Influence of process parameters on the immobilization of commercial porcine pancreatic lipase using three low-cost supports

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Inﬂuence of process parameters on the immobilization of commercial

porcine pancreatic lipase using three low-cost supports

Robison P. Scherer

a

, Roge´rio L. Dallago

b

, Fa´bio G. Penna

b

, Francile Bertella

b

, De´bora de Oliveira

a,n

,

J. Vladimir de Oliveira

a

_{, Sibele B.C. Pergher}

c

a

Departamento de Engenharia de Alimentos, Universidade Regional Integrada do Alto Uruguai e das Miss ~oes, Campus Erechim, Av. Sete de Setembro, 1621, 99700-000 Erechim RS, Brazil

b

Departamento de Quimica, Universidade Regional Integrada do Alto Uruguai e das Miss ~oes, Campus Erechim, Av. Sete de Setembro, 1621, 99700-000, Erechim RS, Brazil

c

Departamento de Quimica, Universidade Federal do Rio Grande do Norte, Av. Senador Salgado Filho, 3000, Lagoa Nova, 59078-970, Natal RN, Brazil

a r t i c l e

i n f o

Article history: Received 3 April 2012 Received in revised form 5 June 2012

Accepted 5 June 2012 Available online 15 June 2012 Keywords:

Porcine pancreatic lipase Immobilization KSF

Natural montmorillonite Pillared montmorillonite

a b s t r a c t

The use of lipases as biocatalysts has attracted much attention recently, but industrial application has been hindered mainly due to the high production cost, determined by the production yield and enzyme stability. Immobilization of biocatalysts in inert supports can ensure their use for several batches, making possible to build cost-effective processes. The objective of this work was to investigate the influence of immobilization time and enzyme to support mass ratio on the yield of immobilization and esterification activity of porcine pancreatic lipase. Higher immobilization yields (38.2%) were obtained for pillared montmorillonite, 120 min of immobilization and enzyme to support mass ratio of 2:0.5. The highest esterification activity (1403 U/g) was achieved using the montmorillonite, after 180 min of immobilization and enzyme to support ratio of 2:1. The characterization of the supports, free and immobilized enzyme on different supports made possible to elucidate the immobilization results through the knowledge of support features.

1. Introduction

There has been a growing interest in lipid modification through the use of lipases as biocatalysts over the last years. The attractive aspects of this catalyst over chemical methods include the high specificity of some lipases, the mild conditions required for the reactions to take place, thereby requiring mini-mal energy inputs, reduced levels of by-products generated during the reaction and more efficient conversion of thermo sensitive substrates. Lipases catalyze hydrolysis of long chain, insoluble triglycerides and other insoluble esters of fatty acids with varying chain length specificity. The ability demonstrated by lipases to catalyze reactions in micro-aqueous ambient explains their large use to produce esters from fatty acids and alcohols (Abbas et al., 2002;Treichel et al., 2010;Sun and Xu, 2008).

Despite the potential of using lipases as biocatalysts for a wide variety of reactions with great interest, the high production cost of the catalyst and reduced stability under adverse conditions has limited related industrial applications. The enzyme stabilization is thus a desirable step from an economic point of view. Enzymes in soluble form (free enzyme) can lose the catalytic activity in batch

reactions, making difﬁcult their reuse and additionally the pre-sence of residual enzyme in the reaction medium may thus represent an undesirable contamination (Villeneuve et al., 2000;

Sebr~ao et al., 2007;Dumitriu et al., 2003). Nevertheless, immobi-lization of biocatalysts in inert supports can ensure the use for several batches, hence resulting cost-effective industrial pro-cesses (Dalla-Vecchia et al., 2004).

