Food Chemistry 120 (2010) 799804

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Purication and characterisation of Saccharomyces cerevisiae external

invertase isoforms
Uro Andjelkovic a,*, Srdjan Picuric b, Zoran Vujcic c
a
Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
b
Institute of Biochemistry II, University Clinic Johann Wolfgang Goethe-University Frankfurt, 60590 Frankfurt, Germany
c
Faculty of Chemistry, Department of Biochemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia

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

Article history: Four external invertase isoforms (EINV1, EINV2, EINV3 and EINV4) from Saccharomyces cerevisiae were
Received 23 March 2009 highly puried by isoelectric precipitation, ethanol precipitation, ion-exchange on QAE-Sephadex and
Received in revised form 3 November 2009 gel ltration using Sephacryl S-200. Unlike previously published procedures for external invertase puri-
Accepted 9 November 2009
cation, a specially designed step elution was applied on QAE-Sephadex which enabled the separation of
four isoforms. The isoforms have the same molecular mass and catalytic properties: Km for sucrose
(25.6 mM), pH optimum (3.55.0) and temperature optimum (60 C), but they exhibit signicant differ-
Keywords:
ence in pI values, thermal stability and chemical reactivity. Deglycosylation studies showed that the
Invertase
Isoforms
observed differences between isoforms arise from posttranslational modications. Results showed that
Saccharomyces cerevisiae external invertase is a mixture of at least four isoforms, but in order to improve the efciency of food
Enzyme stability industry processes, only the most stable isoform (EINV1) should be puried and utilised. Substantially
Deglycosylation different chemical reactivity of the isoforms could be used to improve the yield of covalent immobiliza-
Imobillization tion procedures.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
External invertase (b-fructofuranosid fructohydrolase, EC
3.2.1.26) from Saccharomyces cerevisiae is an important enzyme
in the food industry; it catalyses the hydrolysis of sucrose into an
equimolar mixture of glucose and fructose, known as invert sugar.
Invert sugar obtained by this enzyme reaction is colourless and has
a higher yield of conversion than the product that is obtained by
acid hydrolysis (Danisman, Tan, Kaar, & Ergene, 2004). Invert su-
gar has a lower crystallinity then sucrose, which is important in the
food industry to ensuring that the products remain fresh and soft
for a long time. In the production process of high-test molasses,
the addition of external invertase is essential to prevent crystalli-
zation (Piggot, 2003). External invertase is also important in the
production of high fructose syrup (Tomotani & Vitolo, 2007). Addi-
tion of external invertase increases the yield production of ethanol
from molasses (Doelle & Doelle, 1989) and high-test molasses
(Piggot, 2003). Invertase has been isolated from the different
microorganisms (Bhatti, Asgher, Abbas, Nawaz, & Sheikh, 2006;
Haq, Baig, & Ali, 2005). However, the external invertase from S.
cerevisiae is commonly used in food industry because this yeast
is non-pathogenic and non-toxicogenic (Food and Drug Adminis-
* Corresponding author. Tel.: +381 11 3282393; fax: +381 11 2636061.
E-mail address: uros@chem.bg.ac.rs (U. Andjelkovic).
tration, 2001). External invertase is also the most commonly used
model enzyme to study different immobilization matrices for the
food industry due to its stability, high degree of glycosylation, no
need for any cofactors and its commercial signicance (Danisman
et al., 2004; Milovanovic, Bozic, & Vujcic, 2007). On the other hand,
the application of the suitably immobilized external invertase in
enzyme reactors for sucrose hydrolysis, on the industrial scale, is
still in the development phase with search for more stable external
invertase preparations ongoing.
The main functional state of the external invertase is a homody-
mer with a molecular mass of 270 kDa (Neumann & Lampen,
1967). It is a very stable glycoprotein that dissociates only under
denaturation conditions. External invertase dimers can also associ-
ate to form tetramers, hexamers and octamers (Esmon, Esmon,
Schauer, Taylor, & Schekman, 1987). Approximately 50% of the
external invertase mass is polymannan and 3% is glucosamine
(Neumann & Lampen, 1967). The carbohydrate component is
organised in the 910 asparagin linked oligosaccharide subunits
of different lengths (Ziegler, Maley, & Trimble, 1988). External
invertase also contains phosphate groups covalently bound to
mannose (Trimble, Maley, & Chu, 1983). The external invertase
carbohydrate moiety increases its thermal stability (Wang, Eufemi,
Turano, & Giartosio, 1996), resistance against protease (Chu &
Maley, 1980), solubility (Gascon, Neumann, & Lampen, 1968) and
makes the enzyme extremely stable at the room temperature

