Journal of Thermal Analysis and Calorimetry

, Volume 117, Issue 1, pp 301–306

Use of polyethylene glycol in the process of sol–gel encapsulation of Burkholderia cepacia lipase

Authors

    • UNIT, Universidade Tiradentes
  • Emanuelle L. P. Faria
    • UNIT, Universidade Tiradentes
  • Renan T. Figueiredo
    • UNIT, Universidade Tiradentes
    • ITP, Instituto de Tecnologia e Pesquisa
  • Alini T. Fricks
    • UNIT, Universidade Tiradentes
    • ITP, Instituto de Tecnologia e Pesquisa
  • Gisella M. Zanin
    • UEM, Universidade Estadual de Maringá
  • Onélia A. A. Santos
    • UEM, Universidade Estadual de Maringá
  • Álvaro S. Lima
    • UNIT, Universidade Tiradentes
    • ITP, Instituto de Tecnologia e Pesquisa
  • Cleide M. F. Soares
    • UNIT, Universidade Tiradentes
    • ITP, Instituto de Tecnologia e Pesquisa
Article

DOI: 10.1007/s10973-014-3663-0

Cite this article as:
Souza, R.L., Faria, E.L.P., Figueiredo, R.T. et al. J Therm Anal Calorim (2014) 117: 301. doi:10.1007/s10973-014-3663-0

Abstract

Lipases from Burkholderia cepacia were encapsulated using polyethylene glycol (PEG, Mw 1500) at various concentrations (0.5–3.0 %, w/v) as an additive during the sol–gel immobilisation process. Matrixes immobilized in the presence and absences of additives were characterized by thermal analysis [thermogravimetric (TG) and differential scanning calorimetry (DSC)], scanning electron microscopy (SEM), enzymatic activity, and total activity recovery yield (Ya). The addition of PEG increased the activity values, with Ya just above 1.0 % (w/v) in the presence of PEG. The additional of 1.0 % (w/v) PEG increased enzyme activity from 33.98 to 89.91 U g−1 and the values of recovery yield were 43.0–91.4 %, compared to values of the samples without PEG. PEG enhanced the thermal stability of the matrix structure in the temperature range 50–200 °C, as confirmed by TG and DSC analyses. This was influenced by the presence of water bound to the matrix. The SEM micrographs clearly showed an increase in the number of deposits on the material surface, producing matrices with greater porosity.

Keywords

LipaseImmobilizationSol–gelProtic ionic liquids

Introduction

Applications of lipases in industrial processes are becoming increasingly important. The commercial values of these enzymes may be enhanced by using immobilisation techniques that maintain catalytic efficiency and operational stability. Therefore, numerous efforts have been focused on preparing immobilized lipase in ways that involve both a variety of support materials and immobilisation methods [1, 2]. Several different approaches have been used to immobilize the lipase from Burkholderia cepacia on various supports [35]. In the literature, the use of additives in lipase immobilization on the surface of a solid support through hydrophobic interactions has been widely reported for the applications of lipase in industrial processes [68]. Activation at the hydrophobic interface is a characteristic property of lipases, which was first reported by Sarda and Desnuelle in 1958 [9].

One alternative for the immobilisation of enzymes is gel entrapment, which involves lesser structural strain for the enzyme as it is captured in its native form in a specific microenvironment that can enhance the stability and activity of the lipase. Hydrolysis and polycondensation of alkoxide precursors in solution at a low temperature is based on the sol–gel process, leading to the formation of a polymer network that allows the incorporation of a variety of compounds [1012]. This is an alternative to produce materials exhibiting stable physical properties that cannot be obtained by other processes [13, 14]. A very large number of biomolecules, including enzymes, antibodies, microorganisms, plant, and animal cells, have been immobilized by the sol–gel technique [1518]. This generally gives better enzyme activity and stability, however, there are some disadvantages in the process of sol–gel immobilisation [19, 20].

The use of additives such as aliquat [21] and protic ionic liquid [22] in the process of sol–gel immobilisation is one way of circumventing the negative effects caused during the shrinkage of the gel [11, 2325]. The use of macromolecules such as polyethylene glycol (PEG) in the process of immobilisation has been reported to elicit a better distribution of the lipase on the mounting surface, allowing better contact between the water/oil interface, and favouring necessary conditions for the hydrolytic activity of the immobilized lipase [11, 26, 28].

Through the careful selection of additives, these materials can be designed for specific applications and can produce useful, robust devices. Despite reports in the literature about the use of PEG for lipase immobilisation, none have studied the effects of varying the concentration of the additive on immobilized lipases.

Therefore, the objective of this work was to use PEG as an additive at different concentrations during the sol–gel immobilisation of lipase from B. cepacia. Moreover, morphological structure [scanning electron microscopy (SEM)], thermal properties differential scanning calorimetry (DSC) and thermogravimetric analysis (TG), and full recovery of activity were characterized.

