 12, 117–163.
Klein, R., Deibel, M., Sarcich, J., Zurcher, H., Reardon, I., Heinrik-
son, R., 1989. Purification and characterization of invertase from a
novel industrial yeast, Schwanniomyces occidentalis. Biochem. J.
172, 15–22.
Kinetic constants Km and Vmax were determined by
Lineweaver–Burk plots, as a function of the sucrose
concentration (0.05 to 1 M) at pH 4.5.
3.8. Substrate specificity
Kronenburg, N., Mutter, M., Visser, H., Bont, J., Weijers, C., 1999.
Purification of an epoxide hydrolase from Rhodotorula glutinis.
Biotechnol. Lett. 21, 519–524.
The substrates used in the reaction mixtures were:
0.02 M cellobiose, lactose, maltose, melezitose, raffi-
nose, stachyose, sucrose, trehalose and 1% inulin.
Prescott, S., Dunn, C., 1962. Produccion de grasa por Rhodotorula
glutinis. In: Garcia, J., Palasi, V. (Eds.), Microbiologia Industrial,
3rd ed. Version espanola de Aguilar, Madrid. Cp III, pp. 95–109.
Rose, A., Harrison, J., 1970. Rhodotorula glutinis, fat synthesizing
yeast. In: Rose, A., Harrison, J. (Eds.), The Yeasts, Vol. 3. Aca-
demic Press, London/New York, p. 433.
3.9. Effect of sucrose concentration on
transfructosylating activity
Romero-Gomez, S., Augur, C., Viniegra-Gonzalez, G., 2000. Inver-
tase production by Aspergillus niger in submerged and solid-state
fermentation. Biotechnol. Lett. 22, 1255–1258.
Invertase was incubated in the reaction mixture with
different sucrose concentrations (0.15, 0.29, 0.58, 0.70
and 1 M) for 3 h at pH 4.5. Aliquots (100 ml) were
removed and spotted directly on the paper. Paper chro-
matography was carried out with n-butanol:pyridine:
water (52:33:15) as solvent for 30 h. Sucrose, d-glucose
and d-fructose were used as migration markers. The
sugars were detected by dipping the chromatogram in an
alkaline silver nitrate solution (Trevelyan et al., 1950).
Rubio, M., Maldonado, M., 1995. Purification and characterization of
invertase from Aspergillus niger. Curr. Microbiol. 31, 80–83.
Rubio, M., Maldonado, M., Navarro, A., 1997. Effect of culture con-
ditions on invertase production in strain of Aspergillus niger.
Microbiologie Aliments Nutrition (MAN) 15, 17–22.
Somogyi, M., 1952. Notes on sugar determination. J. Biol. Chem. 195,
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Trevelyan, W., Procter, D., Harrison, J., 1950. Detection of sugars on
paper chromatograms. Nature (London) 166, 444–445.
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