Sucrose hydrolysis by invertase immobilized on functionalized porous silicon

Article abstract of DOI:10.1016/j.molcatb.2011.01.011

A successful recipe for the production of immobilized invertase/porous silicon layer with appropriate catalytic behavior for the sucrose hydrolysis reaction is presented. The procedure is based on support surface chemical oxidation, silanization, activation with glutaraldehyde and finally covalent bonding of the free enzyme to the functionalized surface. The catalytic behavior of the composite layer as a function of pH, temperature, and the current density applied in the porous silicon (PS) preparation is investigated. Interestingly, V_max undergoes a substantial increase (ca. 30%) upon immobilization. The value of K_m increases by a factor of 1.53 upon immobilization. The initial activity is still preserved up to 28 days while the free enzyme undergoes a 26% loss of activity after the same period. Based on the outcomes of this study, we believe that tailored PS layers may be used for the development of new bioreactors in which the active enzyme is immobilized on the internal walls and is not lost during the process.

Full text of DOI:10.1016/j.molcatb.2011.01.011

Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Sucrose hydrolysis by invertase immobilized on functionalized porous silicon
Mehrnoosh Azodia, Cavus Falamakia,∗, Afshin Mohsenifarb
a Chem. Eng. Dept., Amirkabir University of Technology, 15875-4413, Tehran, Iran
b Department of Toxicology, Tarbiat Modares University, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 October 2010
Received in revised form 24 January 2011
Accepted 24 January 2011
Available online 28 January 2011
A successful recipe for the production of immobilized invertase/porous silicon layer with appropriate
catalytic behavior for the sucrose hydrolysis reaction is presented. The procedure is based on support
surface chemical oxidation, silanization, activation with glutaraldehyde and finally covalent bonding of
the free enzyme to the functionalized surface. The catalytic behavior of the composite layer as a function
of pH, temperature, and the current density applied in the porous silicon (PS) preparation is investi-
gated. Interestingly, Vmax undergoes a substantial increase (ca. 30%) upon immobilization. The value of
Km increases by a factor of 1.53 upon immobilization. The initial activity is still preserved up to 28 days
while the free enzyme undergoes a 26% loss of activity after the same period. Based on the outcomes
of this study, we believe that tailored PS layers may be used for the development of new bioreactors in
which the active enzyme is immobilized on the internal walls and is not lost during the process.
© 2011 Elsevier B.V. All rights reserved.
Keywords:
Porous silicon
Invertase
Immobilization
1. Introduction
tion resulted in improved stability of the enzyme [13–18], while
the enzyme–substrate affinity and/or enzyme–substrate complex
Porous silicon (PS) is a very attractive candidate for the pro-
duction of immobilized enzyme bioreactors. Relevant published
reports are relatively still limited [1–10] but the future is promising.
Enzymes like glucose-oxidase [2–4], ascorbate-oxidase [3], glu-
curonidase [5], gluthathion-s-transferase [6], urease [9,10], azurine
and laccase [10] have been immobilized on PS so far. Studies on the
development of biochemical process monitoring micro-systems
based on PS have used microreactors consisting of etched micron-
sized trench structures [1–4]. In other cases, plain thin films of PS
have been subject of investigation [5–10].
The present work focuses on the immobilization of invertase
(␤-froctofuranosidase) on PS thin films. Invertase is industrially
used for the production of glucose/fructose equimolar solutions
by catalyzing the hydrolysis of sucrose. The latter product has a
wide application in food industries, especially for the production
of colorless, low crystallinity confectionary sweeteners: fructose
syrups [11]. Due to the vast use of this enzyme in the food indus-
tries, several attempts have already been performed to immobilize
it on different supports in order to extend its stability, providing
re-usage possibility and preventing its loss upon processing. Many
different supports have been investigated [11–18].
intermediate to product “transformation reaction rate constant”
reduced in most of the cases. The reported improved enzyme sta-
bility upon immobilization may be attributed to the occurrence of
enzyme multipoint or multi-subunit attachment (preferably cova-
lent) to the support [19,20]. Torres et al. [21] studied the stability
of invertase immobilized by ionic adsorption on Sepabeads. Their
work clearly shows that the state of oligomerization of the enzyme
depends strongly on the solution pH. In addition, release of the
enzyme subunits of the immobilized enzyme depends on the solu-
tion pH too. Therefore, it goes without saying that a successful
immobilization of invertase is a tricky task. To the authors’ knowl-
edge this is the first study on the immobilization of invertase on
PS.
