The effect of pretreatment with substrates on the activity of immobilized pancreatic lipase

Abstract

Porcine pancreatic lipase was covalently immobilized on polyvinyl alcohol using adipoyl dichloride as a cross-linker. The effect of pre-treatment of lipase previously with various types of oils on immobilization efficiency was investigated. The increment in immobilization efficiency was observed after pre-treatment of lipases with oils. The highest immobilization efficiency obtained was 20% (v/v) for olive oil pre-treated lipase, which was 8 times higher than that of non-pretreated immobilized lipase. Immobilized lipase had better stability and had some advantages in comparison with free enzyme.

Keywords::

• adipoyldichloride
• immobilization
• polyvinylalcohol
• porcine pancreatic lipase
• substrate pre-treatment

Introduction

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) catalyze chemo-, regio-, and/or stereoselective hydrolyses of esters in largely aqueous solution, and their synthesis under low water activities (Kilinc et al. 2002, Reetz 2002). Thus, lipases are important biocatalysts in several applications, such as synthesis of chiral drug intermediates and nutraceutical lipids, bioconversion of oils and fats, and production of biodegradable polymers based on the ability of lipases for esterification, transesterification, aminolysis, and hydrolysis reactions (Kartal et al. 2009, Villeneuve et al. 2000, Thakar and Madamwar 2005). These enzymes are found in animals, plants, and microorganisms, in which they play a key role in lipid metabolism, and lipases from a variety of sources are commercially available (Krishna and Karanth 2002).

Due to the high cost of the enzymes, they need to be used in an immobilized state in order to enhance the stability and enable the recovery and reusability for their industrial applications. Many immobilization methods and support materials have been used and reviewed recently (Mateo et al. 2007, Soares et al. 2003, Fernandez-Lafuente et al. 1998, Sharma et al. 2001, Rodrigues et al. 2008) for lipases. Lipase attachment to a matrix by covalent binding is not common as the physical adsorption, but it presents the advantage of avoiding the desorption phenomenon. However, enzymes to be immobilized using this technique usually lose part of their initial activity because amino acids in the catalytic site can also attach to the matrix as amino acids which are not located in the catalytic region (Kartal et al. 2009). The main concern is to carry out the covalent attachment between the carrier and the lipase region far from the active site.

A unique feature of lipases is their activation at interfaces. Activation of lipases at interfaces is a result of conformational change in lid, α-helix structure that covers the active site. At the interface, the lid opens and the active site becomes accessible to substrate molecules. More active immobilization of lipases is possible by mimicking this unique feature of lipases. Possible ways to increase the lipolytic activity include the manipulation of the lipase structure, the method of immobilization, the medium conditions, and the use of additives (Rocha et al. 1998). The effect of additives on the enzymatic activity is not well understood yet. Probably, they act by a combination of various effects, including (i) enzyme protection from inactivation during the immobilization step, (ii) retention of water layer around the catalyst, and (ii) dispersing effects of the enzyme molecules that facilitate mass transport when the additives are used together with immobilizing matrixes (Soares et al. 2003, Rocha et al. 1998). In these techniques, the geometry of the enzyme activity center is perturbed by additives such as ligands, substrate, substrate analogues, and other components (salts and polymers) in aqueous solutions (Cao 2005). Polar organic solvents have also been used with this aim (Talukder et al. 2005).

Polyvinyl alcohol (PVA) is a polymer that is frequently used as a matrix for the immobilization of various enzymes and cells because of its easy availability, low price, and hydrophilic character and hydroxyl groups on the surface capable of chemical reaction (Kartal et al. 2006).

The aim of this work is to improve the immobilization efficiency of porcine pancreatic lipase (PPL) by means of pre-treatment of enzymes with their substrates (olive oil, soybean oil, and corn oil) in organic solvent. In previous studies, pre-treatment with substrates and immobilization of lipase were carried out in hydrophilic solution, and diversely, we investigated the influence of performing pre-treatment and immobilization in an organic solution on the activity of lipase.

