Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar

Article abstract of DOI:10.1021/jf072646h

The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized enzyme was 5, and the optimal reaction temperature was 40°C. The apparent Michaelis constant and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose was 78 ± 5 mM and 5 × 10⁴ ± 3 × 10² s^-1, respectively, while for the free enzyme, it was 98.0 ± 4 mM and 1.2 × 10⁴ ± 2.5 × 10² s^-1, respectively, suggesting a better apparent affinity of the enzyme for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized re-INVB also showed good thermal stability and good operational stability (40% of the initial activity remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per 2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol are an appropriate support for re-INVB immobilization and the production of invert sugar.

Full text of DOI:10.1021/jf072646h

1392 J. Agric. Food Chem. 2008, 56, 1392–1397
Immobilization of Recombinant Invertase (re-INVB)
from Zymomonas mobilis on D-Sorbitol Cinnamic
Ester for Production of Invert Sugar
VANESSA VALLEJO-BECERRA,† MARÍA ELISA MARÍN-ZAMORA,‡
JAZMIN MAGDALENA VÁSQUEZ-BAHENA,† FRANCISCO ROJAS-MELGAREJO,‡
MARÍA EUGENIA HIDALGO-LARA,† AND PEDRO ANTONIO GARCÍA-RUIZ*,‡
Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional
No. 2508, D.F. CP 07360, México, and Grupo de Química de Carbohidratos y Biotecnología de
Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de
Murcia, E-30100 Espinardo, Murcia, Spain
The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto
the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative
initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows
maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical
adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and
related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were
important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized
enzyme was 5, and the optimal reaction temperature was 40 °C. The apparent Michaelis constant
and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose
was 78 ( 5 mM and 5 × 104 ( 3 × 102 s-1, respectively, while for the free enzyme, it was 98.0 (
4 mM and 1.2 × 104 ( 2.5 × 102 s-1, respectively, suggesting a better apparent affinity of the enzyme
for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized
re-INVB also showed good thermal stability and good operational stability (40% of the initial activity
remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per
2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol
are an appropriate support for re-INVB immobilization and the production of invert sugar.
KEYWORDS: INVB; Zymomonas mobilis; immobilization; cinnamic carbohydrate esters; enzyme kinetics;
invertase INVBre stability
INTRODUCTION
D-sorbitol (SOTCN) on glass beads, which allowed immobiliza-
tion by adsorption, with little change in enzyme conformation,
and provided an active immobilized re-INVB. This support was
previously used to immobilize other enzymes including tyro-
sinase and peroxidase with good results (4–7). More specifically,
the effect of immobilizing re-INVB on its catalytic activity and
on its stability in the face of different factors was studied.
Invertase enzyme catalyzes the hydrolysis of sucrose to
produce invert sugar. In this study we have recombined
invertase, producing recombinant invertase (re-INVB) from
Zymomonas mobilis to obtain higher activities than with standard
invertase. Invert sugar is used as fructose-rich syrup, principally
in the beverage industry, and its production can be increased
by immobilizing invertase in an active state (1). Recombinant
enzymes are active in their folded and soluble forms and may
be completely inactivated if produced as insoluble aggregates
(2). It is therefore important for industrial purposes to find a
method to immobilize the enzyme in an active state (3).
In this study, the polymeric support used for re-INVB
immobilization was the totally cinnamoylated derivative of
MATERIALS AND METHODS
The inVbpetK plasmid was obtained from CINVESTAV-IPN (Mexico)
(2). Sucrose and D-sorbitol were purchased from Sigma (Spain). All
other chemicals were of analytical grade and supplied by Fluka (Spain),
Panreac (Spain), J. T. Baker (Holland), Sigma (Spain), and Laboratory-
Scan (Ireland). Ultrapure water from Milli-Q system (Millipore Corp.)
was used throughout this research.
Recombinant Protein Production. Inoculum. A fresh clone of
Escherichia coli BL21 (DE3) harboring the inVbpetK plasmid, was
grown in 500 mL shake flasks containing 100 mL of 2TY medium (16
g/L peptone, 10 g/L yeast extract, and 5 g/L NaCl at pH 7.5). Cultures
* To whom correspondence should be addressed. Telephone: +34-
96-83-64-814. Fax: +34-96-83-64-147. E-mail: pagr@um.es.
† CINVESTAV-IPN.
‡ Universidad de Murcia.