Porous silicon is a versatile material in the sense that it can
be produced in a wide range of porosity, pore size distribution,
specific surface area and thicknesses. If produced through elec-
trochemical etching, these properties may be tailored by choosing
the appropriate controlling parameters (electrolyte composition,
current density, time, etc.) [22].
PS has many advantages for being used as a support: (a) its
microstructure can be tailored, (b) it provides large outermost sur-
face, (c) its surface may be properly functionalized and (d) it is
not subject to microbial attack. The structure of PS and the immo-
bilization method embrace innumerable choices, thus probably
providing the possibility of achieving an immobilized enzyme with
desired properties.
The immobilization process has been mostly realized by cova-
lent bonding of the amino-groups of the protein to the proper
functional groups of the support. Generally, invertase immobiliza-
This study presents a successful recipe for the production of
immobilized invertase (from Candida utilis)/PS layer with appro-
∗
Corresponding author. Tel.: +98 21 64543160, fax: +98 21 66405847.
E-mail address: c.falamaki@aut.ac.ir (C. Falamaki).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.01.011

M. Azodi et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
155
Fig. 1. Scheme of the PS layer functionalization and immobilization procedure.
priate catalytic behavior for the sucrose hydrolysis reaction. The
2.2. PS functionalization
general immobilization procedure adopted in this research com-
prehends the following steps: (a) PS fabrication by electrochemical
etching, (b) chemical oxidation of surface, (c) silanization (with ␤-
(triethoxysilyl)-propylamine), (d) activation with glutaraldehyde
and (e) enzyme immobilization. Each step is followed by FT-IR
spectroscopy to monitor the functionalization or enzyme immo-
bilization steps. Glutaraldehyde activated supports have been
reported to provide multipoint or multi-subunit immobilization of
enzymes [23]. This is while the chemistry behind is still a matter
of debate [24,25]. It will be shown that the glutaraldehyde route
is successful in providing an immobilized enzyme with improved
stability. The catalytic behavior of the composite layer as a func-
tion of pH, temperature, and the current density applied in the
PS preparation is investigated. It should be noted that except the
work of Maia et al. [8], published reports [1–7,9,10] on enzyme
immobilization on PS did not investigate the pH and temperature
dependency of the catalytic behavior and hence, did not obtain the
optimum process conditions. The kinetic parameters Km and Vm
are determined and compared with their free enzyme counterparts.
The effect of current density on the immobilized enzyme activity is
also investigated.
Fig. 1 shows the general scheme used for the PS layer function-
alization and immobilization steps. After electrochemical etching,
the PS samples were washed with a 96 wt.% ethanol aqueous solu-
tion and immediately subjected to a chemical oxidation treatment
based on the work reported by Liu et al. [26]. For this means,
two solutions were prepared. Solution A consisted of a mixture of
NH3 solution (25 wt.%), deionized water and H2O2 (30 wt.% aque-
ous solution) with volume proportions 1:1:5. Solution B consisted
of a mixture of HCl solution (32 wt.%), deionized water and H2O2
(30 wt.% aqueous solution) with volume proportions 1:1:6. All
chemicals were reagent grade and purchased from MERCK. The
compositions of the A and B solutions had been optimized as to
obtain maximum surface density of Si–OH groups. First, the PS was
treated with solution A at 80 ◦C for 80 s. The treated PS was then
put into contact with solution B at 80 ◦C for 240 s. The obtained PS
was washed with deionized water.