Experimental

Materials and reagents

Lipase from B. cepacia (Amano Lipase) was purchased from Aldrich. The silane precursor tetraethoxysilane (TEOS) was supplied by Across Organic (New Jersey, United States) and used without further purification. Ethanol (minimum 99 %), ammonia (minimum 28 %), hydrochloric acid (minimum 36 %) and gum Arabic were obtained from Synth (São Paulo, Brazil). PEG with a nominal molecular mass of 1,500 g mol−1 was purchased from Sigma Chemical (Co., USA). Water was purified by reverse osmosis and deionized through a Milli-Q four-cartridge organic-free water purification system. Other chemicals were of analytical grade and used as received.

Encapsulation of lipase from Burkholderia cepacia in sol–gel matrices

The methodology previously established by Patent PI0306829-3 [27] was used and is briefly described as follows: 30 mL of TEOS were dissolved in 36 mL of absolute ethanol under an inert nitrogen atmosphere. To this, 0.22 mL of hydrochloric acid dissolved in 5 mL of ultra-pure water was slowly added and the mixture was shaken (200 rpm) for 90 min at 35 °C. The enzyme (2693.25 U) and additive [PEG, Mw 1500, used at concentrations from 0.5 to 3.0 % (w/v)] were added to 10 mL of ultra-pure water; at the same time, 1.0 mL of ammonium hydroxide dissolved in 6.0 mL of ethanol was added (hydrolysis solution) and the mixture was kept under static conditions for 24 h to complete polycondensation. The bulk gel was washed with heptane and acetone and dried under vacuum at room temperature for 72 h. For comparison, the encapsulated lipase from B. cepacia was prepared similarly in the absence of PEG (EN-AP) and pure silica sol–gel (PS) was prepared in the absence of both the enzyme and additive. The samples with the presence of PEG were designated as EN-P0.5, EN-P1.0, EN-P2.0, and EN-P3.0.

Enzymatic activity

The determination of enzymatic activity of lipases with and without PEG was performed by the method described by Soares et al. [6], with some modifications. The substrate was prepared by mixing 50 mL of the olive oil with 50 mL of gum Arabic solution (7 % w/v). The reaction mixture containing 5 mL of the oil emulsion, 4 mL of sodium phosphate buffer (0.1 M, pH 7.0) and either free (1.0 mL, 0.1 mg mL−1) or immobilized (≈250 mg) lipase was incubated in a thermostated batch reactor for 5 min (free lipase) or 10 min (immobilized lipase) at 37 °C, with stirring 80 rpm. A blank titration was done with a sample where the enzyme was replaced with distilled water. The reaction was stopped by the addition of 2 mL of acetone–ethanol–water solution (1:1:1). The liberated fatty acids were titrated with potassium hydroxide solution (0.04 M) in the presence of phenolphthalein as an indicator. All reactions were carried out in triplicate. One unit (U) of enzyme activity was defined as the amount of enzyme that liberated 1 μmol of free fatty acid per min (μmol min−1) under the assay conditions (37 °C, pH 7.0, and 80 rpm).

Analyses of hydrolytic activities carried out in the lipase loading solution and encapsulated preparations were used to determine the total activity recovery yield, Ya (%), according to Eq. (1).
$$ \left( {Ya = U_{\text{s}} /U_{\text{o}} \times 100} \right) $$
(1)
in which Us is the total enzyme activity recovered on the support and Uo is the enzyme units offered for immobilization.

Sample characterization

Scanning electron microscopy (SEM; model Hitachi SU-70) was used to characterize the surface of pure silica matrices and immobilized lipase samples. The thermogravimetric (DTA/TG) curve was obtained in a Shimadzu DTG-60H Simultaneous DTA-TG apparatus, under a nitrogen atmosphere that started from room temperature and went up to 1,000 °C at a heating rate of 20 °C min−1. The DSC curve was obtained in a Shimadzu DSC-60, under a nitrogen atmosphere that started from room temperature and went up to 500 °C at a heating rate of 10 °C min−1.

