immobilization and characterisation of a lipase from a new source, bacillus sp. itp-001
Post on 10-Dec-2016
215 Views
Preview:
TRANSCRIPT
ORIGINAL PAPER
Immobilization and characterisation of a lipase from a newsource, Bacillus sp. ITP-001
Rebeca Y. Cabrera-Padilla • Matheus Albuquerque • Renan T. Figueiredo •
Alini T. Fricks • Elton Franceschi • Alvaro S. Lima • Onelia A. A dos Santos •
Daniel P. Silva • Cleide M. F. Soares
Received: 24 July 2012 / Accepted: 13 December 2012
� Springer-Verlag Berlin Heidelberg 2013
Abstract A new source of lipase from Bacillus sp. ITP-
001 was immobilized by physical adsorption on the poly-
mer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV)
in aqueous solution. The support and immobilized lipase
were characterised, compared to the lyophilised lipase, with
regard to the specific surface area, adsorption–desorption
isotherms, pore volume (Vp) and size (dp) by nitrogen
adsorption, differential scanning calorimetry, thermo-
gravimetric analysis, chemical composition analysis, Fou-
rier transform infrared spectroscopy and biochemical
properties. The immobilized enzyme displayed a shift in
optimum pH towards the acidic side with an optimum at pH
4.0, whereas the optimum pH for the free enzyme was at pH
7.0; the optimum temperature of activity was 80 and 37 �C
for the free and immobilized enzyme, respectively. The
inactivation rate constant for the immobilized enzyme at
37 �C was 0.0038 h-1 and the half-life was 182.41 h. The
kinetic parameters obtained for the immobilized enzyme
gave a Michaelis–Menten constant (Km) of 49.10 mM and a
maximum reaction velocity (Vmax) of 205.03 U/g. Fur-
thermore, the reuse of the lipase immobilized by adsorption
allowed us to observe that it could be reused for 10 suc-
cessive cycles, duration of each cycle (1 h), maintaining
33 % of the initial activity.
Keywords Lipase Bacillus sp. � Immobilization � PHBV �Adsorption � Kinetic parameters
Introduction
Lipases from microbial sources are currently receiving
considerable attention due to their potential applications in
industry such as in detergents, oleochemicals, organic
synthesis, dairy, fat and oil modification, tanning, phar-
maceuticals and sewage treatment [1, 2]. In general, lipases
from Bacillus sp. have not been thoroughly studied, and in
most cases only the basic characteristics of the enzymes
have been reported. However, there are a small number of
these enzymes have been tested in chemical reactions, their
structures have been elucidated or theoretically modelled,
and they have been subjected to immobilization [3].
Some studies have been related to the production of
lipase from Bacillus sp. isolated from petroleum-contami-
nated soil in Brazil [4, 5] and others throughout the world,
such as lipases from Geobacillus thermocatenulatus
(BTL2), Bacillus subtilis (lipA) and Geobacillus zalihae
(T1) [6–8]. Two isoforms of the Geobacillus stearother-
mophilus lipase (L1 and P1) [9, 10] and the monoacyl-
glycerol lipase from Bacillus sp. H-257 [11] have been
resolved in relation to the optimal conditions for their
production and catalytic reactions. This provides the basis
for the identification of directions for further investigation
and possible applications of these enzymes [3].
R. Y. Cabrera-Padilla � M. Albuquerque �R. T. Figueiredo � A. T. Fricks � E. Franceschi �A. S. Lima � D. P. Silva � C. M. F. Soares (&)
Universidade Tiradentes, Av. Murilo Dantas, 300,
Bairro Farolandia, 49032-490 Aracaju-SE, Brazil
e-mail: cleide.soares@pq.cnpq.br
R. T. Figueiredo � A. T. Fricks � E. Franceschi �A. S. Lima � D. P. Silva � C. M. F. Soares
Instituto de Tecnologia e Pesquisa, Av. Murilo Dantas,
300-Predio do ITP-Bairro Farolandia,
49032-490 Aracaju-SE, Brazil
O. A. A dos Santos
Departamento de Engenharia Quımica, Universidade Estadual de
Maringa, Bloco D-90 Avenida Colombo, 5790,
87020-900 Maringa-PR, Brazil
123
Bioprocess Biosyst Eng
DOI 10.1007/s00449-012-0875-1
Immobilization is a powerful tool for fine modifications
to the catalytic properties of enzymes for industrial pur-
poses. Immobilized enzymes have the advantages of
enhanced thermal and chemical stability, ease of handling,
easy recovery and repeated use as compared with free
forms. Immobilization can also enhance activity and even
reverse selectivity. Some approaches have been applied to
the immobilization of Bacillus and Geobacillus lipases on
supports, providing more evidence for the enormous
unexplored potential of this group of biocatalysts [12–14].
For example, a lipase from Geobacillus thermocatenulatus
(BTL2) was covalently immobilized on CNBr-agarose or
glyoxyl-agarose [14], an extracellular alkaline lipase of the
thermotolerant Bacillus coagulans BTS-3 was immobilized
onto glutaraldehyde-activated nylon-6 by covalent binding
[13] and an alkaline/thermotolerant bacterial lipase from
Bacillus coagulans MTCC-6375 was purified and immo-
bilized on a methacrylic acid and dodecyl methacrylate
(MAc-DMA) hydrogel [12].
According to the literature, natural and synthetic polymers
belong to a class of very important supports in the field of
biocatalyst immobilization [15]. Synthetic polymers exhibit a
variety of physical forms and chemical structures that can be
combined to form an ideal support; however, natural polymers
have some advantages compared to synthetic ones because
they are generally low in cost and are easily degradable, and
thus cause no damage to the environment [16].