In continuation, the PS was subjected to a silanization process
using reagent grade ␤-(triethoxysilyl)-propylamine (APTES) pur-
chased from MERCK. The silanization process was performed a
liquid phase route [27]. For the latter treatment, a 95 wt.% ethanol
aqueous solution was prepared with a pH between 4.5 and 5.5 using
acetic acid to which APTES was added (10 vol.%). After 10 min, 3 mL
of the latter solution was added to a plastic container consisting of
the oxidized PS. The latter was shaken for 24 h at RT with a Pars
Azma apparatus with an agitation rate of 100 rpm. The treated PS
was then rinsed with a 96 wt.% ethanol solution and dried in an
oven at 110 ◦C for 30 min.
2. Experimental
2.1. PS fabrication
Highly boron doped silicon wafers (0.002 and 0.01 ꢀ cm resistiv-
ity) were used throughout this study. Electrochemical etching was
performed using a setup reported elsewhere [21]. Electrochemi-
cal etching was performed with a CVTM V1.00 galvanostat. Three
current densities of 51, 76 and 127 mA cm−2 have been used. The
composition of the electrolyte was 1:1 (v/v) (40 wt.% HF (BDH),
99.99 wt.% ethanol MERCK). Specific surface area, pore size dis-
tribution and pore volume determination of raw and modified PS
layers were performed using an Autosorb-1 (Quanta Chrome) appa-
ratus.
The final product was used immediately for the next step with-
out rinsing. In the next step, the silanized surface was activated with
glutaraldehyde (GA) (25 wt.%, MERCK) according to the following
procedure: (a) an aqueous GA solution with known concentration
(2.5 wt.%) was prepared; (b) di-nitro-pyridine (DNP) was dissolved
separately in hot water (1 mg DNP per 1 mL water) and then added
drop-wise to the solution obtained in (a) (4 mg DNP solution per
20 mg glutaraldehyde solution); (c) 3 mL of the final solution was
transferred to the container containing the silanized PS and shaken

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M. Azodi et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
at RT for 20 min; (d) solid NaBH4 (reagent grade, MERCK) was dis-
solved in the aqueous solution of the vessel containing the treated
PS (4 mg NaBH4 per 20 mg solution); (e) the whole assembly was
shaken at RT for 70 min; (f) the treated PS was washed with plenty
of water (15 times rinsing with 4 mL deionized water).
2.3. Immobilization of invertase on PS
The final wet PS was then used for the enzyme immobilization
step. For this step, first 15 mg invertase (␤-fructofuranosidase, EC
3.2.1.26, grade X, Candida utilis, SIGMA) were dissolved into 3 mL
phosphate buffer (pH = 7). Each PS sample was then exposed to a
solution containing 3 mL of buffer solution and 20 ␮L of the enzyme
solution. DNP and NaBH4 solutions were added as in the previous
section. The container containing the PS and supernatant solution
was then shaken at 0 ◦C for 1.5 h with an agitation rate of 100 rpm.
The final product was washed with plenty of the buffer solution
with predetermined pH. The samples were maintained in the buffer
at 5 ◦C solution for the kinetic experiments.
Fig. 2. FT-IR spectrum of PS layers subject to different treatments using a wafer
of 0.002 ꢀ cm resistivity, 76 mA cm−2 current density and 10 min electrochemi-
cal etching time (PS = as synthesized PS, OPS = oxidized PS, SOPS = silanized OPS,
GSOPS = SOPS reacted with glutaraldehyde, and IE/PS = immobilized enzyme on
GSOPS composite layer).
