Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry

Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry

G Model ARTICLE IN PRESS PRBI-10325; No. of Pages 10 Process Biochemistry xxx (2015) xxx–xxx Contents lists available at ScienceDirect Process Bi...

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ARTICLE IN PRESS

PRBI-10325; No. of Pages 10

Process Biochemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry Marita Gimenez Pereira a , Fernanda Dell Antonio Facchini b , Luiz Estevam Cavenage Filó a , Aline Moraes Polizeli a , Ana Cláudia Vici b , João Atílio Jorge a , Glória Fernandez-Lorente c , Benevides Costa Pessela c , Jose Manoel Guisan d , Maria de Lourdes Teixeira de Moraes Polizeli a,∗ a

Departamento de Biologia – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – Universidade de São Paulo, Brazil Departamento de Bioquímica e Imunologia – Faculdade de Medicina de Ribeirão Preto – Universidade de São Paulo, Brazil Departamento de Biotecnología y Microbiologia de los Alimentos, Instituto de Ciencias de la Alimentación, CIAL-CSIC, Calle Nicolás Cabrera 9, Campus UAM, Cantoblanco, 28049, Spain d Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica – CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain b c

a r t i c l e

i n f o

Article history: Received 18 September 2014 Received in revised form 22 December 2014 Accepted 23 December 2014 Available online xxx Keywords: Lipase Immobilization Stability Organic solvents Hydrophobic supports Ionic supports

a b s t r a c t Hypocrea pseudokoningii purified lipase was immobilized on hydrophobic supports (phenyl-sepharose, butyl-sepharose, octyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite, Decaoctyl sepabeads) and ionic supports (Duolite, DEAE-agarose, PEI-agarose, MANAE-agarose, and Q-sepharose). The immobilization processes resulted in derivatives with excellent thermal stabilities, increasing the half-life up to 500-fold. The derivatives had excellent stability to organic solvents compared to the crude lipase. In the presence of 50% ethanol, hexyl and Decaoctyl derivatives increased by about 6- and 3.5-fold their stability to organic solvents, respectively. When tested for methanol, phenyl-sepharose derivative also increased their stability to organic solvents in approximately 2-fold. Octyl-sepharose derivative was fully stable for 48 h in the presence of propanol, which showed a half-life of about 7.5 h. The greater activation of the derivatives occurred using 50% cyclohexane, in which the hexyl derivative obtained an increase in the activity of 9-fold and phenyl and octyl derivatives had their activity increased by 6-fold. The lipase showed activity on different oils. Therefore, the adsorption of lipases in low ionic strength and highly hydrophobic supports is shown to be a simple and rapid tool for the immobilization of H. pseudokoningii lipase. These derivatives strongly increase the chances of this biocatalyst for industrial application. © 2015 Published by Elsevier Ltd.

1. Introduction Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are hydrolases that catalyze the hydrolysis of triglycerides to glycerol and free fatty acids. Lipase also catalyzes the hydrolysis and transesterification of esters as well as the synthesis reaction of esters with enantioselective properties [1].

∗ Corresponding author at: Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeirão Preto, SP, Brazil. Tel.: +55 16 3602 4680; fax: +55 16 3602 4886. E-mail address: [email protected] (M.d.L.T.d.M. Polizeli).

They are considered as the third largest enzyme group, after proteases and carbohydrases, based on their market value [2]. Lipases are applied as additives in foods, pharmaceuticals, medical assay, cosmetics, leather processing, dairy industry, fine chemicals, detergents, paper manufacturing and waste-water treatment [3]. Lipases are ubiquitous in nature, produced by several plants, mammals and microorganisms. Fungi that are able to produce lipases can be found in various habitats, including soils contaminated by oils, wastes of vegetable oils, dairy product industries, seeds and deteriorated food. Microbial lipases represent the most widely used class of enzymes in biotechnological applications and organic chemistry [1]. More and more strains of lipolytic microorganisms are being identified and used by industries. Most chemical processes catalyzed by enzymes require the stabilization, reuse or continuous use of the biocatalyst for a very long

http://dx.doi.org/10.1016/j.procbio.2014.12.027 1359-5113/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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time both for technical and economic reasons. The immobilization of lipases on chemical supports can occur either by means of simple adsorption or covalent bond. The most common immobilization still is through adsorption, because it is low-cost and causes few deleterious effects on the activity and selectivity of the enzyme [4]. The adsorption of lipases is favored when using low ionic strength [5]. The ability of enzymes to act as catalysts has been recognized for many years in various segments of the industry [6,7]. The immobilization of enzymes is one of the most important techniques in applying enzymatic catalysis for synthetic reactions in organic solvents. For practical and economic reasons, it is often advantageous to use immobilized enzymes since, with few exceptions, they are insoluble in organic solvents [8]. One of the biggest advantages of immobilization is to use the catalyst repeatedly without a significant loss of catalytic activity. At times, the enantioselectivity is increased [7]. Besides, the immobilization lipases through simple adsorption do not require much energy, nor do they affect the properties of the enzyme. This process generates very stable derivatives and the selected supports can increase the half-life of the immobilized enzymes. However, an unwise support choice can greatly affect the enzyme activity. Therefore, the present paper contributes to a better understanding of lipase from Hypocrea. The adsorption of Hypocrea pseudokoningii lipase in low ionic strength and high hydrophobic supports is shown to be a simple and rapid tool for the immobilization and purification of an enzyme with increased chances of industrial application due to thermal and organic solvent stabilities.