Currently, eco-friendly supports are used for the immo-
bilization of enzymes, but some have not yet been tested for
Bacillus lipase, for example poly(3-hydroxybutyrate-co-
hydroxyvalerate) (PHBV). Based on other applications
(agroindustry and medical) for PHBV [17, 18], our group
recently immobilized Candida rugosa lipase (CRL) in
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and obtained
an efficient immobilization yield, improved thermal sta-
bility and operational stability; this work is unique in the
literature with lipase immobilized on PHBV [19]. Conse-
quently, PHBV has become a good alternative to be used as
a support for the immobilization of enzymes due to its
biocompatibility, biodegradability, strength, easy absorp-
tion, as well as its eco-friendly and non-toxic properties.
In this sense, the aim of this study was to immobilise
lipase from Bacillus sp. ITP-001 on PHBV by physical
adsorption. The biochemical and physico-chemical prop-
erties were also investigated.
Materials and methods
Enzyme and chemicals
Lipase from Bacillus sp. was obtained by fermentation; the
bacterium was recently isolated from petroleum-
contaminated soil by the Institute of Research and Tech-
nology (Aracaju, Sergipe, Brazil). Enzyme production was
performed under optimal fermentation conditions at a
temperature of 37 �C with a substrate inductor oil (palm
oil), as described by Feitosa et al. [5]. The lipase obtained
from the fermentation medium was purified using an
aqueous two-phase system containing a salt and a polymer
(PEG-8000) as described by Souza et al. [20]. The purified
lipase from Bacillus sp. was lyophilised and their nominal
activity was 906.67 U/g.
Poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), a
natural biopolymer, was used as an immobilization support
for the lipase from Bacillus sp. ITP-001. PHBV was kindly
supplied by PHB Industrial S.A. Hexane and acetone were
obtained from Isofar (Rio de Janeiro, Brazil); 99 % ethanol
was obtained from Vetec (Rio de Janeiro, Brazil); gum
arabic was obtained from Cromoline (Sao Paulo, Brazil);
olive oil was purchased at a local market. Other chemicals
were of analytical grade and used as received.
Activity of lipase in the hydrolysis
of emulsified olive oil
The hydrolytic activities of free and immobilized lipase
were assayed by the olive oil–water emulsion method,
according to the modification proposed by Soares et al.
[21]. The substrate was prepared by mixing 50 mL of olive
oil with 50 mL of a gum arabic solution (7 % w/v) with
initial concentration of 1,860 mM. The reaction mixture
containing 5 mL of the emulsion, 2 mL of 0.1 M sodium
phosphate buffer (pH 7.0) and either free (100 mg) or
immobilized (100–250 mg) enzyme was incubated for
5 min for the free enzyme or 10 min for the immobilized
enzyme at 37 �C with stirring. The reaction was stopped by
the addition of 2 mL of an acetone-ethanol–water solution
(1:1:1 v/v). Liberated fatty acids were titrated with a
0.04 M potassium hydroxide solution in the presence of
phenolphthalein as an indicator. One unit (U) of enzyme
activity was defined as the amount of enzyme that liberated
1 lmol of free fatty acid per min (lmol/min) under the
assay conditions (37 �C, pH 7.0, 150 rpm).
Enzyme immobilization
The lipase from Bacillus sp. ITP-001 was immobilized by
physical adsorption on PHBV using a modified procedure
from Soares et al. [22]. Briefly, 20 mL of hexane was
added to 2 g of the support with vigorous agitation at room
temperature for 2 h, then 20 mL of the enzymatic solution
(mass of the enzyme solubilised in 20 mL of 0.1 M sodium
phosphate buffer, pH 7.0) was added to the hexane and
support suspension and agitated for another 2 h. The
enzyme-support system was then incubated for 24 h at
Bioprocess Biosyst Eng
123
4 �C. The immobilized lipase was recovered by vacuum
filtration coupled with repeated hexane washes. The water
content (dry weight) and enzyme activity of the immobi-
lized biocatalyst were then quantified. Filtrates and washes
were collected and used for activity determination. The
prepared immobilized lipase was then stored at 4 �C.
In order to determine the optimal amount of mass of the
enzyme, we tested the effect of various enzyme/support
ratios (0.150, 0.225, 0.300, 0.375 and 0.450) on
immobilization.
Analyses of hydrolytic activities performed on the free
and immobilized lipases were used to determine the cou-
pling yield g (%) according to Eq. (1).
g %ð Þ ¼ US
U0
� 100 ð1Þ
in which US corresponds to the total enzyme activity
recovered on the support and U0 represents the enzyme
units offered for immobilization.
Biochemical properties of the immobilized lipase
The biochemical properties of the immobilized enzyme
were compared with its free form determined previously by
our research group Barbosa et al. [23].
Effect of pH and temperature on activity
The effect of pH on the relative activity of the immobilized
lipase was determined in the pH range of 3.0–9.0 using
0.1 M phosphate buffer. The hydrolytic activity of the
immobilized enzyme was determined as described
previously.
The effect of temperature on the relative activity of the
immobilized lipase was determined at pH 4.0 in a tem-
perature range varying from 30 to 90 �C, using an olive oil
emulsion as the substrate. Relative activities were calcu-
lated as the ratio of the enzyme activity measured at dif-
ferent temperatures compared to the maximal activity of
the enzyme measured as described previously.