2.4. Determination of enzyme activity
PS and 1619 cm−1 of the treated PS are attributed to adsorbed
water OH bending mode. Considering the oxidized PS treated with
a 10 vol.% APTES solution, a new broad and high intensity zone
comprehending many peaks appears after silanization in the range
1400–1650 cm−1. The peaks at 1639 and 1564 cm−1 are attributed
to the bending and scissor mode of NH bond, respectively. The
peaks at 1467 and 1457 cm−1 are attributed to CHx bonds. Before
silanization, it is imperative to transform SiHx entities into Si–OH
ones. Accordingly, it is deduced that APTES resides on the PS outer
surface. At this stage we cannot deduce the formation of cova-
lent bonds between the Si atom of APTES molecule and the Si
atom of PS on the outer surface (in the form of Si–OH). It is not
possible to deduce the formation of new Si–O–Si bonds by con-
sidering the decrease of surface Si–OH groups due to the large IR
adsorption of the OH groups of adsorbed water. Such a deduction
has been reported previously by Liu et al. [26] but is scientifi-
cally not very robust. Also, a clear increase in the intensity of the
peaks in the range 2810–2980 cm−1 due to CHx bonds is observed.
Recall that invertase is a carboxylic group rich acidic protein [17].
Considering the silanized PS layer treated with a 2.5 wt.% GA solu-
tion, the appearance of a distinct peak at 1710 cm−1 is attributed
to the presence of carbonyl group of GA molecules. This might
be a good sign showing that GA molecules did not react with
all their carbonyl groups with the amine groups of the silanized
PS, leaving free carbonyl groups for further reaction with the
enzyme molecules. Comparing the FT-IR spectrum of the immobi-
lized enzyme/PS (IE/PS) layer with the previous one, no observable
change due to enzyme immobilization is observed. However, we
cannot deduce the absence of the enzyme, as the latter sample
showed high enzyme activity. The persistence of the 1710 cm−1
peak is attributed then to the carbonyl stretching vibration of
the enzyme molecule. An important point is worth mentioning.
Referring to Fig. 2, it is observed that SOPS (silanized–oxidized PS)
contains a clear peak at 1564 cm−1, which belongs to the free amine
groups located on the exposed surface. Upon treatment with GA,
the latter peak disappears (GSOPS curve in Fig. 2). This shows that
the amine groups have been eliminated due to the eventual pro-
duction of amide groups. Upon enzyme immobilization, if ionic
adsorption of the enzyme molecules on the surface takes place,
we should be able to observe the free amine groups of the enzyme
in the FTIR spectrum of the final product as they do not convert to
amide groups. However, it is clearly observed that the final prod-
uct does not show any peak at 1564 cm−1 (or near it). Hence, any
ionic adsorption of the enzyme, if existent, is scarce. Recall that we
To determine the immobilized enzyme activity, 30 mL of a
50 mM sucrose (MERCK) aqueous solution buffered at pH = 4.5
(phosphate buffer) was put into contact with the enzyme-
immobilized PS. For this means, the wetting liquid of the PS was
sucked up with paper tissue and dried at RT prior to the addition of
the substrate solution. After addition of the substrate solution, the
vessel was shaken in an incubator (311DA Labnet) at the desired
temperature (55 ◦C) for 20 min with an agitation rate of 100 rpm.
Afterwards, the inhibitor solution (0.5 M Na2CO3, 200 ␮L per mL
buffer solution) was added according to [23] and shaking was con-
tinued for 5 min. The concentration of glucose (equal to fructose)
in the final solution was determined using an enzymatic colori-
metric method using a Glucose PAP SL diagnosis kit (ELI diagnosis
TECH). Absorbance measurements were performed using a spec-
trophotometer (CECIL CE7250 Bio Aquarius) at the wavelength of
500 nm. For obtaining the Lineweaver–Burk plot at the optimum
pH and temperature, 1, 2, 3, 4 and 5 mM sucrose buffer solutions
were used.
2.5. FT-IR and FESEM analysis
FT-IR measurements were performed using a NICOLET (Nexus
B70 FT-IR) apparatus. Field emission scanning electron microscopy
(FESEM) was performed using a S-4160 Hitachi instrument.