2. Materials and methods 2.1. Microorganism and culture conditions H. pseudokoningii was isolated from the soil samples collected from the different regions of the São Paulo State – Brazil. The fungus strains were taxonomically identified and deposited at the Federal University of Pernambuco, PE, Brazil. The fungus was maintained at 30 ◦ C in 1.8% Potato Dextrose Agar [9]. 2.2. Enzyme production A volume of 1.0 mL (final concentration of 105 spores) of a conidial suspension of H. pseudokoningii was inoculated into 125 mL Erlenmeyer flasks containing 25 mL of liquid Adams medium [10] added to 1 mL olive oil. The cultures were incubated in a rotational shaker (110 rpm), for 96 h, at 30 ◦ C. After that, the mycelia were separated from the liquid medium by means of vacuum filtration on Whatman filter paper number 1, and the crude filtrate was used as a source of extracellular lipase activity. 2.3. Measurement of lipase activity Extracellular lipase activity was determined using pnitrophenyl butirate as substrate. Standard assay conditions were 50 ␮l of enzymatic sample, 2.5 mL of 25 mM sodium phosphate buffer, pH 7.0 containing 20 ␮l of p-nitrophenyl butyrate 50 mM diluted in acetonitrile. The mixture was incubated at 25 ◦ C for different periods. The assay was carried out for measuring the absorbance at 348 nm, using a spectrophotometer equipped with a thermostatized chamber and continuous magnetic stirring keeping the immobilized enzyme homogenously suspended. The presence of the support during the assay only produced a marginal increase in the noise of the readings and did not affect the measurement of absorbance. One unit (U) was defined as the amount of enzyme

necessary to hydrolyze 1 ␮mol of pNPB per minute under the previously described conditions. 2.4. Measurement of protein Proteins were measured using the Bradford method [11], using standard curve 0–200 ␮g of bovine serum albumin with intervals of 0.05 ␮g among the points. The values of protein were expressed as mg of protein per mL of solution. Specific activity was expressed as units/mg of protein. 2.5. Purification of H. pseudokoningii lipase The extracellular lipase was purified from 200 mL culture supernatant using 20 g of octyl-sepharose support. The purification occurred in the batch process, overnight, at 4 ◦ C, with mechanical stirring. This process resulted in 100% of lipase activity and 36% of total proteins were immobilized to the octyl-sepharose support. The derivative (enzyme immobilized on support) was washed with about 10-fold volume of 10 mM sodium phosphate buffer, pH 7. After this process, electrophoresis was carried out to verify the purity. A single band was observed. Hence, this process was used for the selectivity for lipase and only one lipase was eluted from the hydrophobic support using 2% Triton X-100 in 10 mM sodium phosphate buffer. 2.6. Support preparation The support Polyethyleneimine (PEI)-agarose was prepared with the amination of glyoxyl-agarose with primary amino groups of PEI, as described by Mateo et al. [12]. A solution of 9 mL of 10% polyethylenimine prepared in bicarbonate solution 0.1 M, pH 11, was added of 1 g of glyoxyl agarose gel. This mixture was kept under stirring for 3 h, at 25 ◦ C. Finally, the gel was rinsed with plenty of distilled water and stored at 4 ◦ C. Monoaminoethyl-N-ethyl-agarose (MANAE-agarose) was prepared according to Fernandez-Lafuente et al. [13]. Afterwards, 27 mM ethylenediamine, with pH adjusted to 10, was mixed with 35 g glyoxyl agarose under stirring for 2 h. After that, 2 g of sodium borohydride was added and kept under the same conditions. The final step was to wash the gel with 1 L of 100 mM sodium acetate buffer, pH 4.0 and 1 L of 100 mM sodium borate solution, pH 9.0. DEAE-agarose, Q-sepharose and CM-sepharose were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Lewatit VP OC 1600 was purchased from Bayer (Leverkusen, Germany). Purolite MN200 was a generous gift of Purolite, Romania. 2.7. Hydrophobic immobilization and ionic immobilization of H. pseudokoningii lipase One g of hydrophobic supports (butyl-sepharose, phenylsepharose, octyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite and Decaoctyl sepabeads), and ionic supports (MANAE-agarose, DEAEagarose, Q-sepharose, PEI-agarose, and Duolite) was added to 10 mL of the purified lipase (2.2 mg of protein) in 25 mM sodium phosphate buffer, pH 7, at 4 ◦ C. Before use, supports were washed with abundant distilled water and 25 mM sodium phosphate buffer, pH 7. The activities of the suspension and supernatant were periodically assayed as described. After immobilization, the adsorbed lipase derivatives were extensively washed with distilled water. A blank suspension was prepared by adding of 1 g of Sepharose 4BCL. The parameters of the immobilization procedures were defined as: yield of immobilization (YI), which is the ratio between the amount of immobilized enzyme and the amount of enzyme offered to immobilization (% immobilization). The activity recovery (AR) is