10.1021/jf072646h CCC: $40.75
2008 American Chemical Society
Published on Web 02/01/2008

Immobilization of re-INVB from Z. mobiliz
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1393
were incubated at 37 °C and 250 rpm for 2 h in an orbital shaker up
to an optical density of 0.8 at 600 nm (OD600). For recombinant protein
expression, a 25% (v/v) inoculum ratio was used to seed a 1.0 L
fermenter containing 0.4 L of 2TY medium. Dissolved oxygen was
manually controlled at 20–30% saturation by means of an airflow
(0.2–0.25 vvm) and altering the stirring speed (400–900 rpm). Protein
expression was induced by adding isopropyl-ꢀ-D-thiogalactopyranoside
(IPTG) to a final concentration of 1 mM.
The cells contained in 1 mL of culture broth were harvested by
centrifugation (4000g and 4 °C for 20 min) and resuspended in Laemmli
sample buffer prior to analysis of total protein extract by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8). Soluble
protein was prepared from the bacterial pellet of the remaining culture
broth resuspended in lysis buffer (20 mM Tris-HCl at pH 7.5) at 20:1
(w/v) ratio. Cells were lysed with lysozyme (10 mg mL-1) at 4 °C for
30 min and by means of a AMINCO HA6027 Press at 16 000 psi.
Cell debris was removed by centrifugation (10000g at 4 °C for 15 min),
and the supernatant was used to analyze invertase activity and proteins
by SDS-PAGE. The enzyme was purified by nickel-affinity chroma-
tography “Ni Sepharose High Performance” (Amershan Pharmacia).
Fractions (4 mL) were collected and analyzed by SDS-PAGE.
Chromatography was made in a BioLogic LP (BioRad) chromato-
graph.
Preparation of Totally Cinnamoylated Derivative of D-Sorbitol
(SOTCN). A modified version of the method proposed by Van Cleve
(9) was used (4). For this, 0.02 mol of D-sorbitol was dissolved in 100
mL of pyridine by heating at 60 °C for 1 h. The resulting solution was
cooled to room temperature before adding 0.15 mol of cinnamic acid
chloride. The reaction was allowed to proceed at room temperature for
4 h, after which the resulting mixture was poured into vigorously stirred
water at 4 °C. The precipitate obtained after decanting and filtering
this mixture was dissolved in chloroform (the minimum quantity
necessary to dissolve the precipitate) and purified by dropwise addition
to vigorously shaken hexane. The solid obtained after redissolving and
reprecipitating was dried on P2O5 at reduced pressure.
Re-INVB Immobilization. MICROPERL Industrial (Type A) glass
beads of 0.6–1.0 mm diameter manufatured by SOVITEC IBERICA
S.A. (Barcelona, Spain) were supplied by JAQUE (Murcia, Spain).
Before use, the glass beads were washed and degreased (6, 10). A
chloroform solution of SOTCN at 15 g/L was prepared, into which the
glass beads were immersed. A film of prepolymer was formed on the
beads, and the solvent was eliminated by evaporation and low-pressure
suction. After drying, the prepolymer film was polymerized by
irradiation in the ultraviolet zone for 15 min using an Osram HOL-
125W mercury vapor lamp providing a power of 1.6 mW/cm2, as
determined by a Nover-Laser power/energy monitor (Ophir Optronics
Ltd.).
Figure 1. SDS-PAGE (10%) analysis of total protein extracts of
transformed E. coli BL21(DE3). For induction and purification, 20 µL of
sample containing 350 mg of protein was injected. Lane 1, MW markers;
lanes 2 and 3, noninduced; lanes 4 and 5, induced with IPTG; lane 6,
proteins in the washed fraction; lanes 7–10, pure re-INVB enzyme.
Minipuls 3 peristaltic pump. The enzymatic activity was expressed as
UI (micromoles of sucrose hydrolyzed per minute) per milligram of
immobilized protein (UI/mg). In many cases, the results were normal-
ized, considering the highest enzymatic activity value obtained in each
of the series of measurements made as 100%.
Protein Determination. The amount of protein in a solution was
determined by the Lowry method (12), and the amount of immobilized
protein was determined by measuring protein in the immobilization
medium before and after the immobilization process.
Study of the Enzyme Concentration, Immobilization Time, and
Irradiation Time. The effect of the protein concentration in the
immobilization medium (0.1–4 mg/mL), immobilization time (1–70 h),
and irradiation time (2.5–30 min) of the support was studied. For each
measurement, a syringe containing enzyme immobilized on SOTCN
derivative was used, as described in the Materials and Methods.