Thermal stability
Immobilized lipase preparations were stored in sodium
phosphate buffer solutions (0.1 M, pH 4.0) for 4 h at 37
and 60 �C. Samples were periodically withdrawn for
activity assays as described previously. Residual activities
were calculated as the ratio of the activity of enzyme
measured after incubation compared to the maximal
activity of the enzyme.
The thermal inactivation rate constant (kd) and half-life
(t1/2) of the immobilized ITP-001 lipase were calculated
using Eqs. 2 and 3, respectively according to the literature
[19, 24]
Ain ¼ Ain0exp �kd � tð Þ; ð2Þ
t1=2 ¼ lnð0; 5Þ=�kd: ð3Þ
Determination of Km and Vmax
To calculate the Michaelis–Menten constant and maximum
reaction velocity (Km and Vmax, respectively), reaction
systems were prepared containing fatty acids at concen-
trations ranging from 37 to 2,604 mM, obtained from
emulsions containing different proportions of olive oil
(1–70 %) and an aqueous solution of gum arabic (7 % w/v).
Initial hydrolysis reaction rates, catalysed by the free and
immobilized ITP-001 lipase, were determined according to
the methodology described previously. The apparent values of
Km and Vmax were calculated by non-linear fitting using the
programme Origin� 8.0.
Reusability of the immobilized lipase
The operational stability and reusability of the immobilized
system were determined by conducting hydrolysis reac-
tions in consecutive batches using the same immobilized
enzyme. Each batch consisted of a hydrolysis reaction for
1 h at a temperature of 37 �C and pH 4.0. After each batch,
the immobilized enzyme was washed with hexane once and
reused for the next cycle of hydrolysis. This procedure was
repeated for twelve cycles.
Morphological and physico-chemical properties
Nitrogen adsorption–desorption measurements
The adsorption–desorption isotherms of the N2 specific
surface area, pore size distribution, pore volume (Vp) and
mean diameter (dp) of the PHBV used as the support and
the immobilized biocatalyst (lipase from Bacillus sp. ITP-
001 supported on PHBV) were determined from nitrogen
adsorption–desorption measurements, which is a widely
used method for the characterisation of microporous and
mesoporous materials. The surface area of the PHBV and
immobilized lipase samples was calculated using the
Brunauer-Emmett-Teller (BET) method [25]. Pore vol-
ume, pore size distribution and average pore diameter,
based on BJH calculations [26], were evaluated by the
BET apparatus software (Model NOVA 1200-Quanta-
chrome Analyser) using N2 adsorption at 77 K. Before
the analysis, the samples were dried in an oven at 120 �C
for 48 h to eliminate any water within the pores of the
solids.
Bioprocess Biosyst Eng
123
Differential scanning calorimetry (DSC)
In this work, the free enzyme, PHBV used as the support
and immobilized biocatalyst (ITP-PHBV) samples were
analysed using a differential scanning calorimeter appara-
tus (SHIMADZU-Model DSC 60). About 4–6 mg of the
sample were sealed in aluminium pans and submitted to a
heating rate of 20 �C/min from room temperature to
200 �C; nitrogen was used as an inert gas at a flow rate of
30 mL/min.
Thermogravimetric analysis (TGA)
The free enzyme (ITP-001), support (PHBV) and immo-
bilized biocatalyst (PHBV-ITP) samples were analysed in a
simultaneous DTA-TG apparatus (SHIMADZU-Thermo-
gravimetric Analyser Model DTG-60 H). In each analysis,
approximately 4 mg of the sample were sealed in platinum
pans and submitted to a heating rate of 10 �C/min from
room temperature to 600 �C; nitrogen was used as an inert
gas at a flow rate of 30 mL/min.
Analysis by Fourier transform infrared spectroscopy
(FTIR)
The free enzyme (ITP-001), support (PHBV) and immo-
bilized biocatalyst (PHBV-ITP) samples were prepared in
KBr pellets and submitted to FTIR analysis (FTIR BO-
MEM MB-100 spectrophotometer) in transmission mode.
Spectra were obtained in the wavelength range from 500 to
1,800 cm-1.
Results and discussion
Immobilization of lipase by physical adsorption
on PHBV
In order to determine the optimal amount of lipase, we
studied the immobilization of various amounts of enzyme
on 2.0 g of the PHBV support. The effect of the enzyme/
support mass ratio on activity is shown in Fig. 1. The
activity of lipase from Bacillus sp. ITP-001 was signifi-
cantly increased when the enzyme/support ratio was
increased from 0.15 to 0.30 (w/w), while above a ratio of
0.30 (w/w), the activity decreased substantially, due to
saturation of the PHBV support with lipase. Because the
highest activity was achieved when a ratio of 0.3 was used,
this enzyme/support (w/w) ratio was selected for the
immobilization of lipase from Bacillus sp. ITP-001 on
PHBV. Under these conditions, the immobilization yield
was 25 %, considered to be a satisfactory result when
compared with other enzymes immobilized on several
types of support such as commercially available acrylic
polymers (recoveries from 8 to 20 %) [27]. Similar results
were reported by Montero et al. [28] that obtained 21 % of
immobilization yield for Candida rugosa lipase immobi-
lized on microporous polypropylene by adsorption physi-
cal. Yesiloglu [29] reported 30 % of immobilization yield
for Candida rugosa lipase non-covalently immobilized on
bentonite and also Ghiaci et al. [30] for Candida rugosa
lipase immobilized on bentonite with bilayer surfactant
obtained 28 % of immobilization yield.