The major contribution to achieve a good performance of immobilized catalyst is primarily provided by the strategy employed for immobilization (Cardias et al., 1999) and by the characteristics of the support. According toGomes et al. (2006), the choice of the appropriate support can enhance the half-time of the enzyme and increase the global performance of the process. Several organic, inorganic and natural materials with different characteristics have been used for lipases immobilization (Villeneuve et al., 2000;

Dumitriu et al., 2003;Dalla-Vecchia et al., 2004;Gomes et al., 2006;

Herna´ndez-Ju´stiz et al., 1998; Watanabe et al., 2000). However, appraisal of literature information on the subject showed that just a few works regarding the use of low-cost inorganic supports for lipases immobilization are available (Yesiloglu, 2004;Yesiloglu and Yesim 2005; Rahman et al., 2005; Sanjay and Sugunan, 2006;

Gopinath and Sugunan, 2007; Meunier and Legge, 2010;Secundo et al., 2008;Zaidan et al., 2010).

Considering the relevance of developing new methods towards enzyme immobilization and the lack of information in the Contents lists available atSciVerse ScienceDirect

journal homepage:www.elsevier.com/locate/bab

Biocatalysis and Agricultural Biotechnology

n

Corresponding author. Tel.: þ55 54 3520 9000; fax: þ 55 54 3520 9090. E-mail address: odebora@uricer.edu.br (D. de Oliveira).

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specialized literature concerning the evaluation of the effects of process parameters on the immobilization of porcine pancreatic lipase using low-cost supports, the aim of this work was to assess the inﬂuence of immobilization time and enzyme to support mass ratio on the amount of protein adsorbed, yield of immobilization and esteriﬁcation activity for three inorganic supports. The partial characterization of free enzyme, each support and immobilized lipases were also carried out in attempt to interpret the data obtained in the adsorption process.

2. Material and methods 2.1. Enzyme and supports

Lipase (EC 3.1.1.3 Type II, crude; from porcine pancreas) was purchased from Sigma-Aldrich. Three different low-cost supports were used for the evaluation of conditions of immobilization of porcine pancreatic lipase: natural montmorillonite (Colorminas SA), montmorillonite KSF (Sigma-Aldrich) and pillared montmorillonite. 2.2. Immobilization of commercial lipase in the different supports

The lyophilized preparation was solubilized in phosphate buffer (pH 7.0) (2 g of enzyme and 60 mL of buffer) and submitted to preferential immobilization by physical adsorption on each support presented before. Enzyme immobilization was performed at different enzyme to support mass ratios (2:1, 2:2 and 2:0.5) and contact time (60, 120 and 180 min). Immobilization was carried out with magnetic stirring in an ice cooler and aliquots were sampled periodically for determination of protein content, yield of esteriﬁcation and esteriﬁcation activity.

2.3. Analytical methodology

2.3.1. Protein content and yield of immobilization

The protein content in the inlet and outlet solutions was measured by the methodology proposed by Bradford (1976). Samples were analyzed in spectrophotometer (Agilent Tecnolo-gies 8453) at 595 nm and the protein content was determined.

The yield of immobilization was calculated by:

Z

ð%Þ ¼Pa

Po

100 ð2Þ

where

Z

¼yield (%), Parepresents the amount of protein adsorbed

(difference between the initial and stable content), Podenotes the

amount of protein used in the immobilization (inlet solution). 2.3.2. Lipase esteriﬁcation activity

The enzyme activity was determined as the initial rates in esteriﬁcation reactions between oleic acid and ethanol at a mole ratio of 1:1 using 0.4 g of enzyme (immobilized in each support tested or in its free form) at 40 1C and 150 rpm for 40 min. One lipase activity unit (UE) was deﬁned as the amount of enzyme necessary to consume 1

m

mol of oleic acid per minute at the established experi-mental conditions presented previously. All enzymatic activity deter-minations were replicated at least three times. Results presented were in fact mean values of the measurements performed. Standard errors lower than 5% were obtained in all determinations.

2.4. Characterization of the supports, free and immobilized enzymes The free and immobilized enzymes and supports were partially characterized in terms of Speciﬁc surface area, Small-angle X-ray powder diffraction (SAXRD), Infrared spectroscopy and Scanning electronic microscopy (SEM). The textural characterization of the

samples was carried out using an Autosorb-1 (Quantachrome Nova-2200e) by the low-temperature N2 adsorption–desorption

experi-ments. The speciﬁc surface area was determined by the BET method. The SAXRD analyses were carried out in a Diffractometer model D5000 (Siemens) using ﬁlter of Ni and radiation Cu-k

a

(

l

¼1.54 ˚A). The SEM analyses were performed in a JEOL-JSM 5800 microscopy with acceleration voltage of 20 KV. The Infrared spectroscopy of diffuse reﬂectance Fourier transform (DRIFTS) was carried out in a spectrophotometer FTIR Shimadzu, model 8300.