Results and discussion

Enzymatic activity of the immobilized lipase

The motivation for the use of additives in the process of sol–gel immobilization arose from the possibility of preserving the catalytic activity of the lipase from B. cepacia during the process of gel formation. Therefore, in this study, we evaluated the effect of PEG concentration on the process of encapsulation of lipase by measuring the catalytic activity of the enzyme (Table 1).
Table 1

Influence of PEG concentration used for lipase encapsulation on enzyme activity

Sample

Dry mass/g

Enzymatic activity/U g−1

Total activity/U

EN-AP

14.11

33.98 ± 0.00

374.00

EN-P0.5

7.53

30.56 ± 5.25

184.69

EN-P1.0

9.65

89.91 ± 6.51

795.47

EN-P2.0

8.68

76.65 ± 4.75

602.42

EN-P3.0

8.21

76.84 ± 4.17

583.39

The use of low-molecular-mass additives such as 1.0 % (w/v) PEG enhances catalytic activity, according to the literature, when the Candida rugosa lipase is covalently immobilized on silanized-controlled pore silica (CPS) [29, 30]. We also observed the same trend; increasing PEG concentration during the sol–gel immobilization of the lipase increased enzyme activity from 33.98 to 89.91 U g−1, when compared to the sample without PEG (EN-AP). The same effect was observed for the total activity yield recovered (43.0–91.4 %), as seen in Fig. 1. Raising the concentration of PEG induced favorable condition for enzymatic activity. Decreases in catalytic activity and yield of enzymatic activity (Ya) were observed when the amount of PEG used was above its optimal level (1.0 % w/v). This might have been due to reduced pore size and substrate accessibility. In accordance with our results, Soares et al. [11] also observed an increase in the yield of recovery of enzymatic activity of immobilized Candida rugosa lipase, reaching values of 60 % in the presence of PEG, however, their values were lower than ours. Mohidem and Mat [26] found that the catalytic activity of laccase was enhanced using PEG of low-molecular mass (PEG, Mw 600) during sol–gel immobilization.
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-014-3663-0/MediaObjects/10973_2014_3663_Fig1_HTML.gif
Fig. 1

Total activity recovery yield of lipase from Burkholderia cepacia encapsulated in the absence and presence of different concentrations of PEG

Our results showed the positive effect of PEG on the encapsulation process of lipase, confirming the literature data that indicated low-molecular-mass additives such as PVA, PEG, and APTS to increase the catalytic activity of enzymes [26, 30]. The presence of PEG during the immobilization process probably affects the moisture level of the lipase by modifying the hydrophobicity of the microenvironment, similar to that proposed Mohidem et al. [26].

The additive can also act as an agent model or modify the morphology of the pores of the gel, thereby facilitating the internal mass transfer and providing better accessibility of the substrate, similar to that reported by Yi et al. [31].

Thermal characterization

The thermal behavior of the matrices obtained by sol–gel method, can be analyzed using the DTA/TG and DSC techniques which allows for different engine that form gels [32], providing a thermal characterization of immobilized biocatalysts.

Thermogravimetric analysis: DTA/TG

Figure 2 shows the TG curves for samples of immobilized enzyme in the presence and absence of PEG. The mass loss obtained after heating the samples to 1,000 °C is reported as TG mass loss in Table 2.
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-014-3663-0/MediaObjects/10973_2014_3663_Fig2_HTML.gif
Fig. 2

TG curves of samples without PEG (EN-AP) and with PEG at 20 °C min−1 under a nitrogen atmosphere

Table 2

Loss of mass peaks associated with the degradation of lipase samples with or without PEG

Sample

Water content/%

Temperature rangea/°C

Mass lossb/%

Temperature rangea/°C

Mass lossb/%

Temperature rangea/°C

Mass lossb/%

Total mass loss/%

EN-AP

21.88

26.94–194.7

22.22

204.85–604.3

18.93

657.49–812.6

2.10

45.12

EN-P0.5

19.72

31.57–191.5

16.23

203.16–647.6

18.31

679.85–840.2

2.79

38.37

EN-P1.0

8.29

38.36–187.8

10.20

194.38–617.6

20.06

651.37–873.3

3.77

34.74

EN-P2.0

9.46

33.38–186.0

8.18

200.75–617.7

25.68

633.90–910.4

0.84

35.18

EN-P3.0

7.48

33.48–183.2

9.26

199.93–531.9

46.59

545.59–701.9

6.25

63.48

aInitial temperature and final the main peak of degradation determined by analysis of the DTA curve

bSamples of mass loss related to temperature range

The TG curves were divided into three regions to illustrate which groups were lost in specific temperature ranges. Region I was mainly associated with dehydration, the decomposition of amino groups, and the solvent adsorbed onto the surface of the material, usually organic groups. This region comprised temperatures close to 200 °C. We could observe that increasing the amount of PEG, which has hydrophobic characteristics, induced a lower loss in mass in this temperature range compared to the sample without the additive (EN-AP), as shown in Table 2. Rao and Kalesh [33], in agreement with the observed results, reported that only materials prepared with TEOS presented hydrophilic groups such as Si–OH on their surfaces. These groups can absorb moisture from the environment and therefore, undergo a significant loss of mass at temperatures between 50 and 200 °C. This was observed in the sample EN-AP which exhibited the highest loss in mass (22.22 %) and water content (21.88 %) in this temperature range. This could have been due to the evaporation of water and/or alcohol groups that are produced from condensation reactions between the Si–OH groups. Thus, the presence of a higher amount of Si–OH groups in the matrix immobilized matrix does not promote enzymatic activity, as seen previously in Fig. 1. This may be linked to the fact that a higher content of water is directed into the matrix, increasing the hydration shell of the enzyme and consequently enhancing enzymatic activity. In fact, the greatest loss in mass in this region (Region II in Fig. 2) occurred when the lipase was immobilized with PEG.