3. Result and discussion
3.1. FT-IR analysis
Fig. 2 shows the FT-IR spectrum of PS layers subject to differ-
ent treatments using a wafer of 0.002 ꢀ cm resistivity, 76 mA cm−2
current density and 10 min electrochemical etching time. Consid-
ering the raw PS layer, the strong peaks in the wave number range
of 2000–2300 cm−1 show that the outermost surface of the PS layer
consist of SiHx species. It is observed that after the chemical oxida-
tion step, the intensity of the SiHx peaks reduces. In addition, the
relative intensity in the range of 2500–3700 cm−1 increases sig-
nificantly. This phenomenon is attributed to the transformation of
the Si–Hx into Si–OH species, making the surface hydrophilic [26].
The FT-IR spectrum of the raw PS showed three peaks at 2845,
2930 and 2952 cm−1 which is attributed to adsorbed hydrocar-
bon species from air before measurement. These persist, although
with some shift in the spectrum of the chemically oxidized PS layer
(2850, 2916 and 2963 cm−1). The peaks at 1625 cm−1 of the raw

M. Azodi et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
157
Fig. 3. (a) Pore size distribution of the IE/PS layer using a current density of
76 mA cm−2 and an etching time of 10 min based on the adsorption branch of the
isotherm. (b) The same based on the desorption branch of the isotherm. (c) Pore
size distribution of the PS layer after chemical oxidation using a current density of
76 mA cm−2 and an etching time of 10 min based on the adsorption branch of the
isotherm.
Fig. 4. FESEM pictures of the EI/PS layer (a) cross section, (b) top view and (c) both.
tion and desorption steps. The general trend resembles the one
simulated by Tadvani and Falamaki [22] for boron-doped (1 0 0)
Si wafers, but with a resistivity of 0.01 ꢀ cm. Both pore size distri-
butions do possess three main peaks (1.9, 3.7 and 9.0 nm for the
adsorption step and 2.2, 4.6 and 15 nm for the desorption step).
The difference in the pore size distributions is due to the real shape
and connectivity of the pores which do not obey exactly the main
assumptions made in the BJH evaluation method. Based on the lat-
ter method, the average pore size is 5.1 nm for the adsorption and
4.9 nm for the desorption branches. An important point is the pres-
ence of a broad peak extending to sizes as large as 200 nm. Fig. 4a
shows the corresponding FESEM picture of the cross-section of the
EI/PS layer. Fig. 4b shows the surface of the PS layer under consid-
eration. It is observed that large crater like pores with openings as
large as 137 nm are present. Referring to Fig. 4c and considering the
washed the final product several times with the buffer solution in
order to exclude any ‘adsorbed’ species. By the way, if mere wash-
ing is not able to exclude adsorbed enzyme entities, the remaining
molecules should be detectable by FTIR analysis, and this has not
been the case.
3.2. Nitrogen adsorption and FESEM analysis
Fig. 3a and b shows the pore size distribution of the IE/PS layer
using a current density of 76 mA cm−2 and an etching time of
10 min. The pore size distribution has been calculated based on
the BJH (Braunauer, Emmett, Teller) methodology for the adsorp-

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M. Azodi et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
Fig. 5. The variation of the IE/PS and free enzyme activity versus temperature in the
range of 35–80 ◦C.
Fig. 6. pH dependency of the free enzyme and IE/PS activity.
indicated zone in the picture, it may be stated that the depth of the
large surface pores might be as large as 1000 nm. This is in accor-
dance with the results of the N2 adsorption experiments. Such a
non-uniformity of the pore-microstructure has also been reported
for electrochemically etched n-type substrates by Letant et al. [5].
It is the opinion of the authors of this work that the main pores
involved in the multi-step functionalization/enzyme immobiliza-
tion process are those possessing a size larger than 20 nm. Such
pores are scarce within the core of the PS layer, but constitute the
majority of pores on the exposed surface of the PS layer. Theoret-
ically, as shown by Tadvani and Falamaki [22], the pores initiated
on the exposed surface of silicon upon electrochemical etching are
usually at least one order of magnitude larger than the average
pore size of the whole porous body. The SEM picture shown in
Fig. 4b confirms the above explanation. Fig. 3c shows the pore size
distribution of the PS layer under consideration just after the chem-
ical oxidation step. Compared with Fig. 3a, it is observed that the
pore size distribution remains approximately intact upon enzyme
immobilization. This phenomenon can be explained considering
the presence of large pores on the exposed surface of the PS layer.
curve narrows significantly. The curve is skewed towards the right
showing that the IE/PS exhibits high activity in the span of pH equal
to 5–6. The trend of the curve is significantly different from that
reported in the literature [11–18,28].