3. Results and discussion

3.1. Inﬂuence of process parameters on the immobilization yield and esteriﬁcation activity

Table 1(a) presents the protein adsorbed on the surface of each support, yield of immobilization and esteriﬁcation activity of lipase immobilized on different supports for different immobili-zation times for a ﬁxed enzyme to support mass ratio of 2:1. It was observed from this table that the natural montmorillonite presented an increase in the immobilization yield with increasing time, whereas KSF and pillared montmorillonites did not present the same behavior, probably due to a possible saturation of the enzyme on the surface of the support.

Data related to the enzyme to support mass ratio of 2:0.5 are presented inTable 1(b) and in this case in it was veriﬁed that the saturation of the enzyme on the supports surface occurred around 120 min of immobilization, which may be attributed to the high amount of enzyme used. After this immobilization time, the enzyme was desorbed from the support to the reaction medium. A comparison of the three supports after 120 min of immobiliza-tion showed that higher yields of immobilizaimmobiliza-tion were achieved for pillared montmorillonite, which might be probably related to the higher speciﬁc surface area of this support compared to the others.

Table 1(c) presents the results achieved for each support using an enzyme to support mass ratio of 2:2. An enhancement in the immobilization yield as immobilization time increases was observed for natural and pillared montmorillonites. The use of KSF as support led to high yields after 1 h of immobilization, but after this period, a desorption process was observed. The pillared montmorillonite provided higher immobilization yields, presum-ably due to its high speciﬁc surface area.

Literature pointed out that the support, the amount of enzyme and the contact time between enzyme and support presented high inﬂuence on the immobilization yields (Tzialla, 2010;

Li et al., 2009;Li et al., 2010). It was reported in these works that the yield of immobilization, depending on the support and the amount of enzyme used, increased up to reaching the saturation. Comparison of the results obtained for the three different sup-ports, in different immobilization times and also enzyme to support mass ratios, showed that distinct behavior was observed depending on the process parameter values and type of support. The use of pillared montmorillonite as support led to good immobilization yields for all experimental conditions studied. This result can be associated to its high speciﬁc surface area, good adsorption capacity and favorable chemical interactions with the enzyme. Based on these facts and considering the low-cost and high availability of this support, it can be considered a potential immobilization adsorbent for porcine pancreatic lipase.

Table 1(a, b and c) also presents the esteriﬁcation activities of immobilized lipases in the supports for different immobilization times and enzyme to support mass ratio of 2:1, 2:0.5 and 2:2, respectively. For enzyme to support mass ratio of 2:1, higher enzyme activities were obtained when natural montmorillonite was used as support, leading to activities higher than those

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obtained for free porcine pancreatic lipase. Using enzyme to support mass ratio of 2:0.5, Table 1(b), higher esteriﬁcation activities were achieved for natural and KSF montmorillonites after 180 min of immobilization, while for a ratio of 2:2,

Table 1(c), shows that higher activities were obtained after 180 min using KSF montmorillonite as adsorbent.

3.2. Characterization of supports and enzymes 3.2.1. Drifts

Fig. 1 (a, b and c) presents the FT-IR spectra of free lipase, pillared montmorillonite and lipase immobilized in this support (60 min and enzyme to support mass ratio of 2:2), respectively. In this analysis the presence of vibration bands was related to the characteristic of functional groups, making possible the qualitative identiﬁcation of certain samples (Tres et al., 2012). The infrared analysis of the three samples showed the presence of a band at 3300 cm1_{in spectra of free and immobilized lipases. This band}

corresponded to stretching (NH) groups of peptide bounds, typical of enzymes. A presence of a band at 1635 cm1_{, corresponding to a}

carbonyl group of low intensity in both spectra, characteristic of a symmetric angular deformation in plan of group (NH) was also observed. The visualization of bands at 3300 cm1_{and 1635 cm}1_,

both in the infrared of free and immobilized lipases, demonstrated the efﬁciency of the immobilization process. The same behavior presented here for lipase supported in pillared montmorillonite was observed for natural montmorillonite and KSF as well as lipases immobilized in these supports.