Region II, which comprised temperatures between approximately 200 and 600 °C, was associated with the loss of organic components (C, H, O, and N), including lipases, in the form of volatile compounds present or formed at the beginning of organic decomposition, as well as the loss of unreacted silanol groups of TEOS present in the silica because of incomplete sol–gel reactions [34] and the removal of water molecules that were strongly linked to the silica matrix [35]. The results presented in this temperature range justified the use of PEG in lipase immobilization, as the lipase-PEG samples displayed greater mass loss in this region II than in region I (Table 2), demonstrating that the particles of cross-linked PEG formed a protection to the enzymes. Furthermore, we can see that the results agree with those observed by Suay and coworkers [36] when the process of degradation of the Si–O–Si network is achieved from 450 °C. This phenomenon can best be seen in Fig. 3 at the samples immobilized with PEG above 2 % (w/v).
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-014-3663-0/MediaObjects/10973_2014_3663_Fig3_HTML.gif
Fig. 3

DSC curves at 10 °C min−1 under a nitrogen atmosphere of lipase samples in the presence of PEG at concentrations of 0.5–3.0 % (w/v) (EN-P0.5, EN-P1.0, EN-P2.0, and EN-P3.0) or without PEG (EN-AP)

The mass loss in region III was associated with dehydroxylation reactions [37], as well as whit the carbonization of organic compounds. At temperatures above 750 °C, the material was completely degraded.

Differential scanning calorimetry: DSC

Data of sol–gel encapsulated samples of B. cepacia lipase in the absence and presence of PEG were submitted to DSC calorimetric analysis to study the phase transitions. These transitions give rise to exothermic or endothermic peaks in a particular temperature range in a DSC scan.

The DSC curves shown in Fig. 3 complemented the TG curves discussed earlier. The reduction of the enthalpy values was related to the increase in PEG concentration; values decreased from 530 to 346, 258, 153, and 197 J g−1 for EN-AP, EN-P0.5, EN-P1.0, EN-P2.0, and EN-P3.0, respectively. These results are consistent, since a greater amount of energy is required for decomposition and dehydration, especially at temperatures up to 200 °C for lipases without PEG.

The endothermic and exothermic peaks (Fig. 3) correlated with the decomposition of organic compounds, including lipase and silanol groups [34], as well as with the removal of water molecules, which were tightly bound to the silica matrix, the so-called “water of hydration”. This is strongly linked to the surface of the silica gel [37], as discussed earlier.

Scanning electron microscopy (SEM)

Micrographs of the immobilized lipases in the presence or absence of PEG are shown in Fig. 4. SEM clearly showed that the coating on the support surface in the presence of PEG had an increased number of deposition on the surface of the material, giving higher porosity due to the large accumulation of particles as crystals (Fig. 4c, d). This effect is possibly associated with the use of the additive during contraction, solidification, and the aging steps of the gel, as observed when compared to the samples immobilized without PEG (Fig. 4a, b).
https://static-content.springer.com/image/art%3A10.1007%2Fs10973-014-3663-0/MediaObjects/10973_2014_3663_Fig4_HTML.jpg
Fig. 4

Scanning electron micrographs for derivatives immobilized in the absence of PEG a magnitude ×500 and b magnitude ×1000 and derivatives immobilized with PEG c magnitude ×500 and d magnitude ×1000

The micrograph of the sample immobilized with PEG (Fig. 4c, d) also showed that after coating, there was no continuous layer of the hybrid material. However, the surface appeared to be smoother with a relatively homogeneous appearance, which facilitates access to the substrate and consequently increases the recovery of enzymatic activity, as previously observed (Fig. 1).

Conclusions

The immobilization of lipase from B. cepacia using PEG as an additive in the sol–gel process was successful. The hydrophobic portion of the additive increased the porosity of the sample, which was verified by SEM micrographs. The addition of 1.0 % (w/v) PEG increased enzymatic activity from 33.98 to 89.91 U g−1 and the recovery of activity from 43.0 to 91.4 % when compared to samples without PEG. Increasing the concentration of PEG made the matrix structure more thermally stable in the temperature range of 50–200 °C, and this was directly influenced by the presence of water bound to the matrix.

Acknowledgements

The authors thank CAPES Pro-Engineering program for financial support (Process 23038-028317/2008-44).

Copyright information

© Akadémiai Kiadó, Budapest, Hungary 2014