Optimization of olive oil hydrolysis process using immobilized Lipase from Burkholderia cepacia sp. in Polyurethane

The aim of this study was to achieve the best conditions for the olive oil hydrolysis process at optimal pH and temperature using Burkholderia cepacia lipase immobilized in situ in rigid polyurethane support. The influences of the temperature (13.85 to 56.5oC) and pH (4.18 to 9.82) were evaluated by a central composite rotational experimental design 2. The operational stability and storage conditions were also studied. The olive oil hydrolysis process was optimized in pH 7.0, at 40°C and 15 min of reaction, with 66 and 93 U g of hydrolysis activity in free and immobilized lipase, respectively, with > 700% yield. The immobilized remained stable for up to 40 days of storage at temperatures of 60C, and for 100 days from 4 to 25°C. The operational stability of the immobilized was 6 continuous cycles. In this way, immobilization showed to be a promising alternative for its application in olive oil hydrolysis, having storage stability and reuse capability.

In order to overcome these difficulties, industries have invested in efficient and environmentally sustainable waste treatment systems.In this context, we highlight the enzymatic treatments that correspond to the most recent technology for the biological treatment of effluents (Buss & Henkes, 2014, Santos et al., 2015, Silva, Silva, Gois, Almeida, & Abud, 2015).The lipolytic enzymes (lipases) can, by hydrolytic method reduce the concentration and degrade a large number of toxic and persistent substances (Liu et al., 2011, Baldo et al., 2013, Chen, Zhang, Xu, & Yan, 2013, Zadinelo et al., 2013, Costa et al., 2014).
Immobilization processes of different materials are described in the literature, especially the entrapment (with or without binding) during support synthesis.Within this context, the polyurethane (PU) support is highlighted, demonstrating the possibility of lipase in situ during the polymerization stage, presenting excellent activity and yield results in terms of esterification (Nyari et al., 2016).In addition, it has presented chemical compatibility with lipase, resistance to pH, temperature and organic solvents (Simões, Mori, Faria, Castro, & Mendes, 2011, Cui, Tao, Li, Chen, & Tan, 2013, Cipolatti et al., 2015, Nyari et al., 2015).In this context, this study aimed at evaluating the hydrolytic capacity of Burkholderia cepacia sp.lipase immobilized in PU support, as well as the pH and temperature effects on its activity.The immobilization process, stability, storage, and reuse have also been evaluated The chosen reaction model was the olive oil hydrolysis.

Materials
The Burkholderia cepacia sp.lipase used in this study was obtained from Amano Pharmaceuticals Co., Ltd.(Nagoya, Japan).The commercial polyol and isocyanate monomers used in this work were produced for a specific formulation for mattresses and foam injected by the Flexible Polyurethanes -Mannes Company (Erechim, State Rio Grande do Sul, Brazil).The solvents used were acetone (FMaia), ethanol (Merck), olive oil, arabic gum, sodium phosphate (monohydrate and heptahydrate -Nuclear) and sodium hydroxide (Nuclear).

Burkholderia cepacia sp. lipase
The lipase protein content was determined by Bradford (1976), using bovine serum albumin as standard.The specific activity (U mg -1 ) was determined by the enzyme activity quotient (U mL -1 ) and total protein content (mg mL -1 ).
Immobilization of Burkholderia cepacia sp.lipase in polyurethane (PU) The lipase from Burkholderia cepacia sp.immobilization on PU was performed using 6 mL of polyol and 4 mL of isocyanate (60-40%, v v -1 ), with 2 mL of the enzymatic solution, corresponding to 0.2 g of the enzyme (1 g of the enzyme in 10 mL of distilled water) (Nyari et al., 2015).The enzymatic solution was added to the polyol and homogenized (5 s) and after that, isocyanate was added at constant stirring (10 s).The in situ polymerization was conducted at 20°C, until the growing of PU foam (5 min), remaining stationary for 3 hours and then crushed, producing a homogeneous product.

Morphology characterization using scanning electron microscope (SEM)
The morphology of PU support and Burkholderia cepacia sp.lipase in polyurethane (PU) were evaluated by scanning electron microscope (SEM) (DSM960 Zeiss).The samples were mounted on double-sided conductive carbon tape adhered to an aluminum sample port and sputter coated with gold in a sputtering unit (SCD050/ LEICA) at 40 mA for 90 s.Next, they were characterized with SEM operating at 10kV.