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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the ratio between the measured derivative activity and the theoretical immobilized activity (difference between the initial activity and the activity measured in the final supernatant – hyperactivation or hypoactivation). 2.8. Immobilization of the lipase on CNBr-activated support The CNBr support was prepared using the protocol from Pharmacia, at pH 7. The immobilization occurred through the most reactive group: the amino terminal [14,15]. One gram of CNBractivated support was added to a solution of 10 mL of the purified lipase (0.22 mg/mL) in 25 mM sodium phosphate buffer at pH 7.0. After 15 min, 100% of lipase became immobilized on the support. The enzyme immobilization was ended by blocking the amine reactive groups in the support with 1 M ethanolamine at pH 8. After 2 h, the immobilized preparation was washed with abundant distilled water. 2.9. Desorption of lipase from the hydrophobic supports The octyl-sepharose, phenyl-sepharose, butyl-sepharose, Hexyl Toyopearl, Lewatit, Purolite and Decaoctyl sepabeads derivatives obtained from hydrophobic immobilization were suspended in 25 mL of 5 mM sodium phosphate buffer, pH 7.0. Increased concentrations (0–2%) of Triton X-100 were added and samples were taken from the supernatant after 45 min, at room temperature. A reference solution with soluble enzyme was submitted to the same treatment to detect any possible effect of the Triton X-100 on the enzyme activity. 2.10. Desorption of lipase from the ionic supports The DEAE-agarose, PEI-agarose, MANAE-agarose, Duolite and Q-sepharose derivatives obtained from ionic immobilization were suspended in 25 mL of 5 mM sodium phosphate buffer, at pH 7.0, at room temperature. Increased concentrations (0–5 M) of NaCl and (0–5%) Triton X-100 were then added, and samples were taken from the supernatant after 45 min. A reference solution with soluble enzyme was submitted to the same treatment to detect any possible effect of the NaCl or Triton X-100 on the enzyme activity. 2.11. Thermal stability studies In order to study the enzyme thermal stability, 1 IU/mL lipase was used. The inactivation was carried out at 40 ◦ C, 45 ◦ C, 50 ◦ C and 60 ◦ C, at pH 7. At different times, samples were withdrawn and their activities were determined as previously described. The remaining activity was calculated as the ratio between activity at a given time and activity at zero time of incubation. 2.12. Studies on the stability of organic solvents In order to study the lipase stability at organic solvents, 2.34 U/mL lipase were incubated with 50% (v/v) ethanol, methanol, propanol, and cyclohexane. The inactivation was carried out at pH 7.0 and room temperature. At different times, samples were withdrawn and their activities were tested. The remaining activity was calculated as the ratio between the activity at a given time and activity at zero time of incubation. 2.13. Hydrolysis of oils The hydrolysis of sardine oil was performed in a water–organic solvent two-phase system [16] as described: 4.5 mL of cyclohexane, 5 mL of 0.1 M Tris buffer pH 6 and 0.5 mL of sardine oil. The reaction was initiated by adding 0.4 g of lipase derivatives, and the mixture

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Table 1 Immobilization on hydrophobic supports at pH 7.0. Derivatives

% immobilization

Relative activity

Butyl-sepharose Phenyl-sepharose Octyl-sepharose Hexyl Toyopearl Decaoctyl sepabeads Lewatit Purolite

97 97 100 100 93 100 100

1.55 1.58 1.12 0.56 0.59 0.36 0.25

Relative activity of the immobilized to the free lipase.