Biochemical properties
Effect of pH and temperature on lipase activity
The effect of pH on the activity of an immobilized enzyme
depends of the enzyme, the immobilization method and the
carrier used. According to Guncheva and Zhiryakova [3],
some lipases from Bacillus have a pH optimum in the
acidic pH range (pH \ 6.0). Thus, the effect of pH
(3.0–9.0) on the activity of the free and immobilized
lipases used in this work was also studied. As shown in
Fig. 2a, the free enzyme, as determined by Barbosa et al.
[23], became more active at pH 4.0–7.0, with higher
activity at pH 7, followed by a decrease in activity at
strongly alkaline pH values. The highest immobilized
enzyme activities were achieved in the acid pH range from
3.0 to 5.0, with higher activity at pH 4.0, followed by a loss
of activity at pH greater than 5.0. The immobilized enzyme
displayed an acidic shift with higher stability at pH 4.0.
The microenvironment of an enzyme can influence its
properties. An enzyme in solution may have a different
optimum pH than that of the immobilized enzyme,
depending on the surface charges on the matrix.
0.15 0.20 0.25 0.30 0.35 0.40 0.45
40
50
60
70
80
90
100
110
Rel
ativ
e A
ctiv
ity
(%)
WeighEnzyme
WeighSupport
/
Fig. 1 Relative activity as a function of changes in the enzyme/
support mass ratio during immobilization of lipase from Bacillus sp.
ITP-001 on PHBV. Error bars are standard deviations from triplicates
Bioprocess Biosyst Eng
123
Polycationic carriers tend to shift the optimal pH of
immobilized enzymes to the acidic side and according to
the literature the support PHVB has a radial cationic [31,
32]. Thus, the maximum activity at acidic pH obtained in
this study is probably due to the radical cationic described
by Mendes et al. [32]. For other sources of lipase, similar
behaviour has been observed, e.g. for Thermomyces
lanuginosus lipase immobilized through cross-linking using
glutaraldehyde and hen egg white [33]. However, different
results were found for the lipase from thermophilic Bacil-
lus sp. in aqueous solution and immobilized on silica and
HP-20 beads; the optimum pH (pH 8.5) was nearly the
same for both the free and immobilized enzymes [34].
Dosanjh and Kaur [35] reported that the lipase from
Bacillus sp. immobilized by cross-linking on a hydropho-
bic surface and the free form had the same optimum pH
(8.0).
The resistance of an immobilized lipase to temperature
is an important potential advantage for practical applica-
tions of this enzyme. The temperature dependence of the
relative activity of the immobilized lipase from Bacillus sp.
ITP-001 was studied at pH 4.0 in the range of 30–90 �C.
The activity profiles of the free lipase as determined by
Barbosa et al. [23] compared with the results obtained in
this work for the immobilized lipase at a different tem-
perature are presented in Fig. 2b. The highest activity was
achieved for the free enzyme at 80 �C [23], while the
immobilized enzyme showed similar activities and higher
than the free enzyme between 37–60 �C. At 80 �C, free
enzyme showed relative activity slightly higher to the
immobilized enzyme. These results show greater tolerance
to heat for immobilized enzyme compared with free lipase.
A similar result was reported in our previous study, indi-
cating slight changes in the optimum temperature for the
Candida rugosa lipase (CRL) immobilized on the same
support (PHBV) (37–45 �C) [19]. The literature reports
that for immobilized lipases from Bacillus, small or any
difference in optimum temperature, compared to the free
enzyme, are observed. The optimum temperatures for
lipase from thermophilic Bacillus sp. to be 60 and 65 �C
for the free and immobilized enzyme on silica and HP-20
beads, respectively [34]. In other study, the optimum
temperature for the lipase from thermotolerant Bacillus
coagulans BTS-3 immobilized on glutaraldehyde-activated
nylon-6 by covalent binding was 55 �C for both forms of
the enzyme [13]. Palomo et al. [36] reported the adsorption
of the lipase from Bacillus thermocatenulatus (BTL2) on
octadecyl-Sepabeads, which permitted an increase of 10 �C
for the optimal temperature of the enzyme.
Thermal stability
The immobilization of lipases onto a solid support
increases their thermostability and extends their biotech-
nology potential, since running bioprocesses at elevated
temperature is advantageous due to higher diffusion rates,
lower substrate viscosities, increased reactant solubilities
and reduced risk of microbial contamination [37].
The thermal stability of immobilized (PHBV-ITP) was
determined by measuring the residual activity as a func-
tion of time at the temperatures of 37 and 60 �C (Fig. 3).
Compared with data from the free enzyme [23], better
thermal stability was observed with the immobilized lipase
at both temperatures. At 37 �C, the residual activity of the
free enzyme was 81 % [23], while for the immobilized
lipase, the residual activity was 98 % after 4 h of exposure
at this temperature. At 60 �C, a greater difference was
observed, as the residual activity of the free enzyme
was 71 % [23], while for the immobilized enzyme, this
was 96 % at the end of the time period studied. These data
2 3 4 5 6 7 8 9 1010
20
30
40
50
60
70
80
90
100
110R
elat
ive
Act
ivit
y (%
)
pH
a
20 30 40 50 60 70 80 90 100 11010
20
30
40
50
60
70
80
90
100
110
Rel
ativ
e ac
tivi
ty (
%)
Temperature (C)
b
Fig. 2 Effect of pH (a) and temperature (b) on the relative activity
of free and immobilized lipase from Bacillus sp. ITP-001 (filledsquare immobilized ITP-001 and filled triangle free ITP-001). Errorbars are standard deviations from triplicates
Bioprocess Biosyst Eng
123
indicate that the thermal stability of the lipase was enhanced
by the described immobilization method and support used.