0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.11.013
800 U. Andjelkovic et al. / Food Chemistry 120 (2010) 799804
(Chu, Trimble, & Maley, 1978). External invertase exhibits a high
degree of microheterogeneity, with respect to the carbohydrate
component, and variable state of oligosaccharide chain phosphor-
ylation (Colonna, Cano, & Lampen, 1975; Frevert & Ballou, 1982;
Neumann & Lampen, 1967; Smith & Ballou, 1974). There are no ref-
erences as to how this microheterogeneity inuences the catalytic
properties and stability of the external invertase.
Here we show that the microheterogeneous external invertase
preparations that give single bands on SDSPAGE and that were
obtained by frequently used published methods (Andersen & Jor-
gensen, 1969; Neumann & Lampen, 1967), are in fact mixtures of
at least four isoforms which exhibit different stabilities and chem-
ical reactivities. In this paper, we describe the purication and
properties of these four external invertase isoforms. It is shown
that there is no difference between the isoforms in the structure
of the protein part of the molecule. Moreover, we have demon-
strated that these four isoforms are present in the external invert-
ase preparations of several S. cerevisiae industrial strains. Detailed
study of isoforms is important in order to improve the efciency of
the food industry processes as well as for increasing the yield of the
invertase immobilization procedures. Beside commercial benet,
explorations of external invertase isoforms have scientic impor-
tance for understanding the enzyme stability.
2. Materials and methods
2.1. Materials
All chemicals were of analytical grade and obtained from Sigma
Chemical Company (St. Louis, MO, USA). Four different industrial
strains of bakers yeast S. cerevisiae have been examined: Alltech
Fermin (Senta, Serbia), Vrenje (Beograd, Serbia), Budafok (Buda-
pest, Hungary), Ziko (Skopje, FYR Macedonia). Commercial external
invertase (b-fructofuranosid fructohydrolase EC 3.2.1.26, Grade VII
from bakers yeast) from Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Purication of yeast external invertase isoforms
2.2.1. Preparation of yeast external invertase extract
Saccharomyces cerevisiae cells were suspended in deionized
water (1:1 w/w). Toluene (3%) and sodium carbonate (1%) were
added. The suspension was incubated for 4 days at room tempera-
ture allowing cell-autolysis to occur. The crude cell extract was
centrifuged at 4000g for 20 min. The pH in supernatant was ad-
justed to 4.0 with 1 M sulphuric acid and allowed to sediment
overnight at 4 C. The sediment was removed by centrifugation
as above. The external invertase was precipitated at 4 C by addi-
tion of cold 96% ethanol to 50% (v/v) saturation. The pellet which
contained the total invertase activity was completely dissolved in
deionized water and dialysed overnight at 4 C against deionized
water. Material precipitated during dialysis was removed by cen-
trifugation as before. Obtained supernatant, representing puried
extract, has been used for further purication of the external
invertase isoforms.
2.2.2. Ion-exchange chromatography
Isoforms of external invertase were isolated by ion-exchange
chromatography QAE-Sephadex A-50 (Pharmacia, Uppsala, Swe-
den) column (3  15 cm) was equilibrated in 50 mM acetate buffer
pH 4.5 at the room temperature. The puried extract was loaded
on the equilibrated column. Non-bound proteins were washed
out with the equilibration buffer and the external invertase was
eluted with a step elution using equilibration buffer with conduc-