was mechanically stirred at 600 rpm for 24 h. One hundred ␮l of sample present in organic phase was removed and mixed with 400 ␮l acetonitrile. The concentration of polyunsaturated free fatty acids was determined at various times with high-performance liquid chromatography with an ultraviolet detection (HPLC–UV) assay. The hydrolysis of different oils was performed in 25 mM sodium acetate buffer, pH 5, using an automatic titrator. The reaction was described as: 6 mL of oil, 8 mL of 25 sodium acetate buffer, 0.5 mL of Triton X-100. The reaction was initiated by adding 3 mL of purified lipase, and the mixture was mechanically stirred at 250 rpm for 16 h. The reaction was interrupted by the addition of a solution of ethanol:acetone (1:1). 2.14. Reuse of the derivative The derivatives were tested for their reuse. They were washed with 50 mM sodium phosphate buffer, pH 7 and used for the enzymatic hydrolysis reaction of Mandelic butyrate. Syringes containing filters were used, in order to have only Mandelic acid passing through them. After each test, the derivatives were washed again with the same buffer and the hydrolysis process was restarted. After five consecutive reactions, there was no change in the enzymatic activity of the derivatives. This process was chosen for recycling because it is easier to wash the derivatives than the reaction with fish oil. 2.15. Reproducibility of experiments All experiments were performed at least three times or more in order to confirm the results obtained. 3. Results and discussion 3.1. Study of lipase immobilization on hydrophobic supports The purified lipase was incubated with various hydrophobic supports overnight. 100% of immobilization was observed using octyl-sepharose, Hexyl Toyopearl, Lewatit, butyl-sepharose, phenyl-sepharose and Purolite, but minor immobilization levels were observed with Decaoctyl sepabeads (93%) supports (Table 1). The immobilization of lipase from H. pseudokoningii on the hydrophobic support was compared with the free enzyme present in the initial solution. The lipase activation (Table 1) occurred possibly through the lid, which is a highly hydrophobic region and where the lipase catalytic site is located. In supports of smaller chains, the lipase had an increase in the activity, while in supports of larger chains the lipase suffered a slight decline in its catalytic activity. Commercial resins such as Lewatit and Purolite caused a greater inhibition in lipase. Probably, the immobilization in supports with smaller chains allows the active site to be in a more open and stable conformation, facilitating the entry of substrates (Fig. 1). There are reports in the literature showing that the adsorption in octyl-sepharose promoted an increase in the lipase activity

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Fig. 1. Forms of immobilization through simple adsorption. (A) Hydrophobic immobilizations: A. Butyl-sepharose, B. Phenyl-sepharose, C. Hexyl Toyopearl, D. Octylsepharose, E. Decaoctyl sepabeads. (B) Ionic immobilization.

from Rhizopus niveus, Mucor javanicus, Pseudomonas fluorescens, Rhizomucor miehei, Humicola lanuginosa and Candida antarctica, and it has been explained by the fact that the lipase was in its open and dissociated form [5,17,23]. 3.2. Study of lipase immobilization on ionic support The purified lipase of H. pseudokoningii was adsorbed through a load in ionic support and immobilized on them. The lipase was immobilized on Dualite support with 75% of its activity. A high percentage of protein was adsorbed in all other anionic exchanger supports: Q-sepharose, PEI-agarose, DEAE-agarose, and MANAEagarose, all with 100% immobilization (Table 2). That result can

Table 2 Immobilization on ionic supports at pH 7.0. Derivatives

% immobilization

Relative activity

DEAE-agarose MANAE-agarose Q-sepharose PEI-agarose Duolite

100 100 100 100 75

1.44 1.66 1.73 1.63 0.21

Relative activity of the immobilized to the free lipase.

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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Fig. 2. Desorption of the lipase in hydrophobic support using Triton X-100. Symbols: butyl-sepharose (--), phenyl-sepharose (-䊉-), Hexyl Toyopearl (--), octyl-sepharose (--), Decaoctyl sepabeads (--), Lewatit (--), Purolite (--).

Fig. 3. Desorption of lipase in ionic support using NaCl. Symbols: DEAE-agarose (--), Q-sepharose (-䊉-), MANAE-agarose (--), PEI-agarose (--), Duolite (--).

be explained by the higher density of negatively charged groups of the H. pseudokoningii lipase. Using cationic exchanger supports (CM-Sepharose; Sulphopropil-Sepharose), the immobilization did not occur. This result allows us to infer that the surface of H. pseudokoningii lipase is rich in negatively charged amino acid residues due to the greater interaction of the enzyme with anionic supports.

3.5. Effect of temperature on the activity of immobilized lipase

3.3. Desorption of lipase immobilization on hydrophobic supports The aim of the immobilization is that the enzyme becomes strongly attached to the support, so that there is no release of the catalytic to the reactional media, allowing its reuse and preventing the lipases from mixing with the reaction products. Desorption was carried out in all hydrophobic supports, using different concentrations of Triton X-100. It was observed that 100% of the lipase was desorbed on phenyl-sepharose using 1% Triton X-100 while in other hydrophobic derivatives, 100% of desorption occurred at 2% Triton X-100 (Fig. 2). An important parameter in the immobilization of enzymes is the bond strength of the derivative. Derivatives formed with low bond strength allow the easy removal of the enzyme from the support, thus damaging the industrial process. Therefore, it is desired to know the bonding strength of the enzyme to the support. The enzyme of this work showed excellent bond strength, shutting down only with 2% Triton X-100. 3.4. Desorption of ionic support lipase immobilization When ionic supports were used for the immobilization process, only Duolite was 100% desorbed using 4 M NaCl. Other ionic supports did not achieve 100% desorption. Derivatives of DEAE-agarose desorbed only 25%, at 2 M NaCl. Q-sepharose derivative desorbed 27%, at 5 M NaCl. The best desorption percentages were observed with MANAE-agarose (42%), at 1 M NaCl and PEI-agarose (54%), at 5 M NaCl (Fig. 3). The ionic derivatives showed an important characteristic, since it is essential to produce biocatalysts that are resistant to many physical factors such as high salt concentration present in certain processes. When the lipase was immobilized on ionic support, the bond strength was much higher; this way, the enzyme could not be removed from the support even at high salt concentrations or when it used high concentrations of Triton X-100 to remove the enzyme.