This could be due to the location of the lipase inside the
mesoporous support, which offers good protection against
thermal alterations. Nawani et al. [34] found that the ther-
mal stability of the lipase from thermophilic Bacillus sp.
was enhanced after its immobilization on silica and HP-20
beads. Dosanjh and Kaur [35] reported that immobilization
enhanced the thermostability of the lipase from a Bacillus
sp. by cross-linking it on a hydrophobic surface. In other
studies, immobilized preparations were much more stable
than soluble enzymes at higher temperatures; the lipase
from Bacillus coagulans BTS-3, when immobilized on
activated polyethylene, showed 60 % residual activity at
70 �C [38]. The lipase from Bacillus thermocatenulatus,
when immobilized on hydrophobic supports, maintained
100 % activity at 65 �C [36] and the lipase of Bacillus
coagulans MTCC-6375 immobilized on a methacrylic acid
and dodecyl methacrylate (MAc-DMA) hydrogel retained
approximately 50 % of its initial activity at 70–80 �C after
3 h of incubation [12].
The calculated values for the inactivation rate constants
(kd) and half-life (t1/2) for the immobilized biocatalyst
(PHBV-ITP) were kd = 0.0038 h-1 and t1/2 = 182.41 h at
a temperature of 37 �C and kd = 0.0066 h-1 and t1/2 =
105.02 h at 60 �C.
Kinetic parameters
The kinetics of the hydrolytic activity of the immobilized
biocatalyst (PHBV-ITP) were investigated using various
concentrations of an olive oil substrate (at 37 �C, pH 4.0).
The Michaelis–Menten equation was used to fit the kinetic
parameters and data from the initial reaction rate to eval-
uate the constants, Km and Vmax, with the programme
Origin� 8. The Km and Vmax of the free enzyme were
76.85 mM and 110 U/g, respectively, as determined by
Barbosa et al. [23], whereas the apparent Km and apparent
Vmax of the immobilized enzyme were 49.10 mM and 205
U/g, respectively. Lipase immobilized on PHBV showed a
decrease in apparent Km. In addition, we observed an
increase in Vmax for the immobilized biocatalyst; these
changes in the kinetic parameters (Km and Vmax) suggest
that the immobilization of ITP-001 lipase by adsorption
resulted in increased affinity for the substrate and better
accessibility to the active site. Pahujani et al. [13], for the
lipase from Bacillus coagulans BTS-3 immobilized onto
glutaraldehyde-activated nylon-6 by covalent binding,
reported values of Km = 4 mM and Vmax = 10 lmol/min/
ml, while for the free form, the Vmax was 0.72 lmol/min/
ml and the Km was 3.8 mM.
Reusability of the immobilized lipase
The reuse of an enzyme constitutes the main advantage of
the process of biocatalyst immobilization, and is an
important parameter for repeated applications in batch
reactors or for continuous use. Figure 4 shows the variation
in the relative activity of the immobilized biocatalyst after
multiple cycles of reuse. The reusability of the immobi-
lized lipase (PHBV-ITP) was tested and it was found to
retain up to 50 % of its activity after eight reuses. Although
somewhat decreased activity of the immobilized enzyme
was found with each successive reaction, this may be due
to lipase release from the surface of PHBV during the
multiple soaking, separation and washing steps employed
during the recycling reaction, because the enzyme is only
0 1 2 3 4 570
80
90
100
Rel
ativ
e ac
tivi
ty (
%)
Time (h)
Fig. 3 Thermal stability of free and immobilized Lipase from
Bacillus sp. ITP-001. Incubated at 37 �C (filled square PHBV-ITP
and square Free ITP 37 �C) and 60 �C (triangle PHBV-ITP and filledtriangle Free ITP). Error bars are standard deviations from triplicates
0 2 4 6 8 10 12 140
10
20
30
40
50
60
70
80
90
100
110
Rel
ativ
e ac
tivi
ty (
%)
Number of reuse cycles
Fig. 4 Relative activity of immobilized lipase from Bacillus sp.ITP-
001 as a function of reuse. Error bars are standard deviations from
triplicates
Bioprocess Biosyst Eng
123
attached by weak interaction forces [39]. A similar result
was reported in our previous study, where a decrease in
enzymatic activity was also observed for CRL immobilized
on the same support (PHBV) [19]. The literature reports
decreased activity with the reuse of the lipase from ther-
mophilic Bacillus sp. immobilized on silica and HP-20
beads [34] and also for the lipase from Bacillus coagulans
BTS-3 immobilized on glutaraldehyde-activated bylon-6
by covalent binding [13]. Kumar et al. [38] reported that
the Bacillus coagulans BTS3 lipase immobilized on glu-
taraldehyde-activated polyethylene retained more than
50 % of its activity after 10 cycles of olive oil hydrolysis,
while the enzyme on the non-activated carrier was half-
inactivated after eight cycles. This suggests that the ITP-
001 lipase immobilized on PHBV could be more suitable
for industrial applications due to its easy recovery from the
reaction system and efficient reuse.
Physico-chemical properties
Specific surface area and porous properties
The Brunauer-Emmett-Teller (BET) surface area of the
pure PHBV and immobilized (PHBV-ITP) samples as well
as their pore parameters are listed in Table 1. Figure 5a
and b report the nitrogen adsorption–desorption isotherms
of the samples at 77 K for the support (PHBV) before and
after loading of the enzyme. The nitrogen adsorption–
desorption isotherms of PHBV before and after adsorption
of ITP-001 can be classified as a type III isotherm which
does not exhibit any type of hysteresis. These are typical
features of non-porous and macroporous materials [40].