Experimental

Materials

Lipase (EC 3.1.1.3 Type II, crude; from porcine pancreas), PVA type III, and adipoyl dichloride were purchased from Sigma Chemical Co.; cyclohexane and acetone were obtained from Riedel. All other chemicals and oils were of analytical reagent grade.

Preparation of cross-linked polyvinyl alcohol (CL-PVA) matrix

CL-PVA matrix was prepared according to Kilinc et al. (2002). Ten grams of CL-PVA was suspended in 40 ml of 7 M NaOH in a three-neck flask. Two-milliliter volume of adipoyl dichloride was added to the mixture. The reaction mixture was heated under mechanical stirring for 1 h at 50°C and 2 h at 70°C, and then it was neutralized with 3 M HCl. The obtained powder was treated in a soxhelet apparatus with acetone and bidistilled water, respectively. The purified CL-PVA was dried at 90°C overnight and then used as a matrix for immobilization of porcine pancreatic lipase.

Immobilization of lipase

Standard immobilization of porcine pancreatic lipase on CL-PVA process was carried out according to Kilinc et al. (2002); 200 mg of PVA and 40 mg of lipase were suspended in 2 ml cyclohexane, and then 20 μl of adipoyl dichloride was added. The mixture was incubated at room temperature for 30 min with continuous stirring. At the end of the period, the gel was filtered off by suction and washed with 4 ml of cyclohexane, 4 ml of acetone, and 8 ml of bidistilled water, respectively, to remove the unbound proteins. Then, the immobilized enzyme was dried and was ready to use. When not in use, the immobilized enzyme was stored at 4°C.

Lipase pre-treatment with substrates

Substrate pre-treatment of PPL before immobilization was performed in cyclohexane. Different types of oils (olive oil, soybean oil, hazelnut oil, and corn oil) were used as substrates, and the effect of different concentrations (10–35%) of these substrates on immobilization yield was investigated. PPL was incubated with substrates at room temperature for 5 min under constant mixing. Cyclohexane was a solvent for enzyme pre-treatment and immobilization process. CL-PVA was added to the pre-treated enzyme, and immobilization was performed using standard immobilization procedure as mentioned in Section 2.3.

Assay of lipase activity

Hydrolytic activities were determined at 30°C and pH 8.0 by titration method using an automatic titrator (718 Stat Titrino, Metrohm Ltd., Switzerland). The released free fatty acids from tributyrin were titrated with 0.01 M NaOH (Kilinc et al. 2002). One unit of lipase activity was defined as 1 μmol free fatty acid released from tributyrin per minute (Kilinc et al. 2002).

Protein assay

Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The amount of bound protein was determined indirectly from the difference between the amount of protein added to the reaction mixture and the amount of protein present in the filtrate and also in washing solution after immobilization.

Physicochemical properties of free and immobilized lipases

Effect of pH on the activities and stabilities of lipases

The effect of pH on the activity of the free and immobilized enzymes was determined by measuring the hydrolytic activity in a pH range of 6.5–11.0.

The pH stabilities of free and immobilized enzymes were assayed by incubating the enzymes in 50 mM of acetate, sodium phosphate, tris–HCl, and glycine–NaOH buffers having a pH range between 5.5 and 10.5, for 20 min at 4°C, and the remaining activities were determined under the standard activity assay conditions.

Effect of temperature on the activities and stabilities of lipases

The effect of temperature on the activity of enzymes was determined between 20°C and 50°C, and the activities of both the enzymes were assayed under the standard assay conditions.

The thermal stabilities of free and immobilized enzymes were studied in the temperature range of 4–55°C. Both forms of enzyme were incubated in 0.01 M NaCl for 20 min at different temperatures, and after cooling, the remaining activity was assayed under the standard assay conditions.

