Purification Immobilization Hyperactivation and Stabilization of Lipases by Selective Adsorption on Hydrophobic Supports

Yeast Infection Free Forever

Natural Candida Albicans Treatment Systems

Get Instant Access

Jose M. Palomo, Gloria Fernández-Lorente, Cesar Mateo, Rosa L. Segura, Claudia Ortiz, Roberto Fernandez-Lafuente, and Jose M. Guisan


Immobilization of lipases on hydrophobic supports at low ionic strength permits one-step purification, immobilization, hyperactivation, and stabilization of most lipases. This selective adsorption occurs because the hydrophobic surface of the supports is able to promote the interfacial activation of the lipases, yielding enzyme preparations having the open form of the lipases very strongly adsorbed on these hydrophobic supports. At low ionic strength, only proteins having large hydrophobic pockets may become adsorbed on the hydrophobic support, and the only soluble proteins are lipases, which in closed form are fairly hydrophilic, but in open form expose a very hydrophobic pocket. The resulting biocatalysts are therefore hyperactivated, at least with hydrophobic and small substrates (because all the enzyme molecules have the open form). Moreover, the stabilization of the open form of the lipases permits very highly stabilized enzyme preparations.

Key Words: Interfacial activation; hydrophobic supports; stabilization of the open form of lipases; selective adsorption; lipase stabilization.

1. Introduction

Lipases (E.C. have as natural function the hydrolysis of triglycerides. Nevertheless, they may be used in vitro to catalyze many different reactions, in many instances quite far from the natural ones (e.g., regarding conditions, substrates). Thus, lipases are utilized in different industrial applications, such as in the production of modified oils (1,2), cosmetics (3,4), or—the most important one in the last years—production of many different intermediates for organic synthesis (e.g., resolution of racemic mixtures), because they combine broad substrate specificity with a high enantio- and regioselectivity (5-10).

Lipase Adsorption
Fig. 1. Interfacial adsorption of lipases.

However, these enzymes present a very complex catalytic mechanism. In homogeneous aqueous solutions, the lipase is mainly in a closed and inactive conformation where the active site is completely isolated from the reaction medium by an oligopeptide chain (called flap or lid), blocking the entry of substrates to the active site. This polypeptide chain presents several hydrophobic amino acid residues in its internal face, interacting with hydrophobic zones around the active site. This conformation may exist in a partial equilibrium with an open and active conformation, where the lid is displaced, stabilized by ionic interactions or hydrogen bonds with a specific part in the lipase surface allowing the access of the substrate to the active site (11,12).

However, upon exposure to a hydrophobic interface such as a lipid droplet, the lipase can only interact (when it is in the open conformation), via the hydrophobic pocket formed by the internal face of the lid and the surroundings of the active site, consequently shifting the equilibrium towards the open form—the so called interfacial activation (13,14) (see Fig. 1).

This mechanism of action promotes some problems when industrial immobilized lipases are prepared. Inside a porous structure, lipases molecules become inaccessible to any kind of external interfaces, therefore there is no possibility of enzyme interfacial adsorption in aqueous solutions. In fact, conventionally immobilized lipase preparations are usually utilized primarily in anhydrous media, where the lipase may become activated by the direct interaction with the organic solvent phase (15).

However, many interesting reactions catalyzed by lipases may be advantageously carried out in aqueous systems (e.g., hydrolytic resolutions of racemic mixtures) (8-10). The interfacial adsorption of lipases on hydrophobic supports has been proposed as a simple method for preparing immobilized lipase preparations useful in any media (16,17). The hypothesis behind this immobilization strategy is to take advantage of the complex mechanism of lipases (an apparent problem) as a tool that permits the immobilization of lipases via an "affinity-like" strategy (16,17). Using a hydrophobic support (that somehow resembles the sur face of the drops of the natural substrates) and very low ionic strength, lipases become selectively immobilized on these supports. The adsorption involves the hydrophobic areas surrounding the active center and located in the internal face of the flat (no other water-soluble proteins are adsorbed on the support under these mild conditions) (16-18). These adsorbed lipases are able to access the active center; in fact, immobilized enzymes usually exhibit significantly enhanced enzyme activity (by the "interfacial adsorption mechanism"). The result is an immobilized lipase where the open conformation has been "fixed" and does not depend on the presence of external hydrophobic interfaces.

