Use of immobilized lipases for lipase purification via specific lipase–lipase interactions

Palomo, José M.; Ortíz, Claudia; Fuentes, Manuel; Fernández‐Lorente, Gloria; Guisán, José M.; Fernández‐Lafuente, Roberto

doi:10.1016/j.chroma.2004.03.058

Cited by 125 publications

(83 citation statements)

References 48 publications

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“…However, by adding a small concentration of detergents (e.g., 0.5% Triton X-100), adsorption via interfacial activation may be fully prevented 29 , and lipases will become adsorbed by the groups inserted in the matrix. Other possible problems using lipases is their tendency to form lipase-lipase dimers with altered properties; this is also solved using detergents [50][51][52] .…”

Section: Procedurementioning

confidence: 99%

Immobilization of enzymes on heterofunctional epoxy supports

et al. 2007

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Section: Procedurementioning

confidence: 99%

Immobilization of enzymes on heterofunctional epoxy supports

et al. 2007

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“…Immobilization of BTL2-BTL2 immobilization on cyanogen bromide-agarose (CNBr) was performed as previously described (20). This preparation (prepared in the presence of detergent but later thoroughly washed with distilled water) permitted the immobilization of the monomeric form of the lipase, avoiding any intermolecular lipase-lipase interactions that could interfere with the properties of soluble lipases (21).…”

Section: Methodsmentioning

confidence: 99%

Activation of Bacterial Thermoalkalophilic Lipases Is Spurred by Dramatic Structural Rearrangements

Carrasco-López

Godoy

Rivas

et al. 2009

Journal of Biological Chemistry

203

222

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The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60 -75°C and pH 8 -10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the ␣6-and ␣7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the ␣6-helix and the incorporation of short side chain residues to the ␣6 C-terminal end. All these structural changes are stabilized by the Zn 2؉ -binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.

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“…In recent years, Fernández-Lafuente and his coworkers [12][13][14] have developed a new method to immobilize proteases on glyoxyl-agarose via multipoint covalent attachments, and through this a new kind of immobilized enzymes with a high stability were obtained. In their strategy, the enzyme was not stabilized by rigidification but by orienting the active center of the enzyme towards the reaction medium and by an additional modification with poly-aldehyde polymers.…”

Section: Introductionmentioning

confidence: 99%

The Properties of Covalently Immobilized Trypsin on Soap‐Free P(MMA‐EA‐AA) Latex Particles

Kang

Kan

Yeung

et al. 2005

Macromolecular Bioscience

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The covalent immobilization of trypsin onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55 degrees C and 8.5, both of which were higher than that of the free form. It was found that K(m) (Michaelis constant) was 45.7 mg . ml(-1) and V(max) (maximal reaction rate) was 793.0 microg . min(-1) for immobilized trypsin, compared to a K(m) of 30.0 mg . ml(-1) and a V(max) of 5 467.5 microg . min(-1) for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times.

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Use of immobilized lipases for lipase purification via specific lipase–lipase interactions

Cited by 125 publications

References 48 publications

Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes on heterofunctional epoxy supports

Activation of Bacterial Thermoalkalophilic Lipases Is Spurred by Dramatic Structural Rearrangements

The Properties of Covalently Immobilized Trypsin on Soap‐Free P(MMA‐EA‐AA) Latex Particles

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