According to the results presented in Table 1, the
addition of the ITP-001 lipase on pure PHBV did not
significantly influence the values obtained for the surface
area. The value obtained for the specific surface area of the
immobilized (PHBV-ITP) sample was close to that
obtained for pure PHBV, used as the support for the lipase
from Bacillus sp. ITP 001. This is in agreement with the
results obtained for nitrogen adsorption–desorption iso-
therms which demonstrated the same type of isotherm for
PHBV before and after the adsorption of the lipase from
Bacillus sp. ITP-001 (Fig. 5).
However, the slight decrease observed in the specific
surface area (from 6.21 to 5.70 m2/g) with the addition of
the ITP-001 enzyme could be attributed to the blocking of
some micropores, observed in the distribution of pore sizes
(Fig. 6) present on the surface of the PHBV used as the
support. A greater decrease was observed for pore volume,
where the value for the support was 1.447 9 10-2 cm3/g,
while for the immobilized enzyme, this value was
0.994 9 10-2 cm3/g (Table 1). On the other hand, according
to the results presented in Table 1, the addition of the enzyme
to the support exerted a strong influence on the mean pore
diameter. The values obtained for the mean pore diameter of
pure PHBV and the immobilized enzyme were 104 and 61 A,
respectively. The marked decrease in the mean pore diameter
and the decreased surface area and pore volume were not
thought to be mediated by structural collapse caused by the
immobilization of lipase. These results also suggest that
the immobilization of ITP-001 lipase occurred in channels
present in PHBV [41, 42].
Table 1 Textural properties of PHBV before and after adsorption of
ITP-001
Samples Surface area
(m2/g)
Pore volume
cm3/g (9 10-2)
Mean pore
diameter (A)
Pure PHBV 6.2 1.447 104
PHBV-ITP 5.7 0.994 61
0.0 0.2 0.4 0.6 0.8 1.00
2
4
6
8
10
12
Vo
lum
e (c
m3 /g
)
Relative Pressure (P/Po)
a
0.0 0.2 0.4 0.6 0.8 1.00
1
2
3
4
5
6
7
8
Vo
lum
e (c
m3 /g
)
Relative Pressure (P/Po)
b
Fig. 5 Nitrogen adsorption–desorption isotherms of PHBV before
(a) and after (b) lipase from Bacillus sp. ITP-001 loading. (filledsquare adsorption and square desorption)
Bioprocess Biosyst Eng
123
The pore size distributions for pure PHBV and the
immobilized biocatalyst (PHBV-ITP) are shown in Fig. 6.
In samples of pure PHBV and the immobilized biocatalyst
(PHBV-ITP), most of the pores had diameters smaller than
200 A. This is in agreement with the results obtained for
the isotherms of both samples, according to the BET
classification [40], are related to very weak interactions in
systems containing macro and mesopores. Indeed, pure
PHBV and PHBV-ITP are mostly mesoporous materials, as
evidenced by the pore size distribution, although larger
pore diameters were also observed.
Differential scanning calorimetry (DSC)
Figure 7 depicts the DSC curves for the free enzyme
(ITP-001), the support (PHBV) and the immobilized bio-
catalyst (PHBV-ITP). The behaviour of the free enzyme
was slightly different compared to the support and the
immobilized biocatalyst which showed similar behaviours.
It was possible to identify in all the samples a single peak
corresponding to melting in the temperature range studied.
For ITP-001, the melting temperature (Tm = 152.8 �C) and
enthalpy of fusion (DHm = 2.54 J/g) were determined. For
PHBV, the melting temperature (Tm = 166.6 �C) and
enthalpy of fusion (DHm = 78.35 J/g) were also deter-
mined; these values were similar to those reported in the
literature for pure PHBV [43]. For the immobilized bio-
catalyst (PHBV-ITP), Tm = 170.85 �C and DHm = 48.20
J/g were determined. However, according to these data, we
suggest that the enzyme immobilized on the support
absorbed less heat to reach the melting temperature, since
the values obtained with the immobilized enzyme
(ITP-001) on the support (PHBV) were similar to the
values for the support alone, although these values were
slightly higher than for the support alone. This may have
been due to the difference in thermal conductivity of the
immobilized biocatalyst and the support by promoting a
reduction in heat flow. Similar results were observed in our
previous study for a Candida rugosa lipase immobilized on
the same support (PHBV) [19].
TGA results
Thermogravimetric analysis (TGA) is an important tool
which enables the determination of the temperature range
at which a heated sample undergoes a major conforma-
tional change, by means of monitoring its thermal weight
loss profile. The weight loss curves were divided into three
regions: region I (0–250 �C), region II (250–400 �C) and
region III (400–600 �C).
Figure 8 shows that in region I, the free enzyme (ITP-
001) lost mass from the beginning, due to the weakly
adsorbed water on the surface of the enzyme, while for the
support (PHBV) and the immobilized biocatalyst (PHBV-
ITP), the mass loss remained nearly constant. In region II,
thermal decomposition started with considerable mass loss
with increasing temperature for all three samples, although
this was more pronounced for the support and the immo-
bilized biocatalyst. For PHBV, the maximum degradation
occurred at 345 �C, with onset (Tinitial) at 275 �C, consis-
tent with previous reports in the literature stating that
PHBV is thermally unstable above 250 �C [44]. During
PHBV thermal degradation, chain scission and hydrolysis
were shown to lead to a reduction in molecular weight and
the formation of crotonic acid [44]. For PHBV-ITP, the
maximum degradation occurred at 320 �C, with onset at
0 200 400 600 800 1000 1200 1400
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014D
iffe
ren
tial
vo
lum
e (c
m3 /g
))
Pore diameter (Α)
Fig. 6 Pore size distribution for the PHBV before and after lipase
from Bacillus sp. ITP-001 loading. (filled square PHBV and filledtriangle PHBV-ITP)
100 120 140 160 180 200
Hea
t fl
ux
(mW
/mg
)
Temperature (°C)
PHBV
PHBV-ITP
ITP
Fig. 7 DSC curves during heating for free enzyme lipase from
Bacillus sp. (ITP-001), support (PHBV) and immobilized biocatalyst
(PHBV-ITP)
Bioprocess Biosyst Eng
123
260 �C. The immobilized biocatalyst was more stable than
the free ITP-001 lipase, as shown in region II in Fig. 8.