The narrowing of the active ranges due to the immobilization
process may be attributed mainly to conformation effects imposed
by the support on the enzyme molecules.
3.4. Kinetic parameters
The kinetic parameters of the free and immobilized enzyme
were determined by drawing the Lineweaver–Burk plot (Fig. 7) for
the optimal parameters pH = 4.5 and reaction temperature of 57 ◦C.
In both cases, a high correlation coefficient was obtained (>0.99),
confirming the applicability of the Michaelis–Menten equation
for the enzyme-catalyzed reaction. The values of Km and Vmax
were calculated to be 0.0245 M and 0.944 mol min−1 for the free
enzyme, respectively. The corresponding values of Km and Vmax
for the immobilized enzyme were calculated to be 0.0376 M and
1.243 mol min−1, respectively. The value of Km increased by a fac-
tor of 1.53 upon immobilization. Published results on immobilized
invertase report an increase of less than one order of magnitude for
Km in most of the cases [16,30,31]. We suppose that the decrease
in the enzyme affinity versus the substrate upon immobilization
is mainly due to eventual conformational changes. Substrate diffu-
sion resistance is less pronounced as the immobilized enzymes do
reside on the outermost and highly accessible surface of the PS, as
discussed before.
3.3. Effect of temperature and pH on invertase activity
Most of the published reports on enzyme immobilization
[11–18] referred to in Section 1 consider invertase enzyme from
Saccharomyces cerevisiae yeast. The latter studies report an opti-
mum pH in the range of 4–5 and an optimum temperature in the
range of 45–60 ◦C for the enzyme activity.
The enzyme considered in this study is from Candida utilis yeast.
Dickensheets et al. [28] reported an optimum pH for free inver-
tase of Candida utilis equal to 4 and a range of 4–5.4 for the same
immobilized on porous cellulose beads. Their work did not report
an optimum temperature for the catalytic activity. On the other
hand, Santana de Almeida et al. [29] report an optimum pH of 6
and optimum temperature of 70 ◦C for invertase obtained from
Cladosporium cladosporiodes.
An interesting result is the substantial increase (ca. 30%) of Vmax
upon immobilization. All published reports on invertase immo-
bilization indicate a substantial decrease in Vmax. In addition,
Based on the upper discussion, a pH of 4.5 was chosen for inves-
tigating the temperature dependency of the enzyme activity. The
variation of the IE/PS and free enzyme activity versus temperature
in the range of 35–80 ◦C is shown in Fig. 5. It is observed that a dis-
tinct optimum temperature of 57 ◦C exists. It is observed that the
optimum temperature does not change significantly upon immo-
bilization.
Accordingly, the optimum temperature was selected for inves-
tigating the pH dependency of the enzyme activity (Fig. 6). A pH
range of 3–11 was chosen to study the optimum condition. It is
observed that the optimum pH resides in the range of 7–8 for free
invertase. It is observed that the trend of the curve changes abruptly
upon immobilization. The optimum pH shifts from 7.5 to 5 and the
Fig. 7. Lineweaver–Burk plot for the free enzyme and IE/PS kinetic experiments.

M. Azodi et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 154–160
159
Fig. 8. The effect of storage time at 5 ◦C on the activity of free enzyme and IE/PS.
the few published investigations on enzyme immobilization on
PS also show decrease in Vmax (glutathione-s-transferase [6], ␤-
glucuronidase [5]).