Thermal stability of the soluble and the immobilized lipase on different supports were compared at 40 ◦ C, 45 ◦ C, 50 ◦ C and 60 ◦ C. Fig. 4A showed that the lipase from H. pseudokoningii immobilized on hydrophobic supports remained fully active after 24 h of incubation in aqueous media at 40 ◦ C. In this temperature, the uni-punctual immobilized enzyme in CNBr lost about 25% of its residual activity, while other derivatives kept more than 15% of the residual activity. At the end of 24 h, Lewatit derivative maintained approximately 70% of the residual activity (Fig. 4A). At 45 ◦ C, the half-life of CNBr was approximately 1 h and after 24 h it was completely inactivated, while Purolite had a half-life of approximately 18 h (Fig. 4B). At 50 ◦ C, the octyl derivative kept 100% of its residual activity, even after 24 h (Fig. 4C). The biocatalysts prepared were strongly inactivated when incubated at 60 ◦ C, excepted by the immobilized lipase on octyl-sepharose, which showed a half-life of 110 min (Fig. 4D). The thermal stability of the immobilized lipase on different ionic supports was compared at 50 ◦ C, 60 ◦ C and 70 ◦ C. Fig. 5A showed that the H. pseudokoningii lipase immobilized on MANAE-agarose and Q-Sepharose supports remained fully active after 24 h of incubation in aqueous media at 50 ◦ C, while DEAE-agarose derivative remained approximately 80% after 24 h. PEI derivative remained 100% with 2 h. After this period, it kept approximately 75% of residual activity (Fig. 5A). At 60 ◦ C, CNBr derivative was completely inactivated, while MANAE-agarose and Q-sepharose remained 100% up to 2 h. DEAE-agarose and Q-Sepharose derivatives had a half-life of 5 h approximately (Fig. 5B). MANAE derivative had a half-life of 7 h, while PEI had a half-life of 24 h (Fig. 5B). Therefore, the ionic derivatives of H. pseudokoningii lipase were more stable than was CNBr derivative at all temperatures tested, indicating that the immobilized lipase by H. pseudokoningii became much more thermally stable after the procedures than before. It was observed that the immobilization process protected the enzyme against heat inactivation, and that suggests possible perspectives of using this immobilized catalyst in biotechnological process. The purified enzyme had a lower thermal stability when compared with the enzyme without purification [9]. However, the crude enzyme extract is protected by various ions, cofactors and proteins that stabilize it, unlike what happens when the enzyme is purified and immobilized on CNBr. The immobilization of the purified enzyme stabilized, increasing its half-life.

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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Fig. 4. Thermal stability of the lipase adsorbed on hydrophobic supports. (A) 40 ◦ C; (B) 45 ◦ C; (C) 50 ◦ C; (D) 60 ◦ C. Inactivation was performed in 25 mM sodium phosphate, pH 7 at 25 ◦ C, and activity was followed by the pNPB assay. Other specifications are described in Section 2. Free enzyme (--); CNBr derivative (-䊉-); butyl-sepharose derivative (--); phenyl-sepharose derivative (--); octyl-sepharose derivative (--); Hexyl Toyopearl derivative (--); Decaoctyl sepabeads derivative (--); Lewatit derivative (--); Purolite derivative (--).

3.6. Effect of organic solvents on immobilized lipase activity The stability in organic environment is an important property of lipase. The effect of the organic medium depends on the nature of both the enzyme and the solvents [18]. The stability of the immobilized lipase from H. pseudokoningii was investigated in various organic solvents at a final concentration of 50%, at 4 ◦ C. Fig. 6A shows hydrophobic derivatives of the lipase from H. pseudokoningii when incubated in the presence of 50% ethanol. Lewatit derivative had a half-life of 4 h; CNBr derivative showed a half-life of 12 h and Purolite derivative was 32 h. However, some derivatives showed an activation in the presence of ethanol, as Hexyl Toyopearl, which increased the activity to 6-fold. Over the course of 72 h, the lipase still kept the activation to 2-fold. The derivative Decaoctyl sepabeads showed an activation of 3.5-fold, which remained 100% at the end of 72 h. The derivatives of phenyl, butyl and octyl in the presence of ethanol showed an activation of 2-fold, which kept for up to 72 h. Fig. 6B showed that the ionic derivatives as Q-sepharose had a half-life of 2 h; CNBr derivative showed a half-life of 12 h and the half-life of MANAE-agarose and PEI-agarose derivative were 17 h and 24 h, respectively. DEAE-agarose and Duolite derivative had a half-life of 44 and 46 h, respectively. Fig. 8A showed the derivative of H. pseudokoningii when incubated in the presence of 50% of methanol. Some derivatives reached the half-life in the presence of methanol as Lewatit, which reached the half-life after 4 h of incubation. CNBr and Purolite derivatives reached the half-life at 5 and 6 h, respectively. Decaoctyl