Storage stability

The storage stabilities of free and immobilized enzymes were determined by measuring the residual activities of the samples taken from the enzymes that were stored at 4°C. Samples were taken at regular time intervals.

Results and discussion

Lipase was covalently linked to the hydroxyl groups of PVA through its amino groups with adipoyl dichloride. During this process, it seems possible that adipoyl dichloride can either bind enzyme molecules to PVA matrix or produce inter- and intra-molecular cross-links between enzyme molecules and their domains, as well. One reason for the loss of enzyme activity after immobilization process is the prevention of substrate molecules to penetrate the active sites of enzymes. Functional groups placed on enzymes close to the active site were generally responsible for this kind of activity loss. In this work, lipase was previously treated with various types of oils for the prevention of activity loss arising from the immobilization of the enzyme through its amino groups placed near or at the active site. During pretreatment process, cyclohexane was used as an organic solvent for increasing the solubility of oils, and this also facilitated the removal of the excess substrate from the matrix. Varied amounts (10–35%) of olive oil, soybean oil, and corn oil were tested as lipase substrates. Highest specific activities were obtained as 1.84 U/mg, 14.13 U/mg, 8.71 U/mg, and 6.09 U/mg, respectively, for non-pretreated, 20% olive oil-pretreated, 20% soybean oil-pretreated, and 25% corn oil-pretreated immobilized enzyme. The highest activity was obtained with 20% (v/v) olive oil-pretreated immobilized lipase as seen in Table I. As shown in Table I, even though the protein binding yields were nearly the same for all these immobilized enzymes, immobilization yields were distinct. These results showed that the lipase was immobilized more actively after substrate pretreatment comparatively than the non-pretreated counterpart. One explanation for the increment in immobilization efficiency can be stated by the steric hindrance as a result of the blockage of the active site of the enzyme with substrate. This steric hindrance directed the immobilization process to occur with the functional groups placed far from the active site that were not the subject of this hindrance. The increase in immobilization efficiency can be attributed to the interfacial activation mechanism of lipase besides the protection of the active site. To get immobilized lipases with an improved activity, strategies such as adsorption on hydrophobic supports and immobilization in the presence of detergents, substrates, etc., have been developed. The aim for pretreatment of lipases with substrates before immobilization is the activation and immobilization of lipases with activated conformation by opening the lid structure that controls the substrate accessibility of the active site. The immobilization of lipase with higher immobilization efficiency has been tried to be explained in Scheme 1. A few studies showed that the immobilized lipase activity was enhanced by means of substrate pretreatment strategy. For instance, Lee et al. reported that Candida rugosa lipase showed many times higher activity compared to the non-pretreated counterpart when the enzyme was previously treated with soybean oil before covalent immobilization on silica gel (Lee et al. 2006). Ozmen et al. demonstrated that the pretreatment of Candida rugosa lipase with soybean oil before immobilization on β-cyclodextrin-based polymer resulted in the increase in the activity of enzyme which was several times higher than that of the non-pretreated immobilized one (Ozmen and Yılmaz 2009).

Scheme 1. Illustration for immobilization of lipase with higher immobilization efficiency after substrate pretreatment.

Table I. Results of immobilization studies.

Amount of oil (%) (v/v)	Non-pretreated IE		Olive-oil pretreated IE		Soybean oil-pretreated IE		Hazelnut oil- pretreated IE		Corn oil-pretreated IE
	BY (%)	IY (%)	BY (%)	IY (%)	BY (%)	IY (%)	BY (%)	IY (%)	BY (%)	IY (%)
0	37	3	–	–	–	–	–	–	–	–
10	–	–	42	10	48	3	44	3	42	6
15	–	–	38	21	49	6	42	2	45	12
20	–	–	44	24	48	11	33	2	44	10
25	–	–	42	6	47	9	35	2	40	10
30	–	–	34	2	–	–	37	13	39	9
35	–	–	–	–	–	–	36	9	44	1

IE: Immobilized enzyme, BY: Protein binding yield, IY.: Immobilization yield.