Fig. 9. Enzyme activity as a function of current density for a pH = 5 and temperature
of 60 ◦C using etched wafer silicons of 0.002 ꢀ cm (IE/PS1) and 0.01 ꢀ cm (IE/PS2)
resisistivity. The activity of the enzyme immobilized on not-etched silicon is also
shown (0.002 ꢀ cm resistivity).
Reported values for Vmax for free and immobilized enzymes
other than invertase on PS show a sharp decrease (more than one
order of magnitude) upon immobilization. The different behavior
observed for the case of invertase immobilized on PS might be
attributed to several phenomena like favorable change of enzyme
structure upon immobilization and enhanced dissociation of the
product from the product/enzyme complex. Another explanation
may also hold: the raw enzyme may contain an industrial stabilizer.
During the investigation of the free enzyme activity, the inhibitor
leaves the enzyme dissolving into the buffer solution. However, it
is not totally excluded from the system and may thus still induce a
detrimental effect on the enzyme activity. Considering the immobi-
lized enzyme, the inhibitor may be completely washed out during
the ‘cleaning process’ through washing with plenty of the same
buffer solution. In this case, the complete absence of the stabilizer
observed by increasing the current density from 51 to 76 mA cm−1
.
Further increase of the current density to 127 mA cm−1 resulted
in relatively slight increase in enzyme activity (11%). This phe-
nomenon is attributed partly to the enlargement of the pores and
more specifically, to the enlargement of the pores on the outermost
PS layer [22]. The activity of the enzyme immobilized on plain sil-
icon wafer (with the same geometrical exposed surface area) has
been added for comparison. The same current density dependency
of the IE/PS activity is observed when using a silicon wafer with
a resistivity of 0.01 ꢀ cm and a similar preparation procedure as
the chip with the resistivity of 0.002 ꢀ cm. However, the activity
is generally less compared to the etched silicon wafer with the
smaller resistivity of 0.002 ꢀ cm which may be simply attributed
to a smaller pore size.
may result in an increase of activity, i.e., a higher value of Vmax
.
Based on the difference between the activity of the enzyme in
the solution of free enzyme and the same solution after being used
for the immobilization on the functionalized PS support, an approx-
imate value for the immobilization yield was calculated. The latter
value is 91.7, 84.4 and 78.6% for the PS of 0.002 ꢀ cm resistivity
for current densities 51, 76 and 127 mA cm−2, respectively. The
corresponding expressed activity has been 204.8, 185.1 and 53.6,
respectively. The immobilization yield has been estimated to be
93.2, 86.3 and 51% for the PS of 0.01 ꢀ cm resistivity for current
densities 51, 76 and 127 mA cm−2, respectively. The corresponding
expressed activity has been 133.8, 123.2 and 41.1 IU, respectively.
It should be mentioned that the average standard deviation in the
estimation of activity has been 16.7 IU.
4. Conclusion
Invertase enzyme was immobilized successfully and for the
first time on porous silicon. Although the affinity of the enzyme
decreased upon immobilization (increase of Km), the value of Vmax
increased significantly (30%).
The immobilization process enhances considerably the storage
stability of the enzyme.
Based on the obtained experimental results, porous silicon may
be considered a potential support for the industrial immobilization
of invertase. The production of fructose syrup via sucrose hydrol-
ysis is an important chemical process. Based on the outcomes of
this study, we believe that tailored PS layers may be used for the
development of new bioreactors in which the active enzyme is
immobilized on the internal walls and is not lost during the process.
Such reactors may then be used in a continuous mode.
3.5. Storage stability and reusability of IE
Fig. 8 shows the effect of storage time at 5 ◦C on the activity of
IE. It is observed that initial activity is still preserved up to 28 days
while the free enzyme undergoes a 26% loss of activity.
The reusability of the IE/PS was investigated for an operating pH
and temperature of 5 and 60 ◦C, respectively. The initial activity was
maintained intact up to the third treatment. After 7 treatments, the
specific activity reduced to 33% of its original value.
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Fig. 9 shows the enzyme activity as a function of the current
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