sepabeads and Hexyl Toyopearl derivatives reached the half-life with 16 and 42 h, respectively. However, some derivatives were very stable, such as phenyl-sepharose, which undergoes approximately 2-fold activation during the first hour. Butyl-sepharose and octyl-sepharose derivatives remained 120 and 110% of activation, respectively, for up to 72 h. It was possible to observe that phenylsepharose, butyl-sepharose and octyl-sepharose derivatives had similar stability maintaining more than 100% at the end of 72 h. This similar behavior might be explained by the composition of the supports used for the immobilization (Fig. 1) and that possibly the immobilization occurred in the region of the lid; supports are also differentiated by the length of its chain. Butyl, phenyl, hexyl and octyl are made of agarose, but with different lengths of the hydrophobic chain. The stability of the derivatives was tested in the presence of methanol, as shown in Fig. 7. CNBr and Duolite derivatives reached the half-life around 4 h. PEI derivative reached the half-life with 68 h. However, some derivatives were very stable, such as DEAE-agarose derivative, which lost only 20% of its activity after 72 h. MANAE derivative increased 80% during incubation and, at the end of 72 h, the activation was about 50% higher than was it in the control. Q-Sepharose derivative had 75% of increase in its activity, which was kept for up to 8 h; after that, the activation decreased, retaining only 80% of residual activity up to 72 h (Fig. 7B). Lipase derivatives were also incubated in propanol, at a final concentration of 50%. The octyl-sepharose derivative retained 100% activity at 48 h. Butyl Toyopearl and phenyl-sepharose dropped

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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50

100

75

50

25

25

0 0

0 0

2

4

6

8

24

16

24

32

40

48

Time (h)

Time (h) 50

C 40

Residual activity (%)

8

Fig. 6. Ethanol stability of lipase. (A) Adsorbed on hydrophobic supports. Symbols: CNBr derivative (--); butyl-sepharose derivative (-䊉-); phenyl-sepharose derivative (--); octyl-sepharose derivative (--); Hexyl Toyopearl derivative (--); Lewatit derivative (--); Purolite derivative (--); Decaoctyl sepabeads derivative (--). (B) Adsorbed on ionic supports. Symbols: CNBr derivative (--); DEAE-agarose derivative (-䊉-); Q-sepharose derivative (--); MANAE-agarose derivative (--); PEI-agarose derivative (--); Duolite derivative (--). Other specifications are described in Section 2.

30

20

10

0 0

2

4

6

8

Time (h) Fig. 5. Thermal stability of the lipase adsorbed on ionic supports. (A) 50 ◦ C; (B) 60 ◦ C; (C) 70 ◦ C. Inactivation was performed in 25 mM sodium phosphate, pH 7 at 25 ◦ C, and activity was followed by the pNPB assay. Other specifications are described in Section 2. CNBr derivative (--); DEAE-agarose derivative (-䊉-); Q-sepharose derivative (--); MANAE-agarose derivative (--); PEI-agarose derivative (--); Duolite derivative (--).

about 25 and 40%, respectively, at the end of 48 h. Lewatit and Purolite showed a half-life of 3 h each. CNBr derivative had a half-life of 7.5 h, while Hexyl Toyopearl and Decaoctyl sepabead derivative showed a half-life at 36 and 41 h, respectively, as shown in Fig. 8A. Q-Sepharose and MANAE-agarose derivatives had a half-life of 2 and 5 h, respectively. CNBr and DEAE-agarose derivatives had a half-life of 8 and 12 h, respectively, while Duolite and PEI-agarose had a half-life of 29 h (Fig. 8B). Finally, the derivatives were tested for stability in the presence of cyclohexane, at a concentration of 50%. Representing the free enzyme, CNBr derivative had a half-life of 2 h, being completely inactivated at 4 h. Lewatit derivative was the only noncovalent derivative which reached 50% of its activity in 12 h, being completely inactivated within 48 h. All other derivatives suffered activation in the presence of cyclohexane. Decaoctyl sepabeads and Purolite derivatives suffered an activation of about 2-fold and kept over 100% of residual activity at the end of 72 h. Butyl-sepharose derivative suffered activation of 4-fold, which continued for 48 h. After this period, the activity began to decline until it reached 100% at the end of 72 h. Octyl-sepharose and phenyl-sepharose

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200

A

A 100

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Residual activity (%)

Relative activity (%)

175

125 100 75 50

75

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25

25 0 0

8

16

24

32

40

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56

64

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0

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0

8

16

Time (h)

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Time (h)

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B

175

B

150

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Residual activity (%)

100

125 100 75 50

75

50

25

25 0 0

8

16

24

32

40

48

56

64

72

80

0 0

8

Time (h) Fig. 7. Methanol stability of lipase. (A) Adsorbed on hydrophobic supports. Symbols: CNBr derivative (--); butyl-sepharose derivative (-䊉-); phenyl-sepharose derivative (--); octyl-sepharose derivative (--); Hexyl Toyopearl derivative (--); Lewatit derivative (--); Purolite derivative (--); Decaoctyl sepabeads derivative (--). (B) Adsorbed on ionic supports. Symbols: CNBr derivative (--); DEAE-agarose derivative (-䊉-); Q-sepharose derivative (--); MANAE-agarose derivative (--); PEI-agarose derivative (--); Duolite derivative (--). Other specifications are described in Section 2.

