I111111

Fig. 3. Effect of ionic strength in the adsorption of lipase from R. niveus to octyl-agar-ose. 1 mL Octyl-agarose was added to 200 mL of lipase suspension (0.1 mg of protein/mL) at 48°C, pH 7.0), in 10 mMphosphate and different concentrations of ammonium sulfate: without ammonium sulfate (•), 0.1 M ammonium sulfate (■), 1 M ammonium sulfate (A). Activity of supernatant was measured in all cases.

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

3.6. Desorption of the Adsorbed Lipase From the Hydrophobic Support

1. After inactivation of the biocatalyst the lipases may be desorbed through use of detergents or organic buffer/organic solvents if it is required at higher temperatures.

3.7. Effect of the Ionic Strength on the Lipase Adsorption on Hydrophobic Support

Conventional hydrophobic adsorption of proteins is stronger and more rapid when increasing the ionic strength (see Fig. 2). However, adsorption of lipases on octyl-agarose support was slowed down when the ionic strength increased (see Fig. 3) (16). Maximum immobilization rates were obtained at only 5 to 10 mMof sodium phosphate buffer.

3.8. Selectivity of the Immobilization on Hydrophobic Supports

The full activity of lipases from very different sources (Candida rugosa, C.antarcticaB, Humicola lanuginosa, Pseudomonas fluorescens, Rhizopusniveus, Rhizomucor miehei, Thermus aquaticus, Bacillus thermocatenulatus) was quickly adsorbed—less than 10 min in relation to a 1-mL gel and 10 mL of lipase extract— at low ionic strength (5-10 mM sodium phosphate). Furthermore, the other proteins contained in the crude extracts remained in the solution (16,17,19).

In this way, an unique band corresponding to the exact mass of the lipase was observed in the SDS-PAGE for different enzymes adsorbed on the octyl-agarose support (see Fig. 4). Therefore, this easy and quick immobilization methodology can be use to purify lipases.

Lipase Enzyme Sds Page

Fig. 4. Analysis of the adsorption of different lipase extracts on octyl-agarose by SDS-PAGE. Lane 1, molecular weight markers; lane 2, commercial P. fluorescens preparation; lane 5, P.fluorescens lipase adsorbed on octyl-agarose support; lane 4, commercial H. lanuginosa preparation; lane 3, H. lanuginosa lipase adsorbed on octyl-agarose support; lane 6, commercial C. antartica B preparation; lane 7, C. antarctica B lipase adsorbed on octyl-agarose support.

Fig. 4. Analysis of the adsorption of different lipase extracts on octyl-agarose by SDS-PAGE. Lane 1, molecular weight markers; lane 2, commercial P. fluorescens preparation; lane 5, P.fluorescens lipase adsorbed on octyl-agarose support; lane 4, commercial H. lanuginosa preparation; lane 3, H. lanuginosa lipase adsorbed on octyl-agarose support; lane 6, commercial C. antartica B preparation; lane 7, C. antarctica B lipase adsorbed on octyl-agarose support.

3.9. Hyperactivation of Lipases Resulting From the Interfacial Activation Against the Hydrophobic Support

The immobilization of lipases on hydrophobic supports (octyl-agarose or octadecyl-Sepabeads®) promoted a significant increase in the lipase activity (hyperactivation) against completely soluble substrates. Figure 5 shows a typical adsorption immobilization experiment, in which suspension activity increases during immobilization.

3.10. Stabilization of Lipases by Interfacial Activation Against Hydrophobic Supports

Furthermore, these immobilized preparations were very stable biocatalysts, much more that the soluble one, in the thermal-inactivation (see Fig. 6). The enzyme adsorption is so strong that there is no desorption of the protein, even in 40% organic solvents such as dioxane. In fact, in the presence of organic cosolvents the enzyme also exhibited a high increment of stability (see Fig. 7)

3.11. Separation of Lipase Isozymes and Isoforms by Adsorption on Agarose With Different Hydrophobicity

Several lipase isoforms have been described in many organisms. These different isoforms could have different properties when catalyzing organic reactions. However, the separation of very similar enzyme structures may be extremely difficult using conventional chromatographic techniques. Thus, very small modifications on enzyme structure could promote important changes in the mechanism of

Enzyme Immobilization
Fig. 5. Hyperactivation of lipase from H. lanuginosa during its adsorption on octyl-agarose. Supernatant (♦), immobilization suspension (■), blank suspension, with inert agarose (A).
Fig. 6. Thermostability of different preparations of C. antarctica Blipase. Inactivation was performed at 50°C, pH 7.0. Octadecyl-Sepabeads (■), soluble enzyme (•).

activation of lipases. In this way, the rate and intensity of adsorption of different lipase isoforms on tailor-made hydrophobic supports may be dramatically different. In fact, the sequential adsorption of the crude of the C. rugosa lipase on agarose supports with different grade of hydrophobicity, butyl-agarose (low hydrophobicity), phenyl-agarose (medium hydrophobicity), and octyl-agarose (high hydrophobicity) permitted to separate the different isoforms of this lipase (20).

3.12. Reversibility of the Lipase Adsorption

Although it is interesting that the adsorption strength of the lipase to the supports was very high to prevent the desorption of the lipase under industrially rel-

Enzyme Immobilization
Fig. 7. Stability of different preparations of R. miehei lipase against cosolvents. Inacti-vation was performed at 25°C, pH 7.0, in 40% Dioxane. Octadecyl-Sepabeads (■), soluble enzyme (•).

Table 1

Desorption of the Immobilized Lipases From Octyl-Agarose or Octadecyl-Sepabeads Support

Table 1

Desorption of the Immobilized Lipases From Octyl-Agarose or Octadecyl-Sepabeads Support

Lipase

Support

Triton X-100 (%)

C. antartica B

Octyl-agarose

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