Use of polyethylene glycol in the process of sol–gel encapsulation of Burkholderia cepacia lipase
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- 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
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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.
KeywordsLipaseImmobilizationSol–gelProtic ionic liquids
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 [3–5]. 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 [6–8]. Activation at the hydrophobic interface is a characteristic property of lipases, which was first reported by Sarda and Desnuelle in 1958 .
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 [10–12]. 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 [15–18]. 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  and protic ionic liquid  in the process of sol–gel immobilisation is one way of circumventing the negative effects caused during the shrinkage of the gel [11, 23–25]. 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.
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  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.
The determination of enzymatic activity of lipases with and without PEG was performed by the method described by Soares et al. , 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).
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
Influence of PEG concentration used for lipase encapsulation on enzyme activity
Enzymatic activity/U g−1
33.98 ± 0.00
30.56 ± 5.25
89.91 ± 6.51
76.65 ± 4.75
76.84 ± 4.17
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. .
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. .
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 , providing a thermal characterization of immobilized biocatalysts.
Thermogravimetric analysis: DTA/TG
Loss of mass peaks associated with the degradation of lipase samples with or without PEG
Total mass loss/%
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 , 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.
The mass loss in region III was associated with dehydroxylation reactions , 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 , 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 , as discussed earlier.
Scanning electron microscopy (SEM)
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).
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.
The authors thank CAPES Pro-Engineering program for financial support (Process 23038-028317/2008-44).