derivatives increased about 6-fold in the first 4 h, then the activity dropped to 5-fold, which persisted for up to 72 h. Hexyl Toyopearl derivative was the most activated (9-fold), and in the first 8 h it decreased to 7-fold, and kept this for up to 72 h (Fig. 9A). Ionic derivatives were tested for propanol stability: CNBr and DEAEagarose derivative showed a half-life of 2 and 17 h, respectively. Q-Sepharose and Duolite derivatives showed a half-life of 23 and 35 h, while MANAE-agarose and PEI-agarose derivatives showed a half-life of 38 and 48 h, respectively (Fig. 9B). All ionic derivatives were more stable than was the CNBr derivative in propanol. The technological utility of enzymes can be greatly enhanced by using them in organic solvents rather than in their natural aqueous reaction media. Over the past 15 years, studies have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments, enzymes can catalyze reactions which are impossible in water, become more stable and exhibit new behavior such as ‘molecular memory’ [19]. The discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, was of particular importance can be markedly affected, and sometimes, even inverted by the solvent [19].

16

24

32

40

48

Time (h) Fig. 8. Propanol stability of lipase. (A) Adsorbed on hydrophobic supports. Symbols: CNBr derivative (--); butyl-sepharose derivative (-䊉-); phenyl-sepharose derivative (--); octyl-sepharose derivative (--); Hexyl Toyopearl derivative (--); Lewatit derivative (--); Purolite derivative (--); Decaoctyl sepabeads derivative (--). (B) Adsorbed on ionic supports. Symbols: CNBr derivative (--); DEAE-agarose derivative (-䊉-); Q-sepharose derivative (--); MANAE-agarose derivative (--); PEI-agarose derivative (--); Duolite derivative (--). Other specifications are described in Section 2.

Activity of enzymes in organic solvents depends on the concentration of the solvents and the nature of the enzymes [20,21]. The highest activity was achieved against ethanol, methanol, propanol and cyclohexane. In fact, these derivatives were not just stable, but also activated in the presence of organic solvents. The ability of solvents to increase the solubility of substrates, thus facilitating the reaction or maintaining the active structural conformation of the enzyme, might be the cause of higher lipase activity in exposure to organic solvent [20,22]. 3.7. Substrate hydrolysis H. pseudokoningii derivatives were tested for the hydrolysis of fish oil. CNBr, butyl-sepharose and DEAE-agarose derivatives hydrolyzed sardine oil, showing that they were true lipases. We also observed a difference in activity, indicating that the immobilization influenced the activity and specificity in relation to EPA and DHA. Both derivatives hydrolyzed more EPA than DHA (Table 3). Because of its unique chemical and conformational properties, cyclohexane is also used in labs in analysis and as a standard. It

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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M.G. Pereira et al. / Process Biochemistry xxx (2015) xxx–xxx Table 4 Hydrolysis of different oils catalyzed by purified lipase.

1000

A

900 800

Residual activity (%)

9

700 600 500 400 300 200 100

Oils

Activity (U/mL)

Seed Bacuri oil (Latonia insignis) Cupuac¸u oil (Theobroma grandiflorum) Seed Murumuru oil (Astrocaryum murumuru) Nuts Andiroba oil (Carapa guianensis) Nuts of Brazil oil (Bertholletia excelsa) Nuts Babac¸u oil (Orbignya phalerata) Nuts Ucuuba oil (Virola surinamensis) Sunflower oil (Helianthus annuus) Sessame oil (Sesamum indicum) Coconut oil (Cocos nucifera) Palm oil (Elaeis guineensis) Soybean oil (Glycine max) Olive oil (Olea europea)

0.29 0.39 0.30 0.10 0.18 0.15 0.01 0.26 0.28 0.28 0.22 0.29 0.27

± ± ± ± ± ± ± ± ± ± ± ± ±

0.012 0.015 0.012 0.025 0.006 0.010 0.006 0.010 0.012 0.006 0.010 0.012 0.020

The activity was measured by means of titration.