Reaction time study
The reaction time to evaluate the hydrolytic activity was monitored for 40 min.The reaction was conducted at 35°C, 0.2 g enzyme, pH 7 and 160 rpm.An independent test was conducted for each time.

Optimization of hydrolysis process
The hydrolytic activity of the immobilized and free lipase was conducted using an experimental design composite (RCCD) 2 2 , with triplicate at a central point, evaluating the influence of the pH and temperature variables.The reaction time was set at 15 min for the optimization test.
Parallel to this, blank tests have been done using only the emulsion and the acetone-ethanol solution, without the addition of lipase.The definition of hydrolytic activity of the lipase (free and immobilized) has been done according to Equation 1.
( ) where: AH = hydrolytic activity (U mL -1 or U g -1 ); M = NaOH molarity; Va = NaOH volume used in the sample titration after the reaction (mL); Vb = NaOH volume used in the black titration (mL); X = enzymatic extract volume (mL) or mass of immobilized (g) used in the reaction; t = reaction time (min).
One lipase activity (hydrolytic) unit was determined as the amount of enzyme that released 1 μmol of fatty acids per minute.

Yield immobilization
The immobilized yield was calculated considering the total activity of the free lipase in solution given on the immobilization process (that considers the volume of the enzymatic extract employed on the immobilization test and its activity [U mL -1 ]) and the immobilized total activity (that considers the total mass of the produced immobilized and its activity (U g -1 ) according to Equation 2. (2) where: RI (%) = Immobilization yield; UT immobilized = total activity on synthesized immobilized; UT o = total activity of the enzymatic solution offered for immobilization.
The hydrolysis reaction was conducted with 0.2 g of biocatalyst, at 35 o C, pH 7, 15 min reaction time and 160 rpm.

Stability Storage and operational
The immobilized stability during the storage was evaluated in temperatures ranging from -4 to 40°C.The hydrolytic activity was evaluated until it presented residual activity lower than 50%.The immobilized operational stability was studied by consecutive batches, with reuse.

Residual activity (%)
The residual activity was used to evaluate the storage activity (free and immobilized thermal stability) and operational/reuse (immobilized), calculated using the initial activity for each system as a reference, according to Equation 3.
where: RA (%) = Residual activity; U X = Enzymatic activity after reuse and storage; U initial = Initial enzymatic activity.

Statistical analysis
Each experiment was done in triplicate.Data were expressed as means ± standard deviation, and subjected to one-way analysis of variance (Tukey) using Statistic 8.0 (StatSoft) software.A significance level of 95% (p < 0.05) was used.

Results and discussion
Characterization of immobilized Burkholderia cepacia sp.lipase The support morphology with the presence of the Burkholderia cepacia sp.lipase is shown in Figure 1.
It is possible to notice that the pores formed during synthesis are heterogeneous and randomly arranged.

Protein content of Burkholderia cepacia sp. lipase
The enzymatic solution used in this work was characterized in terms of quantification of protein

Study of the reaction time
The results of hydrolytic activity for the free and immobilized lipases, in terms of reaction time, are found in Figure 2.  The free and immobilized lipase forms presented the same behavior, with an increase of the hydrolytic activity on the first 15 min of reaction time, followed by a decrease in subsequent times.It demonstrated, at 40 min of reaction time, activities of 2 and 20 U g -1 for the free and immobilized forms, respectively.This trend suggests the presence of two reaction mechanisms (hydrolyses and esterification), both linked to lipases (Liu & Chang, 2008, Liu et al., 2011).Initially, there is a predominance of hydrolyses reaction on the first reaction minutes, which generates alcohol and carboxylic acid as products.The esterification reaction occurs with the concentration increase of alcohol and acid on the reactive system, consuming both the acid and the alcohol, causing a decrease in the hydrolytic activity.
The greatest hydrolytic activities with 75.5 and 43.7 U g -1 for the immobilized and free forms have been observed at 15 min of reaction time and such time was chosen for the subsequent tests.
It is noted that on all periods the immobilized lipase demonstrated greater activity than the free one.Such behavior suggests a positive effect of the immobilization over the enzyme's activity in terms of the hydrolysis reaction.It is worth mentioning that both tests have been done with the same catalyst mass (free and immobilized).In this context, this effect is sharper when considering that the immobilized mass (0.2 g) employed on the test has, theoretically, 0.0037 g of the enzyme.In other words, 5.4 times less enzyme mass (0.02 g) was present on the test with free enzyme, which was conducted with 0.2 mL of the enzymatic extract prepared with solubilisation of 1 g enzyme lyophilized in 10 mL of water.