Dependence of Enzyme Activity on pH and Temperature. The
effect of reaction pH on the enzymatic activity of the immobilized and
free re-INVB was determined using a pH range of 3.5–7.5 at room
temperature. For the pH range of 3.5–5.5, the sucrose solutions (290
mM) were prepared in both an aqueous solution of 50 mM sodium
phosphate and 50 mM sodium acetate buffer, and for the pH range of
6–7.5, the sucrose solutions were prepared in 50 mM sodium phosphate
buffer. The effect of the reaction temperature on the enzymatic activity
of the samples was studied between 25 and 60 °C.
Thermal Stability. The thermal stability of immobilized re-INVB
on glass bead (3 g) covered with SOTCN was studied by incubating
samples at the desired temperature (25–60 °C) for different lengths of
time.
Operational Stability and Hydrolyzed Sucrose Amount. Opera-
tional stability was studied by submitting a syringe containing 3 g of
glass beads covered with SOTCN and immobilized re-INVB to 45
reaction cycles of 1 min each (washing with distilled water between
each cycle), and the total amount of sucrose hydrolyzed was
calculated.
Re-INVB was immobilized at pH 5.5, which is the optimun pH for
the free enzyme. Moreover, the pI of invertase (4.9) is very near the
immobilization pH chosen, with this close relation between pI and
optimum immobilization pH (in the range of 4.5–5.5) having previously
been observed for tyrosinase immobilized on the same support (4, 5).
To immobilize re-INVB, a 2.5 mL solution of this enzyme in sodium
acetate buffer at pH 5.5 was added to a syringe containing 3 g of glass
beads covered with the immobilization support. The immobilization
was allowed to proceed for 12 h at 4 °C. After immobilization, the
enzyme solution was withdrawn and the immobilized enzyme was
thoroughly rinsed in distilled water.
Invertase Activity Assay. Spectrophotometric measurements were
made with a PerkinElmer, Lambda 35 UV/vis spectrophotometer
controlled by a PC running with the software Lambda 35, adjusted to
the desired wavelength. Soluble and immobilized re-INVB enzymatic
activities were determined by the DNS method (11), and the increase
in absorbance was measured at 550 nm. Equimolar mixture of glucose
and fructose was used as standard. The assay medium contained 290
mM sucrose in 50 mM sodium acetate buffer at pH 5.5 for free enzyme
and pH 5 for immobilized enzyme, the reaction was carried out at room
temperature for 1 min. To assay the immobilized enzyme, syringes
containing 3 g of glass beads with bound re-INVB were used as small
packed bed continuous reactors with recirculation (6 mL) and descend-
ing flow. The substrate solution was pumped at 20 mL/min using a
Steady-State Kinetics and Kinetic Data Analysis. The steady-state
kinetic constants, Vmapapx (apparent maximum steady-state rate) and Kampp
(apparent Michaelis constant), of the immobilized and free re-INVB
were obtained, by measuring in triplicate the initial rates (V0) of the
reaction with different sucrose concentrations and fitting the data by
the Lineweaver–Burk plot (13) using the Sigma Plot 9.0 program for
Windows. In every case kacaptp (apparent catalytic constant) was obtained
app
from the values obtained for V
.
max
RESULTS AND DISCUSSION
Expression of the Recombinant INVB Protein. SDS-
PAGE analysis showed the stages followed from expression to
purification (Figure 1). The extracts from the bacteria harboring
the construct inVBpetK showed a band of 51 kDa, which agrees
with the expected theoretical molecular weight of the recom-
binant INVB (2): lanes 2 and 3 show the total protein profile
from noninduced cells, while lanes 4 and 5 show the total protein

1394 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Vallejo-Becerra et al.
Figure 2. Effect of re-INVB concentration on enzymatic activity retained
on glass beads covered with the cross-linked SOTCN. Representation of
the relative activity, RA (%), versus the re-INVB concentration (mg/mL).
Figure 3. (A) Effect of immobilization time on enzymatic activity retained
by re-INVB immobilized on glass beads covered with the cross-linked
SOTCN. Representation of the relative activity, RA (%), versus the
immobilization time, timmo (h). (Inset B) Effect of irradiation time on
enzymatic activity retained by re-INVB immobilized on glass beads covered
with cross-linked SOTCN. Representation of the relative activity, RA (%),
versus the irradiation time, tirrad (min).
profile from induced cells. Fractions obtained from the nickel-
affinity chromatography were also analyzed (lanes 6–10 in
Figure 1). Lane 6 corresponds to proteins in the washed fraction,
while lanes 7–10 correspond to fractions containing the purified
re-INVB enzyme, after elution with 250 mM imidazol.