Free enzyme-specific activity: 58 U/mg.

Free enzyme was used without cyclohexane pre-treatment for activity measurements and calculation of immobilization yield.

It was seen that the activity of olive oil-pretreated lipase was 8 times higher compared to that of non-pretreated immobilized lipase. Since the 20% (v/v) olive oil-pretreated lipase had the highest immobilization yield, 20% (v/v) olive oil-pretreated immobilized lipase was used for further studies.

The effect of pH on activity and stability of free and immobilized enzymes

As shown in Figure 1, the optimum pH value was 8.0 for free enzyme and was 10.0 for immobilized enzyme, and the pH shift was based on the linking of enzyme via its free amino groups to CL-PVA, meaning that the immobilized lipase gains more polyanionic character. A pH gradient between the domain of immobilized enzyme particles and external solution occurs during the enzymatic hydrolysis reaction, resulting in the shift of the pH optimum to the alkaline region under these conditions as expected. Similar results have been reported previously by different researchers (Kilinc et al. 2002, Kartal and Kilinc 2006, Mina et al. 2010).

Figure 1. The effect of pH on the activity of free and immobilized lipases.

As seen in Figure 2, immobilized form had better stability than free enzyme especially in pH 6–10 region. For this pH range, the free lipase showed a bell-shaped curve whereas the immobilised enzyme activity maintained roughly constant.

Figure 2. pH stabilities of free and immobilized lipases.

The effect of temperature on activity and stability of free and immobilized enzymes

Figure 3 shows the temperature profiles for the free and immobilized lipase preparations. Their responses to the temperature were similar with maximal activities at 30°C. This is in accordance with many reports that optimum temperature values of immobilized enzyme are higher than those of free enzymes (Kilinc et al. 2002, Bayramoglu and Arıca 2008, Bayramoglu et al. 2002, Tang et al. 2007, Goto et al. 2005, Pereira et al. 2003), the catalytic activity of enzymes is related to their conformation, and in general, the activity value increases with a rise in temperature as is usually observed for chemical catalysts. The increment in the activity of the enzyme is observed as long as the stability of the enzyme is maintained. Immobilization serves to stabilize the enzyme structure against environmental conditions such as temperature; consequently, optimum temperature value is expected to increase after immobilization of enzyme. Unfortunately, this principle does not apply in general; sometimes, optimum pH and optimum temperature values remain unchanged after immobilization (Paula et al. 2007, Satoshi et al. 1989, Abbas et al. 2003).

Figure 3. The effect of temperature on the activity of free and immobilized lipases.

The immobilized lipase exhibited higher stability than its free form in the temperature range 4–45°C as shown in Figure 4; after 20-min incubation at 45°C, the immobilized enzyme showed 72% of initial activity, whereas the free enzyme showed 50% of initial activity. After 20-min incubation at 55°C, decrease in the activities of free and immobilized forms was nearly the same.

Figure 4. Thermal stabilities of free and immobilized lipases.

Storage stabilities of enzymes

In 12 days, immobilized lipase activity decreased more slowly than its free form; 70% of free lipase activity was lost at the end of this period while this ratio was 50% for immobilized enzyme as seen in Figure 5.

Figure 5. Storage stabilities of free and immobilized lipases.

Conclusion

This study demonstrated that pretreatment of lipase with substrates, especially olive oil, enhanced immobilized enzyme activity compared to non-pretreated counterpart, probably because the presence of substrate at active site enables the enzyme to immobilize more active conformation. Immobilized enzyme had better properties and stabilities; these are important for biotechnological applications. Furthermore, it is believed that this technique can be applied to the immobilization of different enzymes to greatly increase their activities.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. The authors gratefully acknowledge the support for this research provided by the Scientific Research Foundation of Ege University (BAP - Grant Number Sci 2007–011).