3.2.2. N2adsorption–desorption isotherms

Table 2 presents the speciﬁc surface area of free enzyme, supports and lipases immobilized in each support. A detailed

inspection of this table revealed a signiﬁcant area reduction of each support after the immobilization process, which indicated the adherence of the enzyme in the cavity of the supports, hence demonstrating the efﬁciency of the immobilization process.

3.2.3. SAXRD

Fig. 2(a, b and c) presents the SAXRD of free lipase, pillared montmorillonite and lipase immobilized in pillared montmorillo-nite after 60 min and enzyme to support mass ratio of 2:2. Results showed that it was not possible to observe the displacement of

Table 1

Protein adsorbed on the surface of each support, yield of immobilization and esteriﬁcation activity of lipases immobilized on different supports in different times of immobilization and enzyme to support mass ratio of 2:1 (a), 2:0.5 (b) and 2:2 (c).

Sample Time (min) Protein content (mg/mL) Yield of immobilization (%) Esteriﬁcation activity (U/g)

Free lipase – – – 739.2 (a) Natural montmorillonite 60 0.6 14.2 1104.3 Natural montmorillonite 120 1.0 23.9 974.4 Natural montmorillonite 180 1.1 26.2 956.9 Pillared montmorillonite 60 0.9 18.6 408.7 Pillared montmorillonite 120 1.4 33.9 238.7 Pillared montmorillonite 180 0.7 26.5 337.9 KSF 60 0.8 27.5 142.9 KSF 120 0.6 20.3 229.3 KSF 180 0.7 26.6 171.9 (b) Natural montmorillonite 60 0.7 11.8 284.3 Natural montmorillonite 120 2.2 32.5 323.9 Natural montmorillonite 180 0.6 22.8 679.0 Pillared montmorillonite 60 0.4 20.9 233.4 Pillared montmorillonite 120 0.8 38.2 102.6 Pillared montmorillonite 180 0.8 32.4 332.9 KSF 60 0.8 27.9 46.9 KSF 120 0.9 30.2 226.9 KSF 180 0.3 11.3 881.4 (c) Natural montmorillonite 60 0.6 17.6 398.3 Natural montmorillonite 120 0.7 19.8 492.1 Natural montmorillonite 180 1.2 29.7 164.0 Pillared montmorillonite 60 0.5 20.5 710.9 Pillared montmorillonite 120 0.6 21.9 504.2 Pillared montmorillonite 180 0.8 33.1 430.7 KSF 60 0.8 34.8 119.2 KSF 120 0.6 15.8 135.5 KSF 180 0.3 12.7 1403.8 4000 3000 2000 1000 0 80 82 84 86 88 90 92 94 96 98 100 102 c b Transmittance % Wavenumber cm-1 a

Fig. 1. FT-IR spectra of free lipase (a), pillared montmorillonite (b) and lipase immobilized in pillared montmorillonite (c) after 60 min and enzyme to support mass ratio of 2:2.

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the first reflection (001) since the structure of the material is pillared and the interlamellar space is fixed by the insertion of pillars, so this clay cannot be expanded. However, it can be observed that the enzyme is probably in the interlamellar region, since peaks related to the free enzyme cannot be observed and also a reduction occurred on superficial area. The KSF clay did not present displacement of reflection 001, as it is low intense and this material is disorganized. Based on this statement, it seems that the immobilization took place in the interlamellar region and also kept the material disorganized, so that the expansion of the clay did not occur. The SAXRD of KSF clay and lipase immobilized in KSF after 180 min and enzyme to support mass ratio of 2:2 showed that the structure of KSF clay was kept after the immobilization process, indicating that the incorporation of the lipase occurred with no modification in the structure of the support. As peaks of free lipase were not observed, it can be supposed that the catalyst was immobilized in the clay and does not present a physical mixture with the support. The SAXRD of natural montmorillonite and lipase immobilized in natural mon-tmorillonite after 180 min and enzyme to support mass ratio of 2:1 showed that after the immobilization process, a displacement of 001 reflection for lower angles was observed, indicating that the enzyme may be in the interlamellar clay region, provoking the laminas expansion and, accordingly, an increase in the basal space. For the support, after the immobilization, reflections regarding the enzyme were not observed, indicating that the catalyst is probably located in the interlamellar region and is not present as a physical mixture of enzyme and support.