Cinnamic Carbohydrate Esters as Immobilization Sup-
ports. SOTCN was synthesized and used as support for re-INVB
immobilization. This support was previously used to immobilize
other enzymes, such as tyrosinase and peroxidase (4–7) with
good results. One of the characteristics of this support is that
all of the hydroxyl groups are esterified with cinnamoyl groups,
as can be deduced from various experimental analyses. 1H
nuclear magnetic resonance (NMR), 13C NMR, distortionless
enhancement by polarization transfer (DEPT) spectra, and two-
dimensional experiments (COSY and C/H ratio) of the prepared
compound pointed to its complete esterification, similar to that
seen for other cinnamate derivatives. The infrared spectra of
the prepared compound also indicated full esterification (4).
Differential thermal analysis showed sharp heat-absorption peaks
(probably melting), which disappeared in repeat runs because
of oligomerization of the sample (4). Therefore, the support is
very heat-stable but requires cross-linking prior to use. The
cross-linking of SOTCN was achieved by irradiation in the
ultraviolet region, where the cinnamic carbohydrate esters
showed maximum sensitivity.
The beads (i.e., the inert matrix) were covered with a film of
the cinnamoyl derivative (i.e., the immobilization support) by
solvent evaporation. The best results were obtained at support
concentrations close to 15 g/L (10), when approximately 0.52
( 0.03 mg/g of glass beads was obtained (4). It was assumed
that the re-INVB enzyme was immobilized on the glass beads
covered with the SOTCN derivative through a process of
physical adsorption involving intense hydrophobic interactions
between the cinnamoyl groups of the support and related groups
of the enzyme.
showed a lineal increase of the activity with the enzyme
concentration up to 2.5 mg/mL. More concentrated enzyme
solutions were not appropriate because activity did not in-
crease further and even decreased slightly for 4 mg/mL. The
increase in the activity of the immobilized enzyme with an
increasing enzyme concentration in the immobilization medium
was similar to that obtained for invertase immobilized on other
supports (1, 14) and for other enzymes, such as tyrosinase,
immobilized on the same support (4). The slight decrease in
the activity at high concentrations is probably due to enzyme
packaging, which would slightly inactivate the enzyme. An
initial soluble re-INVB concentration of 2.5 mg/mL, which
provided the greatest immobilized activity, was chosen as the
optimum immobilization concentration. The values obtained for
enzymatic activity were sufficiently high to be correctly
measured, and good reproducibility was achieved for the
different assays.
Another important feature was the effect of immobilization
time on the retained enzymatic activity. In this assay, the
immobilization times were varied between 1 and 70 h. The
results (Figure 3A) indicated that maximum enzyme activity
was achieved for immobilization times of 12 h, above which
activity decreased. Similar behavior has been observed for
invertase immobilized on other supports (1, 3, 14) and for other
enzymes, such as tyrosinase, immobilized on the same support
and for oxidases immobilized on other supports (4, 15).
The effect of irradiation time on the initial activity retained
was studied (Figure 3B), irradiating the SOTCN derivative for
2.5-30 min. The activity increased sharply during the first 5
min of irradiation, after which no further effect was observed
(5–20 min) probably because cross-linking was complete and
the support had already reached its maximum possible molecular
weight. A similar behavior was observed for tyrosinase im-
mobilized on the same support (4) and for HRP immobilized
Invertase Immobilization. Several aspects including (i) re-
INVB concentration, (ii) immobilization time, and (iii) irradia-
tion time were studied to find the best immobilization conditions.
The amount of immobilized protein was also determined.
When the most suitable initial concentration of re-INVB was
studied for immobilization on SOTCN (Figure 2), the results

Immobilization of re-INVB from Z. mobiliz
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1395
Figure 5. Thermal stability of re-INVB. Representation of residual
enzymatic activity, RA (%), versus time (min). I, immobilized re-INVB; F,
free re-INVB. Temperatures were I (25 °C, 30 and 40 °C) and F (30 and
40 °C). (Inset) Representation of residual enzymatic activity RA (%) versus
time (min), I (4 °C).