In region III, it was observed that after the thermal
decomposition of the support and the immobilized biocata-
lyst (ITP-PHVB), a slight variation of the mass occurred. For
the free ITP-001 lipase, the mass loss was still substantial in
region III. This was probably associated with the decompo-
sition of organic compounds from the biocatalyst [22]. Our
previous study showed similar behaviour for a Candida
rugosa lipase immobilized on the same support [19].
FTIR analysis
The FTIR spectra obtained for the free enzyme (ITP-001),
the support (PHBV) and the immobilized biocatalyst
(PHBV-ITP) are shown in Fig. 9.
The free enzyme displayed a typical FTIR protein spec-
trum; with weak bands in the range of 1,650–1,540 cm-1
associated with characteristic primary and secondary amino
groups (CONH). Although those bands were very faint in the
FTIR spectrum of the immobilized biocatalyst, they revealed
the presence of the lipase amino groups.
The FTIR spectra for PHBV and PHBV-ITP showed
different results, with a substantial increase in the bands for
the immobilized biocatalyst versus the support, consistent
with the presence of the ITP-001 enzyme on the support
surface, verifying once again that the immobilization
technique by physical adsorption was efficient.
In the FTIR spectrum of PHBV, bands assigned to C–O–
C group stretching vibrations between 1,245–1,319 cm-1
were present, as well as bands associated with double bond
C=O stretching at 1,700–1,760 cm-1, in agreement with
the results reported by Goncalves et al. [43]. Bands were
also present at approximately 1,240, 1,220 and 1,160 cm-1
(marked with arrows), which were sensitive to the degree
of crystallisation. In addition, the bands at approximately
1,130, 1,090 and 1,020 cm-1 were also sensitive to the
degree of crystallisation, but to a lesser extent [44]. The
bands of the immobilized biocatalyst (PHBV-ITP) were
stronger compared with the bands of a Candida rugosa
lipase immobilized on the same support reported our pre-
vious study [19].
Conclusion
In the present study, the lipase from Bacillus sp. ITP-001
was immobilized on PHBV by physical adsorption with an
immobilized yield of 25 %. The immobilized lipase on
PHBV displayed better stability than the free form. Indeed,
immobilization of the enzyme primarily enhanced its
thermal stability. The results obtained by N2 adsorption–
desorption isotherms clearly showed that the ITP-001
lipase was adsorbed into the channels of PHBV and the
mesoporous and macroporous support structure was
retained after the adsorption of ITP-001 lipase. The addi-
tion of the ITP-001 lipase onto pure PHBV did not sig-
nificantly influence the values obtained for the surface area
of the biocatalyst. The physical adsorption method used in
the immobilization process produces biocatalysts with a
similar order of magnitude for the specific surface area to
that of the solid used as the support. Consequently, the
nature of the support is preserved during biocatalyst
preparation. The results obtained by DSC showed that the
behaviour of the free enzyme was different compared to the
immobilized biocatalyst. The immobilization of the
enzyme increased the enthalpy of fusion and thus provided
greater thermal stability to the biocatalyst. Indeed, the TGA
profiles clearly showed that the immobilized biocatalyst
100 200 300 400 500 600-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5M
ass
(mg
)
Temperature (°C)
Region II
Region I
Region IIIPHBV
ITP
PHBV-ITP
Fig. 8 Thermogravimetric curves for free enzyme lipase from
Bacillus sp. (ITP-001), support (PHBV) and immobilized biocatalyst
(PHBV-ITP)
1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500
Tra
nsm
itan
ce (
a.u
.)
Wavenumber (cm-1)
ITP
PHBV
PHBV-ITP
C=O C-O-C
Amino I Amino II
Fig. 9 FTIR spectra of the free enzyme lipase from Bacillus sp.
(ITP-001), support (PHBV) and immobilized biocatalyst (PHBV-ITP)
Bioprocess Biosyst Eng
123
was more stable than the free ITP-001 lipase. These results
indicate that PHBV can be used as a support for the
immobilization of lipase for industrial applications, espe-
cially food and oleochemistry industrial.
Acknowledgments The authors acknowledge financial assistance
from the Brazilian research funding agencies CAPES, CNPq and
FAPITEC/SE.