tivity adjusted with 1 M NaCl. The conductivity of buffers for step
elution (determined in preliminary experiments) was: rst step
4.90 mS/cm, second step 8.50 mS/cm, third step 10.00 mS/cm and
fourth step 15.00 mS/cm. Fractions (3 mL) were collected and the
A280 and invertase activity were monitored.
2.2.3. Gel chromatography
Final purication of each of the isoforms was performed on the
Sephacryl S-200 HR (Amersham Biosciences, Uppsala, Sweden) col-
umn XK (2.6  105 cm) equilibrated in 50 mM acetate buffer pH
4.5. The absorbance at 280 nm and the invertase activity were
monitored for each fraction and those with the invertase activity
were analysed with the SDSPAGE. Afterwards, selected fractions
containing only one pure external invertase protein band were
pooled. Samples obtained by gel ltration were further dialysed
against the deionized water overnight at 4 C and afterwards
lyophilised. All following experiments were carried out with those
samples that represent isolated external invertase isoforms. The
retention time was determined with Blue Dextran as void volume
marker.
2.3. Polyacrylamide gel electrophoresis (PAGE)
SDSPAGE was performed on Hoefer SE 600 Ruby vertical sys-
tem, 10% separation gel (pH 8.8) and 3% stacking gel (pH 6.8).
The Tris/glycine buffer with 0.1% SDS was used according to the
discontinuous system; gels were stained with 0.1% Coomassie bril-
liant blue (CBB) R-250 (Laemmli, 1970). Molecular weight was esti-
mated by comparison with marker proteins (a-2-Macroglobulin
(170 kDa), b-galactosidase (116 kDa), Phosphorylase b (97 kDa)
Amersham-Pharmacia and PageRuler Fermentas). Native PAGE
was performed in identical conditions as SDSPAGE omitting SDS
in each buffer and b-mercaptoethanol in sample buffer, and sam-
ples were not boiled.
2.4. Isoelectric focusing
IEF was performed using 4% polyacrylamide gels containing
ampholines of pH range from 3 to 10 (Amersham-Pharmacia, Upp-
sala, Sweden), according to the manufacturers instructions. The
isoelectric point was evaluated by comparison with protein pI
markers (Sigma, USA). Gels were stained by CBB R-250.
2.5. Periodate cleavage of carbohydrate from external invertase
Carbohydrate was cleaved by incubating 0.05 mg of highly puri-
ed external invertase in 5 mL of 25 mM sodium phosphate buffer,
pH 7.0, containing 50 mM sodium meta-periodate. Reaction vessel
was wrapped in foil to protect the reaction mixture from light.
After 6 h of incubation at room temperature (21 C), 10 lL of
glycerol were added and the reaction mixture was desalted on
Sephadex G-25 column. Immediately after salt was removed,
10 lL of b-mercaptoethanol were added and samples were ana-
lysed by SDSPAGE and Native PAGE.
2.6. Invertase activity assay and concentration determination
Invertase (25 lL) was added to 0.3 M sucrose solution in 50 mM
acetate buffer (475 lL), pH 4.5. After 5 min at 25 C reaction was
terminated by addition of 2,4-dinitrosalicylic acid reagent
(500 lL) and mixture was boiled in wather bath for 5 min. Before
mesuring absorbance at 540 nm, 4 mL of deionized water was
added (Bernfeld, 1955). The standard curve was obtained with dif-
ferent concentrations (between 0.5 and 10 mM) of an equimolar
mixture of D-glucose and D-fructose in 50 mM acetate buffer, pH
4.5. One unit of the invertase activity (U) corresponds to the
amount of enzyme that catalyses the hydrolysis of 1 lmol of
sucrose per 1 min under described assay conditions. Invertase
 U. Andjelkovic et al. / Food Chemistry 120 (2010) 799804 801