Figure 4. (A) Effect of reaction pH on re-INVB enzymatic activity. (b)
Re-INVB immobilized on glass beads covered with cross-linked SOTCN
and (2) free re-INVB. Representation of the relative activity, RA (%),
versus the reaction pH. (Inset B) Effect of reaction temperature on re-
INVB enzymatic activity. (b) Immobilized re-INVB on glass beads covered
with cross-linked SOTCN and (2) free re-INVB. Representation of the
relative activity, RA (%), versus the reaction temperature, T (°C).
for a recombinant invertase immobilized on Avicel (16) and
other immobilized invertases (16, 20–22). At high temperatures,
immobilized re-INVB retained more activity than the free
enzyme, which indicates that the immobilization process
stabilizes the enzyme.
on cinnamic carbohydrate esters (7). A total of 15 min was
chosen as optimum irradiation time.
Thermal Stability. The thermal stability of immobilized
enzymes is one of the most important factors for possible further
applications. To study the effect of immobilization on the
thermal stability of re-INVB, samples of immobilized and free
enzyme were incubated at 4 (inset in Figure 5) and 25, 30, and
40 °C in the first case and 30 and 40 °C in the second case
(Figure 5). In both cases, free and immobilized enzyme stability
decreased when the incubation temperature increased, as has
been described in the literature (1, 3, 14). When syringes with
immobilized re-INVB were stored at 4, 25, 30, and 40 °C, 50%
of the initial activity remained after 3600, 250, 100, and 70
min, respectively, resulting in the same order as those obtained
for other recombinant invertase immobilized on Avicel (16).
In the case of free re-INVB, 50% of the initial activity remained
after 20 min of incubation at 30 °C and after 10 min at 40 °C,
meaning that free re-INVB stability is 5-fold lower at 30 °C
and 7-fold lower at 40 °C than the stability of re-INVB
immobilized on SOTCN. The immobilization of re-INVB by
adsorption on SOTCN significantly improved its resistance to
thermal inactivation, especially at high temperatures. Despite
this substantial improvement in the thermal stability of im-
mobilized re-INVB, it is not still as good as the stability of
other native immobilized invertases (1, 3, 20) but certainly
sufficient for it to be considered as of potential use in the
industrial production of invert sugar, especially if we take into
account the high specific activity obtained.
Finally, in the optimal immobilization conditions described
above, the amount of immobilized re-INVB was 0.38 mgprotein
/
mgsupport and the specific activity was 200 UI/mg, which is much
better than that described in the literature; for example, the
specific activity of recombinant invertase immobilized on Avicel
was 20-fold lower than that of our recombinant invertase
immobilized on SOTCN (16), while periodate-oxidized invertase
adsorbed on sepiolite showed a specific activity value of 0.18
UI/mg (lower by a factor of 103) (14); specific activity of
entrapped immobilized invertase on composite gel fiber of
cellulose acetate and zirconium alkoxide was 200-fold lower
than in our case (17). This high specific activity suggest that
re-INVB immobilized on SOTCN could well be used for
industrial applications.
Behavior of Immobilized re-INVB in SOTCN Derivative.
To study the properties of immobilized re-INVB on SOTCN
and to check the applicability of this support for immobilizing
this enzyme and its possible further use in industry, several
aspects were investigated.
Effect of pH and Temperature on Catalytic ActiVity. The effect
of pH on the activity of immobilized and free re-INVB was
studied in the 3.5–7.5 range at room temperature (Figure 4A).
The optimum pH for the hydrolysis of sucrose by immobilized
re-INVB on SOTCN was pH 5, while for the free enzyme, it
was pH 5.5. A small shift in the pH profile (18) to more acid
pH values was observed when the enzyme was immobilized
on SOTCN but not in the case of a recombinant invertase
immobilized on Avicel, with optimum pH value of 5.5 in both
cases (16). The optimum pH value obtained was similar to that
mentioned for other immobilized invertases (1, 3, 14, 19).
