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