0 0

8

16

24

32

40

48

56

64

72

80

Time (h) 100

Residual activity (%)

B Residual activity (%)

100

75

50

75

50

25

25

0 1 0 0

8

16

24

32

40

3

4

5

Number of use

48

Time (h) Fig. 9. Cyclohexane stability of lipase. (A) Adsorbed on hydrophobic supports. Symbols: CNBr derivative (--); butyl-sepharose derivative (-䊉-); phenyl-sepharose derivative (--); octyl-sepharose derivative (--); Hexyl Toyopearl derivative (--); Lewatit derivative (--); Purolite derivative (--); Decaoctyl sepabeads derivative (--). (B) Adsorbed on ionic supports. Symbols: CNBr derivative (--); DEAE-agarose derivative (-䊉-); Q-sepharose derivative (--); MANAE-agarose derivative (--); PEI-agarose derivative (--); Duolite derivative (--). Other specifications are described in Section 2.

was used for the hydrolysis of sardine oil, to demonstrate that it was a true lipase [16]. A difference between activities was observed, indicating that the immobilization influenced the activity and specificity in relation to EPA and DHA. 3.8. Hydrolysis of different oils The purified lipase had activity with all the tested oils. The Cupuac¸u oil was the best hydrolyzed between the oils tested, and it Table 3 Hydrolysis of sardine oil catalyzed by lipase derivatives. a

2

Fig. 10. Operational stability of derivatives lipase in repeated use conditions. Legends: dark gray color, DEAE-agarose derivative; gray color, octyl-sepharose derivative.

was followed by seed Murumuru oil (Table 4). However, the lipase hydrolyzed all oils tested, but mainly rich oils in palmitic (C:16), lauric (C:12), oleic (C:18), and myristic (C:14) acids. 3.9. Reuse and operational stability of the enzyme Reusability studies of the lipase were carried out using the recovered enzyme for subsequent cycles. After each circle, the enzyme was filtered out, washed with fresh buffer and allowed to drain before reuse. Operational stability of the enzyme was observed to be high (Fig. 10). It was found that the two derivatives tested showed good results as the possibility of reuse. The best derivative was octyl-sepharose. This result was expected, since the immobilization takes place through the active site, increasing the lipase stabilization. The DEAE-agarose derivative started to lose activity from the fifth cycle and did not maintain the same yield of octyl. 4. Conclusions

b

Derivative

Activity

Selectivity

CNBr Butyl-sepharose DEAE-agarose

0.018 ± 0.001 0.030 ± 0.002 0.019 ± 0.002

3 4 8

a Activity is expressed as ␮mol of PUFAS (EPA/DHA) released per minute and per gram of the immobilized enzyme. b Selectivity is expressed as the ratio between released EPA and DHA. Selectivity was measured at the first stages of hydrolysis.

Adsorption of lipase, at low ionic strength, on highly hydrophobic supports (e.g. butyl-sepharose, phenyl-sepharose or octyl-sepharose) seems to be a simple and rapid tool for the immobilization of most lipases. The immobilization stabilized and increased lipase activity. Several derivatives were activated in the presence of 50% of organic solvents, over 72 h. The derivatives were more stable in cyclohexane. In summary, the derivatives from

Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027

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H. pseudokoningii were more thermally stable and more stable in organic solvents than in the free enzyme. The derivative also showed good ability to be reused, proving to be economical. Moreover, the results clearly indicate the improvement of derivatives in organic solvents, suggesting the enzyme applicability in industrial processes. Acknowledgments This work was supported by grants from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho de Desenvolvimento Científico e Tecnológico (CNPq process n◦ 406838/2013-5). This project is also part of National Institute of Science and Technology of the Bioethanol (n◦ 574002/2008-1), CNPq – Ciência sem Fronteira (n◦ 242775/2012-8). JAJ and MLTMP are Research Fellows of CNPq. MGP and ACV are supported by CNPq. FDAF was recipient FAPESP Fellowship. We thank Ricardo F. Alarcon, Mariana Cereia and Mauricio de Oliveira for the technical assistance. We also thank teacher Abilio Borghi for the grammar review of the manuscript. References [1] Singh AK, Mukhopadhyay M. Overview of fungal lipase: a review. Appl Biochem Biotechnol 2012;166:486–520. [2] Romdhane IB-B, Fendri A, Gargouri Y, Gargouri A, Belghith H. A novel thermoactive and alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochem Eng J 2010;53:112–20. [3] Basheer SM, Chellappan S, Beena PS, Sukumaran RK, Elyas KK, Chandrasekaran M. Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment. N Biotechnol 2011;28:627–38. [4] Menoncin S, Domingues NM, Freire DMG, Oliveira JV, Di Luccio M, Treichel H, et al. Immobilization of lipases produced by solid state fermentation from Penicillium verrucosum on hydrophobic supports. Cienc Tecnol Aliment 2009;29:440–3. [5] Bastida A, Sabuquillo P, Armisen P, Fernandez-Lafuente R, Huguet J, Guisan JM. A single step purification, immobilization, and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 1998;58:486–93. [6] Homaei AA, Sariri R, Vianello F, Stevanato R. Enzyme immobilization: an update. J Chem Biol 2013;6:185–205. [7] Palomo JM, Fernandez-Lorente G, Mateo C, Ortiz C, Fernandez-Lafuente R, Guisan JM. Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters. Enzym Microb Technol 2002;31:775–83.

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Please cite this article in press as: Pereira MG, et al. Immobilized lipase from Hypocrea pseudokoningii on hydrophobic and ionic supports: Determination of thermal and organic solvent stabilities for applications in the oleochemical industry. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.027