The results obtained when studying the activity of im-
mobilized and free enzyme at temperatures between 25 and 60
°C are shown in Figure 4B. As can be seen, the optimal reaction
temperature for re-INVB immobilized on SOTCN was 40 °C,
the same as for the free enzyme (2) and similar to that obtained
Operational Stability of Immobilized re-INVB and Hydrolyzed
Sucrose. The operational stability of re-INVB immobilized on
glass beads covered with SOTCN derivative was ascertained
by reusing the immobilized enzyme for a total of 45 cycles,
each of 1 min duration (Figure 6). There was a gradually
decrease in activity after each successive use and about 42–49%
of the initial activity remained after 45 cycles. These results
point to the good operational stability of the immobilized
enzyme compared to that described in the literature (23, 24)
and is similar to the stability of tyrosinase immobilized on the

1396 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Vallejo-Becerra et al.
of free enzyme, explaining the higher kacaptp A favorable confor-
mational change occurred when re-INVB was immobilized on
SOTCN, with a better hydrolisis rate being obtained than when
in solution. This result is much better than that obtained for
other invertases mentioned in the literature, for which a decrease
about 24-60% in the activity was observed after enzyme
immobilization (17, 20, 27), even in the case of another
recombinant invertase immovilized on Avicel (16).
To sum up, the re-INVB enzyme immobilized on glass beads
covered with cross-linked SOTCN by means of physical
adsorption provided good enzymatic activity and the results
obtained showed good reproducibility. The best enzymatic
activity of immobilized re-INVB was reached after optimizing
the initial enzyme concentration in the immobilization medium
(2.5 mg/mL), the immobilization time (12 h), and the irradiation
time (15 min). No great differences were observed between
immobilized and free re-INVB wth regard to the optimum
reaction pH and temperature. The Kampp value after immobiliza-
tion was lower than the corresponding value for the free enzyme,
showing better apparent affinity for the substrate. The higher
kacaptp values obtained for the immobilized re-INVB than for the
free enzyme showed that the rate of sucrose hydrolysis is also
better after the immobilization process and better than that
obtained for other immobilized invertases. Good operational
stability was obtained for immobilized re-INVB, and thermal
stability improved after immobilization. These results showed
that the cross-linked cinnamic ester of D-sorbitol represents an
appropriate support for re-INVB immobilization and the re-
INVB immobilized on it could be used for the industrial
production of invert sugar.
Figure 6. Operational stability of re-INVB immobilized on glass beads
covered with cross-linked SOTCN. Representation of residual enzymatic
activity, RA (%), versus number of cycles (N).
same support (5). The fall in the activity of immobilized re-
INVB through reuse might also be due to thermal inactivation
of the enzyme at the assay conditions (25 °C). The amount of
sucrose hydrolyzed after each cycle was calculated, and a total
amount of 90.6 mg of reducing sugars after 45 cycles was
obtained. This was considered a good result and underlines the
potential of the described immobilization process for obtaining
reducing sugars on an industrial scale.
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the immobilized and free re-INVB were determined. A Michaelis-
–Menten type kinetic behavior was observed in both cases. After
fitting the data by Lineweaver–Burk plot, we obtained values
of 78 ( 5 and 98 ( 4 mM for the Kampp of immobilized and free
re-INVB, respectively. The kacaptp values of immobilized and free
enzyme were 5 × 104 ( 3 × 102 and 1.2 × 104 ( 2.5 × 102
s-1, respectively.
The Kampp was affected by the immobilization process and
showed a lower value than free re-INVB, meaning that no
unfavorable conformational changes of the enzyme occurred
during immobilization (24), and the lower Kampp value would be
due to the higher sucrose concentrations near the enzyme
because of substrate adsorption on the support (25, 26). Re-
INVB apparent affinity for the substrate improved when it was
immobilized, the same as observed for another recombinant
invertase immobilized on Avicel (16). However, for other native
invertases, the opposite effect was observed (1, 20–22, 24),
as in the case of invertase immobilized on montmorillonite K-10
(19) when an increase of 14-fold in the Kampp value was observed
after immobilization, indicating an important loss in the affinity
for the substrate.
A better (higher)kacaptp value was obtained for immobilized re-
INVB than for free re-INVB, as was observed for tyrosinase
immobilized by the same method (25, 26). This suggests that,
upon being immobilized on the hydrophobic supports, the
enzyme “opens out”, binding itself to the support by its
hydrophobic part and enabling the substrate to reach the active
center of immobilized enzyme more easily than the active center
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Received for review September 6, 2007. Revised manuscript received
November 26, 2007. Accepted December 5, 2007. This work was
supported by a grant from the Ministerio de Educación y Cultura
(Spain) Project MAT2004-01893, Consejería de Ciencia, Tecnología,
Industria y Comercio (Murcia, Spain) Project 2103 SIU0043, and the
Fundación Séneca (Spain) Projects 04691/bps/06. V.V.-B. gratefully
acknowledges a scholarship from CONACYT, México. M.E.M.-Z.
gratefully acknowledges a working contract with the University of
Murcia.
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