Rosa L. Segura

Purification, Immobilization, and Stabilization of a Lipase from Bacillus thermocatenulatus by Interfacial Adsorption on Hydrophobic Supports

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS‐PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl‐Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl‐Sepabeads‐BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 °C or 30% of dioxane and 45 °C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl‐Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 °C.

Reversible and Strong Immobilization of Proteins by Ionic Exchange on Supports Coated with Sulfate-Dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate‐dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate‐dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic‐dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (β‐galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermus sp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Different Properties of the Lipases Contained in Porcine Pancreatic Lipase Extracts as Enantioselective Biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl‐Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl‐Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (±)‐2‐hydroxy‐4‐phenylbutyric acid ethyl ester [(±)‐1] to produce the corresponding acid [2], the octyl‐25KDa preparation showed the best enantioselectivity ( E) value ( E = 7) at pH 5 and 25 °C, whereas the phenyl‐PPL was the most enantioselective ( E = 10) at pH 5, 4 °C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (±)‐2‐ O‐butyryl‐2‐phenylacetic acid [(±)‐3] with a high E value ( E > 100); for example, with octadecyl‐33 KDa enzyme at pH 8.