References
1. Sharma R, Chisti Y, Banerjee UC (2001) Biotechnol Adv
19:627–662
2. Gupta N, Sahai V, Gupta R (2007) Process Biochem 42:518–526
3. Guncheva M, Zhiryakova D (2011) J Mol Catal B Enzym
68:1–21
4. Carvalho NB, Souza RL, Castro HF, Zanin GM, Lima AS, Soares
CMF (2008) Appl Biochem Biotechnol 150:25–32
5. Feitosa IC, Barbosa JMP, Orelana SC, Lima AS, Soares CMF
(2010) Acta Scienctarum Technol 32:27–31
6. Carrasco-Lopez C, Godoy C, De Las Rivas B, Fernandez-Lorente
G, Palomo JM, Guisan JM, Fernandez-Lafuente R, Martınez-
Ripoll M, Hermoso JA (2009) J Biol Chem 248:4365–4372
7. Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM (2008) J
Mol Biol 381:324–340
8. Matsumura H, Yamamoto T, Leow TC, Mori T, Salleh AB, Basri
M, Inoue T, Kai Y, Rahman RNZRA (2008) Proteins 70:592298
9. Tyndall JDA, Sinchaikul S, FothergillGilmore LA, Taylor P,
Walkinshaw MD (2002) J Mol Biol 323:859–869
10. Jeong ST, Kim HK, Kim SJ, Chi SW, Pan JG, Oh TK, Ryu SE
(2002) J Biol Chem 277:17041–17047
11. Yoneda K, Nishimura T, Katunuma N, Imamura S, Nitta K,
Tsuge H (2002) Acta Cryst D 58:1232–1233
12. Kanwar SS, Chauhan GS, Chimni SS, Chauhan S, Rawat GS,
Kaushal RK (2006) J App Polym Sc 100:1420–1426
13. Pahujani S, Kanwar SS, Chauhan G, Gupta R (2008) Bioresour
Technol 99:2566–2570
14. Godoy CA, De Las Rivas B, Filice M, Fernandez-Lorente G,
Guisan JM, Palomo JM (2010) Process Biochem 45:534–541
15. Mateo C, Abian O, Fernandez-Lafuente R, Guisan JM (2000)
Enzyme Microb Technol 26:509–515
16. Dalla-Vecchia R, Nascimento MG, Soldi V (2004) Quim Nova
27:623–630
17. Costa MS, Duarte ARC, Cardoso MM, Duarte CMM (2007) Int J
Pharm 328:72–77
18. Chen GQ, Wu Q (2005) Biomaterials 26:6565–6578
19. Cabrera-Padilla RY, Lisboa MC, Fricks AT, Franceschi E, Lima
AS, Silva DP, Soares CMF (2012) J Ind Microbiol Biotechnol
39:289–298
20. Souza RL, Barbosa JMP, Zanin GM, Lobao MWN, Soares CMF,
Lima AS (2010) Appl Biochem Biotechnol 161:288–300
21. Soares CMF, De Castro HF, De Moraes FF, Zanin GM (1999)
Appl Biochem Biotechnol 77–79:745–758
22. Soares CMF, Santos OA, De Castro HF, Moraes FF, Zanin GM
(2004) Appl Biochem Biotechnol 113:307–319
23. Barbosa JMP, Souza RL, Melo CM, Fricks AT, Soares CMF,
Lima AS (2011) Quimica Nova (in press)
24. Da Ros PCM, Silva GAM, Mendes AA, Santos JC, De Castro HF
(2010) Bioresource Technol 101:5508–5516
25. Brunauer S, Emmett PH, Teller E (1938) J Am Chem Soc
60:309–319
26. Soares CMF, Dos Santos OAA, Olivo JE, De Castro HF, De
Moraes FF, Zanin GM (2004) J Mol Catal B Enzym 29:69–79
27. ChiralVisao. http://www.chiralvision.com/immobead.htm. Acces-
sed 15 Aug 2011
28. Montero S, Blanco A, Virto MD, Landeta LC, Agud I, Solozabal
R, Lascaray JM, Renobales M, Llama MJ, Serra JL (1993)
Enzyme Microb Technol 15:239–247
29. Yesiloglu Y (2005) Process Biochem 40:2155–2159
30. Ghiaci M, Aghaei H, Soleimanian S, Sedaghat ME (2009) App
Clay Sci 43:289–295
31. Yigitoglu M, Temocin Z (2010) J Mol Catal B Enzym
66:130–135
32. Mendes JBE, Riekes MK, Oliveira VM, Michel MD, Stulzer HK,
Khalil NM, Zawadzki SF, Mainardes RM, Farago PV (2012) Sci
World J 2012:13. doi:10.1100/2012/542937 (Article ID 542937)
33. Karimpil JJ, Melo JS, D’Souza SF (2011) J Mol Catal B Enzym
71:113–118
34. Nawani N, Singh R, Kaur J (2006) Electron J Biotechnol
9:559–565
35. Dosanjh NS, Kaur J (2002) Biotechnol Appl Biochem 36:7–12
36. Palomo JM, Segura RL, FernandezLorente G, Pernas M, Rua
ML, Guisan JM, FernandezLafuente R (2006) Biotechnol Prog
20:630–635
37. Kharrat N, Ali YB, Marzouk S, Gargouri YT, Karra-Chaabouni
M (2011) Process Biochem 46:1083–1089
38. Kumar S, Pahujani S, Ola RP, Kanwar SS, Gupta R (2006) Acta
Microbiol Immunol Hung 53:219–231
39. Yadav GD, Jadhav SR (2005) Micropor Mesopor Mater
86:215–222
40. Gregg SJ, Sing KSW (1982) Adsorption, surface area and
porosity. Academic Press, London, p 4
41. Li Y, Zhou G, Li C, Qin D, Qiao W, Chu B (2009) Colloids Surf
A Physicochem Eng Asp 341:79–85
42. Zhou G, Chen Y, Yang S (2009) Micropor Mesopor Mater
119:223–229
43. Goncalves SPC, Martins-Franchetti SM, Chinaglia DL (2009) J
Polym Environ 17:280–285
44. Singh S, Mohanty AK (2007) Composites Sc Technol 67:1753–
1763
Bioprocess Biosyst Eng
123
top related