concentration was obtained measuring absorbance at 280 nm

(A280nm = 2.25 for 1 mg/mL invertase solution) (Trimble & Maley,
1977).

2.7. Determination of the kinetic constant

The MichaelisMenten constant (Km) for sucrose was deter-

mined using the described conditions of the invertase assay and
a sucrose concentration in the range from 2.5 to 300 mM. The Km
was calculated using the LineweaverBurk transformation of
MichaelisMenten equation.

2.8. pH and temperature optima

To determine the pH optimum of each of the isoforms the de-

scribed invertase assay was performed at 25 C but in McIlvaines
buffer (0.2 M sodium phosphate and 0.1 M citrate) of the different
pH values in the range from 3.0 to 8.0. The temperature optima
were determined using the described conditions of invertase assay
Fig. 1. QAE-Sephadex chromatography of S. cerevisiae external invertase puried
at various temperatures (ranging from 10 to 70 C). extract. Conductivity of the elution buffer was: rst step 4.90 mS/cm, second step
8.50 mS/cm, third step 10.00 mS/cm and fourth step 15.00 mS/cm. Arrows indicate
2.9. Thermal stability position of individual invertase isoforms.

Samples of the external invertase isoforms (15 U/mL) in deion-

ized water were incubated at 60 C. Aliquots (25 lL) were taken Table 1
Purication of the S. cerevisiae external invertase isoforms.
every 5 min and the residual activity was directly measured using
the described conditions of invertase assay. Purication step Volume Total Yield Specic pI
(mL) activity (%) activity
(U) (U/mg)
2.10. Immobilization of the external invertase
Crude lysate 800 785 100
Claried and desalted lysate 1000 773 98.4
The external invertase isoforms were immobilized on Eupergit Acide precipitation 900 641 81.7
C, industry proven carrier porous material containing oxirane Ethanol precipitation 190 622 79.2
groups reactive toward amino and sulfhydryl groups of enzymes QAE-Sephadex
(kindly provided by Rhm GmbH, Darmstadt, Germany). Thirty EINV1 140 203 25.8
milligrams of Eupergit C was added to 2 mL samples of external EINV2 150 254 32.3
invertase isoforms (diluted with deionized water to 32 U/mL) EINV3 90 62 7.9
EINV4 15 30 3.8
and shaken for 1 h at room temperature and afterward placed at
4 C. After 7 days at 4 C with occasional shaking, immobilizate Sephacryl S-200 HR
EINV1 15 165 21.0 3160 4.43
was extensively washed with deionized water to remove the
EINV2 14 211 26.9 2410 4.22
non-bound enzyme. Control samples of invertase were exposed EINV3 17 46 5.9 2470 3.95
to the same conditions omitting Eupergit C. EINV4 21 23 2.9 1480 3.76