Optimization of hydrolytic process: effect of pH and temperature
The hydrolyze results obtained by the experimental design 2 2 showed the best activities for free and immobilized forms with 65.1 and 92.2 U g -1 , respectively, at the optimal range (optimal condition) (pH 7 and 40°C) (Table 1).According to the data, it can be noted that the process optimization condition had been achieved.Equation 4 and 5 show the second order coded model for hydrolytic activity of free and immobilized lipase in terms of temperature and pH, within the ranges studied.
The model was validated by variance analysis and the non-significant parameters were added to the lack of fit for the analysis of variance (ANOVA) test.The correlation coefficient was 0.94 and 0.98, the F calculated value was 16.7 and 55.5 times higher than the F tabulated value for the free and immobilized lipase, allowing the contour curve construction in Figure 3, the free lipase (a) and (b) and immobilized lipase (c) and (d) .The optimal range (optimal condition) for the hydrolytic reaction was at pH 7 and at 40°C.The pH adaptability should be associated with the carriers charge.The presence of cations in polyurethane hydroxyl radicals might have reduced the low pH sensitivity.Furthermore, the structure screen function would play an important role on the adaptability enhancement (Hu et al., 2012).
The results obtained in the current work are similar to those from previous reports in the literature by Souza et al. (2014), using Burkholderia cepacia lipase encapsulated with polyethylene glycol (1% of PEG1500) was immobilized in sol-gel with 89.91 U g -1 activity and 91.4% yield.For Padilha, Ferreira, Castiglioni, Alegre, and Tambourgi (2011), using Burkholderia cepacia lipase the activity showed maximum value (1.85 U mL -1 ) to a specific activity of 29.30U mg -1 and the others Jegannathan, Chan, and Ravindra (2009), Liu and Chang (2008) and Hu et al. (2012), on different supports and consequent industrial processes.
The pH and temperature optimization minimize the enzymes' activity loss since they avoid conditions that would favor their inactivation (Soares, Santana, Zanin, & Castro, 2003, Padilha et al., 2011).The immobilization yield calculations that evaluate the process efficiency were conducted at an optimized condition.

Efficiency of immobilization
The immobilization process' efficiency was evaluated in relation to yield.The free enzyme on the enzymatic extract demonstrates 65.1 U mL -1 of hydrolytic activity that corresponds to the total activity given on the 130.2 U immobilization, where 2.0 mL of enzymatic extract were applied at the immobilization stage.The immobilized (10.8 g) showed 90.2 U g -1 of hydrolytic activity, corresponding to 974.2 U of total activity, in other words, an immobilization yield of 748.2%.Yields greater than 100%, for this same support, on esterification and hydrolysis activities were reported by Nyari et al. (2016) and Bustamante-Vargas et al. (2015) for Candida antarctica lipase and (Rohapect ™ DA6L) pectinase enzymes from Aspergillus niger, respectively, both in situ immobilized in polyurethane.These results suggest a beneficial effect of immobilization on the enzyme activity.This trend may be associated with several factors, such as easy accessibility of new active sites.
On the proposed immobilization process, the support's isocyanate groups (-SCN) might be linking covalently with lipase hydroxyl groups (-OH) during the support's polymerization stage, which occurs with volume expansion.When expanded, the support with the covalently linked lipase cause structural modifications on the lipase, similar to the unwind of a thread ball (a form assigned to lipases that best describes its conformational structure) (Uppenberg, Hansen, Patkar, & Jones, 1994, Uppenberg et al., 1995), displaying new active sites facilitating the substrate dispersion access to it, therefore increasing the activity.
This yield is greater than on reported studies in the literature for other supports.For the Candida rugosa immobilized in silica (CPS) Soares et al. (2003) obtained an immobilization yield of 59.6%.For Liu and Chang (2008), the yield was 42.6% in K-carrageenan.For Hu et al. (2012), the yield was 84.3% in NKA macroporous resin.For Palomino-Romero et al. (2012) the yield was 87% in the SBA-15 mesoporous material.For Shao et al. (2015) the yield was 100% in Accurel MP.