3.2.4. SEM

The micrograph of the free lipase shows that this enzyme presents a morphology based on several particle lengths with amorphous appearance and fiber-like structure. The KSF clay has a lamellar structure and due to the acid treatment used in its synthesis, some exfoliation and partial destruction can also be observed. It can also be noticed the presence of aggregates in face edges, indicating the disorganization of the material. After the immobilization process using KSF as support, the micrographs show an amorphous surface, indicating that the adsorption occurred in a satisfactory way, where the presence of fibers was verified, as noted in the micrograph of free lipase. The micrograph

Table 2

Speciﬁc surface area for samples of free enzyme, supports and immobilized lipases in each support. Sample Time (min) Enzyme to support mass ratio (g/g) Speciﬁc surface area (m2 /g) Free lipase – – 1.1 KSF – – 13.1 Natural montmorillonite – – 58.0 Pillared montmorillonite – – 226.8 Lipase immobilized (KSF) 180 2:2 11.3 Lipase immobilized (natural montmorillonite) 180 2:1 4.1 Lipase immobilized (pillared montmorillonite) 60 2:2 5.9 10 20 30 40 50 60 70 0 500 1000 1500 2000 2500 3000 c b a intensity (a.u) 2 Theta

Fig. 2. SAXRD of free lipase (a), pillared montmorillonite (b) and lipase immobi-lized in pillared montmorillonite (c) after 60 min and enzyme to support mass ratio of 2:2.

Fig. 3. Micrographs of free lipase (a); pillared montmorillonite (b) and lipase immobilized in pillared montmorillonite (c) after 60 min and enzyme to support mass ratio of 2:2.

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of natural montmorillonite presented a typical lamellar morphol-ogy, constituted of lamellas of different lengths. This clay, after the immobilization process of 180 min, using enzyme to support mass ratio of 2:1, kept its lamellar structure, presenting a length reduction, indicating a possible exfoliation during the immobili-zation process. Comparing the micrographs of free and immobi-lized lipase in natural montmorillonite it was not possible to observe the presence of ﬁbers, indicating that the immobilization occurred preferentially in the internal surface of the support.

Fig. 3(a and b) presents the micrograph of free and immobilized lipase in montmorillonite after the pillarization and calcination process. The lamellar structure of the support was preserved, which is in agreement with the results of X-ray diffraction and speciﬁc surface area. This clay was also used as support for porcine pancreatic immobilization, as presented inFig. 3(c). Comparison of

Fig. 3(b and c) showed a modiﬁcation on the morphology of the material and also the presence of ﬁber-like and amorphous particles, as evidenced in the free lipase, making possible to observe the effectiveness of the immobilization process.

4. Conclusions

The KSF, natural and pillared montmorillonites clays showed high potential for use as supports for lipase immobilization. Immobilization yields of 38.2% were obtained for lipase immobi-lized in pillared montmorillonite after 120 min of immobilization and enzyme to support mass ratio of 2:0.5. High esterification activities (about 1400 and 1100 U/g) were obtained for lipase immobilized in KSF and natural montmorillonites (180 and 60 min of immobilization and enzyme to support mass ratio of 2:2 and 2:1, respectively). Results obtained in this work can contribute to the development of immobilization processes of lipases using low-cost supports. In a general sense, the partial characterization of the supports showed significant morphologi-cal differences among them and the characteristics of each support influenced on the amount of enzyme adsorbed, yields of immobilization and enzyme esterification activity.

Acknowledgments

The authors wish to thank CNPq, CAPES and FAPERGS for the ﬁnancial support of this work and scholarships.

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