2. Materials

1. Lipases from Candida antarctica B, Rhizomucor miehei, Humicola lanuginosa (Novo Nordisk, Denmark)

2. Lipases from Rhizopus niveus, Mucor javanicus, and Pseudomonas fluorescens (Amano Co., Nagoya, Japon).

3. Lipase from Candida rugosa, (Sigma, St. Louis, MO).

4. p-Nitrophenyl propionate (p-NPP), (Sigma).

6. Ethyl butyrate (Sigma).

7. Buffer for immobilization: 25 mMsodium phosphate buffer adjusted at pH 6.0, pH 7.0.

8. Assay buffer: 25 mMphosphate, pH 7.0.

9. Butyl-, phenyl-, and octyl-Sepharose 4BCL support (Pharmacia Biotech, Uppsala, Sweden).

10. Octadecyl-Sepabeads® resin (Resindion, SRL; Milan, Italy).

11. Shimadzu UV (UV250PC).

12. pHstat (Mettler Toledo, Madrid, Spain).

3. Methods

3.1. Activity Assay of Lipases

1. A 50-mM stock solution in acetonitrile of the substrate (pNPP) was prepared.

2. 2.5 mL Phosphate buffer and 20 |L of substrate stock solution were added to a spectrophotometry cell and the mixture was preincubated at 25°C for 10 min.

3. Esterasic lipase activity was measured using an ultraviolet spectrophotometer by measuring the increase in the absorbance at 348 nm produced by the release of p-nitrophenol in the hydrolysis of pNPP prepared as described above at pH 7.0 and 25°C.

4. 0.05 mL Lipase solution or suspension (1 U/mL) was added to 2.5 mL of pNPP solution (see Note 1).

3.2. Immobilization of Lipases on Hydrophobic Supports

1. Wash 10 g of support 10 to 15 times with three volumes of distilled water.

2. Mix 1 mg/mL of the enzyme solution and 10 g of swelling support in 5 to 10 mM sodium phosphate immobilization buffer, pH 7.0. Stir gently for 3 h at 25°C (see Note 2).

Lipase Immobilization Adsorption
Fig. 2. Adsorption of proteins/lipases on hydrophobic supports.

3.3. SDS-PAGE of the Immobilized Enzymes

1. Incubate 1 g of immobilized enzyme in 2 mL 4% (w/v) sodium dodecyl sulfate (SDS) in the presence of mercaptoethanol and boil suspension for 5 min. This promotes the release of any adsorbed lipase.

2. Take samples of the supernatant and analyze following standard protocols (see Fig. 2).

3.4. Effect of the Adsorption on Hydrophobic Supports in the Stability of Lipases

1. Resuspend 1 g of the desired lipase immobilized preparation in 25 mMsodium phosphate buffer, pH 7.0, in the desired temperature.

2. Periodically withdraw samples using tip-cut pipets and measure the activity as described in Subheading 3.1.

3.5. Stability of Lipase-Hydrophobic Preparations in the Presence of Organic Cosolvents

1. Different cosolvents at different concentrations are prepared, using Tris-HCl as a buffer in order to prevent pH changes (see Note 3).

2. Wash the enzyme preparation 10 times with three volumes of the desired organic solvent/buffer mixture at 4°C.

3. Resuspend 1 g of the desired lipase immobilized preparation in the previous solution at the desired temperature and measure the activity immediately (see Note 4) as described in Subheading 3.1.

Immobilization Enzyme Organic Solution

Was this article helpful?

0 0
All Natural Yeast Infection Treatment

All Natural Yeast Infection Treatment

Ever have a yeast infection? The raw, itchy and outright unbearable burning sensation that always comes with even the mildest infection can wreak such havoc on our daily lives.

Get My Free Ebook

Post a comment