3. Results and discussion

External invertase was isolated from four different industrial
strains of bakers yeast S. cerevisiae. These preparations were ana-
lysed and here presented results showed the presence of four
external invertase isoforms. Therefore, this appeared to be an
inherent feature of each strain, which is consistent with previously
described external invertase polymorphism in S. cerevisiae strain
X218O (Frevert & Ballou, 1982) and FH4C, LK2G12 and bakers
yeast (Colonna et al., 1975).
3.1. Purication of yeast external invertase isoforms
Extract of the external invertase was prepared by autolysis of
yeast cells. After 4 days of autolysis more than 80% of the external
invertase was released. An extract with low concentrations of con-
taminants was prepared by acid precipitation of contaminants and
the subsequent ethanol precipitation of the external invertase. Iso-
lation of the external invertase isoforms from the puried extract
was achieved on the QAE-Sephadex ion-exchange column. Result-
ing chromatogram is given in Fig. 1. Four different peaks with
invertase activity were obtained applying the step elution (cf. Sec-
tion 2 for detail). Each single peak obtained was separately re-chro-
matographed, on the same QAE-Sephadex column. However,
additional peaks were not obtained and each separated peak exclu-
sively gave a single peak with invertase activity (results not
shown). This simple control experiment conrmed that four differ-
ent samples of external invertase were obtained. The purity of the
four peaks was checked by the SDSPAGE and each peak with
external invertase isoform contained one or two low molecular
weight contaminants. Those contaminants, described earlier
(Trimble & Maley, 1977) were removed by gel ltration on Seph-
acryl S-200 HR column. A summary of the purication and specic
activity of the individual external invertase isoforms are listed in
Table 1. Isoform EINV1 exhibits the highest specic activity
(3160 U/mg) which is in a good agreement with the highest pub-
lished values for external invertase preparation (Andersen & Jor-
gensen, 1969; Neumann & Lampen, 1967; Trimble & Maley,
1977). The achieved nal yield for EINV1 and EINV2 is 21.0% and
26.9%, respectively, which is a very high purication yield.
Previously published procedures (Andersen & Jorgensen, 1969;
Neumann & Lampen, 1967) for external invertase purication did
not segregate individual fractions from ion-exchange chromatog-
raphy. According to these procedures all the fractions with
invertase activity have been pooled together in one sample. This
802 U. Andjelkovic et al. / Food Chemistry 120 (2010) 799804
Fig. 2. Electrophoretic proles of the isolated S. cerevisiae external invertase isoforms. (A) Native PAGE. (B) Isoelectric focusing; arrows indicate position of pI markers.
preparation of external invertase will be identied as total external
invertase (EINV) in further text.
3.2. Electrophoretic analysis of external invertase isoforms
SDSPAGE analysis of external invertase isoforms showed that
all four isoforms have one identical diffuse band. This SDSPAGE
prole is identical with the previously published prole for the
preparation of total external invertase (Trimble & Maley, 1977;
Rodriguez, Gomez, Gonzalez, Barzana, & Lopez-Munguia, 1997).
The diffuse band in the SDSPAGE is characteristic for external
invertase and glycoproteins in general. Native PAGE of four iso-
forms (Fig. 2A) reveals differences between them. Electrophoretic
mobility increases from EINV1 to EINV4, following the order in
which they are eluted from ion-exchange column. Samples of EINV
and the commercially obtained external invertase (EINV Sigma)
have identical broad diffuse bands covering regions of all four iso-
forms (Fig. 2A). This, very broad diffuse band, is characteristic of
external invertase samples prepared following published proce-
dures (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967).
Separation of four external invertase isoforms on an ion-exchange
column was possible due to the difference in the negative charge
density at the surface of isoforms. This difference in negative
charge between isoforms is conrmed by Native PAGE (Fig. 2A).
On the other hand, identical SDSPAGE proles and the same
retention volumes in gel ltration of all four isoforms demonstrate
that there is no signicant difference in molecular weight between
them. Therefore, comparable molecular weight of the isoforms and
differences in quantity of negative charge at the molecule surface is
the main cause of different mass/charge ratio, i.e. mobility in
Native PAGE.
IEF of external invertase isoforms (Fig. 2B) also showed differ-
ences in pI values between four isoforms. The pI values are given
in Table 1. EINV yielded a very broad band in pI range from 3.80
to 4.40. These results are in agreement with the published results
(Colonna et al., 1975) for the preparation of external invertase pre-
pared following the published methods (Andersen & Jorgensen,
1969; Neumann & Lampen, 1967). The acidity of the isoforms in-
creases in the same fashion in which they elute from ion-exchange
column. The differences in pI values between isoforms are higher
than could be predicted on the basis of the amino acid sequence
of the external invertase (Taussig & Carlson, 1983). This difference
between individual isoforms is presumably due to differences in
the covalently bound phosphate to mannose (Frevert & Ballou,
1982; Trimble et al., 1983).
The carbohydrate moiety was removed from all individual
external invertase isoforms by periodate oxidation cleavage. The
resulting proteins were analysed by electrophoresis in order to test
potential differences in the protein part of the molecule. SDSPAGE
analysis of periodate oxidised external invertase isoforms showed
that all four isoforms have one identical band at 60 kDa, which cor-
responds to the mass of a monomer without carbohydrate units
(Fig. 3). The periodate cleavage of the carbohydrate from the exter-
nal invertase gives results that can be compared with the pub-