Stability Thermal stability
The lipase stability is a determining factor for its application in many biotechnological processes and thermal stability is important to determine practical conditions for its use in biotechnology and food processing.However, the lipase characteristics such as its activity and stability in organic solvents must be taken into account for each single process.
The results relating to the thermal stability of immobilized Burkholderia cepacia, conducted at different temperatures (from -4 to 40 o C) are found in Figure 4. Based on the results, a negative effect of temperature can be observed on the thermal stability of the immobilized lipase from Burkholderia cepacia, at 40 o C temperature, producing only 30 days of residual activity greater than 50% when compared to the initial one.In other words, it is approximately 3 times less than the 100 days observed for storage temperatures from -4 to 25 o C.This trend is coherent with the literature, in which it describes that the Lipase inactivation is proportional to temperature increase (Liu & Chang, 2008, Jegannathan et al., 2009).In this respect, the smaller the denaturation the greater the time of denaturation and therefore, it was stable at the temperature analysis.
The thermal stability results of the immobilized lipase from Burkholderia cepacia, stored at ambient (10 to 25 o C), refrigerator (2 to 8 o C) and freezer (-4 to 0 o C) temperatures have shown days 5, 30 and 45 with 100% of residual activity in relation to the initial one (93.3U g -1 ).The residual activities noted at 100 days were 47.6, 62.0 and 80%, respectively.
The results obtained from the current study are similar to those from previous reports in the literature by Chiou and Wu (2004), Liu and Chang (2008), Jegannathan et al. (2009) and Hu et al. (2012) (Table 2).
Results indicated that polyurethane provides better microenvironment to endure high temperatures and it might protect enzyme conformations from destruction by high temperatures.

Operational stability
One of the most important properties of the immobilized is its recovery and reuse abilities, in financial and environmental terms; it may be a limiting factor for industrial applications.In this way, the immobilized's operational stability was evaluated in multiple operating cycles on hydrolytic reactions.The immobilized lipase showed a residual activity of 32%, relative to 93.33 U g -1 initial activity (Figure 5) during 6 continuous cycles.
The results obtained from the current study are similar to those from previous reports in the literature by Jegannathan et al. (2009) and Liu et al. (2011).The immobilization efficiency can be determined by the possibility of recovery, use, and reuse in continuous biocatalytic processes.Among these factors, it is possible to mention the support type used, binding, the reaction medium and processes that will be submitted (Cadena et al, 2010).

Conclusion
According to the results, it was possible to achieve the best conditions for the olive oil hydrolysis process in pH and temperature terms using Burkholderia cepacia lipase in situ immobilized in a polyurethane support.The immobilized achieved a hydrolytic activity of 93 U g -1 in reaction conditions of pH 7, at 40°C for 15 min.The immobilized remained stable for up to 40 days of storage, at temperatures between 40 and 80°C, and 100 days at 4 to 25°C.It also showed the operational stability of 6 continuous cycles.Thus, the immobilized has proven to be a promising alternative for the implementation of olive oil in the hydrolysis process.

Figure 1 .
Figure 1.SEM microscopy of polyurethane foam used as support without Burkholderia cepacia sp.lipase in situ in polyurethane (PU).

Figure 2 .
Figure 2. Hydrolytic activity of free and immobilized lipase from Burkholderia cepacia in terms of reaction time.

Figure 3 .
Figure 3. Response surface and contour curve in accordance with the predictive model for the hydrolytic activity of free (a) and (b) immobilized (Figure 4c and d) lipase in terms of pH and temperature.

Figure 5 .
Figure 5. Operational stability of immobilized lipase in continuous cycles of use.

Table 1 .
The matrix of experimental design 2 2 , the influence of temperature and pH on the hydrolytic activity (U g -1 ) of Burkholderia cepacia lipase immobilized and free.

Table 2 .
Examples of Burkholderia cepacia lipase immobilized in different supports.