lished results of the carbohydrate removal by the endo-b-N-
acetylglucosaminidase H (Chu & Maley, 1980; Trimble & Maley,
1977). Native PAGE analysis showed that differences in the mobil-
ity between the isoforms disappear after periodate treatment (re-
sults not shown). This result suggests that all four isoforms have
the same protein moiety. It seems that the differences between
the isoforms are due to the posttranslational modications, which
is in agreement with the previous reported observations (Colonna
et al., 1975; Frevert & Ballou, 1982).
3.3. Determination of the kinetic constant, pH and temperature optima
The Km was determined to be 25.6 mM, for all four isoforms,
using a substrate range from 2.5 to 300 mM sucrose in 50 mM ace-
tate buffer (pH 4.5). The same value was published for preparation
of EINV (Gascon et al., 1968). All four isoforms exhibit an identical
pH optimum from 3.5 to 5.0 and temperature optimum with the
maximum activity at 60 C, and the results obtained are also in
agreement with those published for the preparation of EINV
(Gascon et al., 1968). The lack of signicant differences in the Km,
pH and temperature optima and the molecular weight between
external invertase isoforms shows that there is no difference in
catalytic activity between isoforms. The absence of a difference
in the enzymatic activity between external invertase isoforms is
one aspect of enzymatic catalysis. Another, important aspect,
particularly when transferred to the industrial application, is how
the observed differences between isoforms affect the enzymes sta-
bility and its chemical reactivity.
 U. Andjelkovic et al. / Food Chemistry 120 (2010) 799804
Fig. 3. SDSPAGE proles of periodate treated external invertase isoforms. Gel is
silver stained.
Fig. 4. Time courses of inactivation of the yeast S. cerevisiae external invertase
isoforms at 60 C.
3.4. Thermal stability
External invertase isoforms exhibit signicant differences in
thermal stability at 60 C (Fig. 4). Isoform EINV1 is the most stable
and had the highest activity (about 30% of the activity) amongst
isoforms even after 35 min of incubation at 60 C. On the other
hand, EINV3 quickly lost its activity and after 20 min almost no
activity could be detected. Thermal stability of EINV is same as
EINV Sigma and was shown for both to be lower than the thermal
stability of the isoform EINV1.
It has been previously published that deactivation of the exter-
nal invertase follows biphasic kinetic (Cavaille & Combes, 1995;
Rodriguez et al., 1997; Vrabel, Polakovic, tefuca, & Bale, 1997).
Our obtained results implied that the cause of biphasic kinetics
of denaturation could be caused by the existence of the four iso-
forms in the preparations of external invertase. Four isolated iso-
forms are different in stability with rst order inactivation
kinetics, showing that inactivation of each isoform is a one-reac-
tion process.
803
Fig. 5. Efciency of immobilization of the yeast S. cerevisiae external invertase
isoforms on Eupergit C.
3.5. Immobilization of the external invertase isoforms
Finally, to test whether the observed differences amongst the
individual invertase isoforms might also lead to differences in
the chemical reactivity, we have used Eupergit C which is com-
monly used matrix for immobilization of enzymes in food industry.
Immobilization of the isoforms on Eupergit C occurs via formation
of covalent bonds between epoxide functional groups of Eupergit C
and amino groups of enzyme. Results (Fig. 5) demonstrate large
differences between external invertase isoforms in the degree of
binding to Eupergit C, revealing their signicantly different chem-
ical reactivities. This result indicated that 72% of the applied activ-
ity of isoform EINV1, 24% of isoform EINV2 and 6% of isoform
EINV4 were bound, whilst the applied activity of EINV3 was not
bound at all. Hence, for the optimisation of the covalent immobili-
zation procedures for the external invertase it is necessary to test
whether the external invertase preparation contains isoforms.
The reactivity of the each isoform with matrix has to be tested.
Loading only the most reactive isoform to the matrix will improve
the yield of immobilization. Therefore it would be possible to re-
duce the amount of matrix, what is especially important when
expensive matrix is applied. Moreover, the existence of external
invertase isoforms with different chemical reactivity could be an
explanation for the low yield of some invertase immobilization
procedures. Differences in surface architecture between isoforms,
i.e. different exposure of amino groups of the protein toward reac-
tive oxirane groups of Eupergit C most probably causes their differ-
ent chemical reactivity resulting in signicant differences in the
yield of immobilization.
4. Conclusions
In the present study the four isoforms of the yeast S. cerevisiae
external invertase were isolated and characterised. Highly puried
preparations were obtained. The procedure presented here is
reproducible, simple, has a low cost and can easily be adapted
for the large scale purication.
The four isolated external invertase isoforms showed substan-
tially different thermal stability and chemical reactivity. The iso-
forms have the same catalytic properties. The deglycosylation
studies showed that the protein part of the four isoforms was
identical.
804 U. Andjelkovic et al. / Food Chemistry 120 (2010) 799804
Isoform EINV1 exhibits the highest stability and this isoform
could be more efciently covalently immobilized in comparison
to other isoforms. This implies that EINV1 should be considered
for application in industrial processes instead of total EINV prepa-
rations in order to improve the efciency of food industry pro-
cesses and invertase immobilization procedures.
Acknowledgments
This work was supported by the Serbian Ministry of Science and
Technological Development, Grant No. 142026B. We thank Dr.
Marijana Petkovic, Vinca Institute of Nuclear Sciences in Bel-
grade, Serbia, for her advices in preparing the manuscript.
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