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Spherezymes: a novel structured self-immobilisation enzyme technology.

Brady D, Jordaan J, Simpson C, Chetty A, Arumugam C, Moolman FS - BMC Biotechnol. (2008)

Bottom Line: Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates.However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts.The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIR Biosciences, Ardeer Road, Modderfontein, 1645 South Africa. dbrady@csir.co.za

ABSTRACT

Background: Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.

Results: Through addition of protein cross-linking agents to a water-in-oil emulsion of an aqueous enzyme solution, structured self-immobilised spherical enzyme particles of Pseudomonas fluorescens lipase were formed. The particles could be recovered from the emulsion, and activity in aqueous and organic solvents was successfully demonstrated. Preliminary data indicates that the lipase tended to collect at the interface.

Conclusion: The immobilised particles provide a number of advantages. The individual spherical particles had a diameter of between 0.5-10 mum, but tended to form aggregates with an average particle volume distribution of 100 mum. The size could be controlled through addition of surfactant and variations in protein concentration. The particles were robust enough to be recovered by centrifugation and filtration, and to be recycled for further reactions. They present lipase enzymes with the active sites selectively orientated towards the exterior of the particle. Co-immobilisation with other enzymes, or other proteins such as albumin, was also demonstrated. Moreover, higher activity for small ester molecules could be achieved by the immobilised enzyme particles than for free enzyme, presumably because the lipase conformation required for catalysis had been locked in place during immobilisation. The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme. This type of self-immobilised enzyme particle has been named spherezymes.

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Lipase spherezymes immobilisation method. Lipase (a) in aqueous medium (left) is added to an excess of a hydrophobic solvent (right), forming a water-in-oil emulsion (b), wherein the lipase migrates to the phase boundary and orientates the hydrophobic face associated with the active site towards the hydrophobic layer. The enzyme is subsequently cross-linked (grey crescents) to make the structure permanent (c) and the solvent removed (d).
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Figure 2: Lipase spherezymes immobilisation method. Lipase (a) in aqueous medium (left) is added to an excess of a hydrophobic solvent (right), forming a water-in-oil emulsion (b), wherein the lipase migrates to the phase boundary and orientates the hydrophobic face associated with the active site towards the hydrophobic layer. The enzyme is subsequently cross-linked (grey crescents) to make the structure permanent (c) and the solvent removed (d).

Mentions: The enzyme sub-class of lipase [EC 3.1.1.3] was chosen as the initial enzyme for investigations due to its biocatalytic importance for chiral resolution of chemicals and pharmaceutical intermediates. Lipases typically possess a hydrophobic surface, often containing a 'lid' region associated with the active site [20] (Fig. 1), and it is believed that this surface associates with the hydrophobic phase at the interface between hydrophilic and hydrophobic fluids. Within a water-in-oil emulsion, these proteins would therefore tend to localise and orientate at the spherical interface of the emulsion droplet. Subsequent cross-linking of the protein would then form a self immobilised particle (Fig. 2).


Spherezymes: a novel structured self-immobilisation enzyme technology.

Brady D, Jordaan J, Simpson C, Chetty A, Arumugam C, Moolman FS - BMC Biotechnol. (2008)

Lipase spherezymes immobilisation method. Lipase (a) in aqueous medium (left) is added to an excess of a hydrophobic solvent (right), forming a water-in-oil emulsion (b), wherein the lipase migrates to the phase boundary and orientates the hydrophobic face associated with the active site towards the hydrophobic layer. The enzyme is subsequently cross-linked (grey crescents) to make the structure permanent (c) and the solvent removed (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266724&req=5

Figure 2: Lipase spherezymes immobilisation method. Lipase (a) in aqueous medium (left) is added to an excess of a hydrophobic solvent (right), forming a water-in-oil emulsion (b), wherein the lipase migrates to the phase boundary and orientates the hydrophobic face associated with the active site towards the hydrophobic layer. The enzyme is subsequently cross-linked (grey crescents) to make the structure permanent (c) and the solvent removed (d).
Mentions: The enzyme sub-class of lipase [EC 3.1.1.3] was chosen as the initial enzyme for investigations due to its biocatalytic importance for chiral resolution of chemicals and pharmaceutical intermediates. Lipases typically possess a hydrophobic surface, often containing a 'lid' region associated with the active site [20] (Fig. 1), and it is believed that this surface associates with the hydrophobic phase at the interface between hydrophilic and hydrophobic fluids. Within a water-in-oil emulsion, these proteins would therefore tend to localise and orientate at the spherical interface of the emulsion droplet. Subsequent cross-linking of the protein would then form a self immobilised particle (Fig. 2).

Bottom Line: Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates.However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts.The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIR Biosciences, Ardeer Road, Modderfontein, 1645 South Africa. dbrady@csir.co.za

ABSTRACT

Background: Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.

Results: Through addition of protein cross-linking agents to a water-in-oil emulsion of an aqueous enzyme solution, structured self-immobilised spherical enzyme particles of Pseudomonas fluorescens lipase were formed. The particles could be recovered from the emulsion, and activity in aqueous and organic solvents was successfully demonstrated. Preliminary data indicates that the lipase tended to collect at the interface.

Conclusion: The immobilised particles provide a number of advantages. The individual spherical particles had a diameter of between 0.5-10 mum, but tended to form aggregates with an average particle volume distribution of 100 mum. The size could be controlled through addition of surfactant and variations in protein concentration. The particles were robust enough to be recovered by centrifugation and filtration, and to be recycled for further reactions. They present lipase enzymes with the active sites selectively orientated towards the exterior of the particle. Co-immobilisation with other enzymes, or other proteins such as albumin, was also demonstrated. Moreover, higher activity for small ester molecules could be achieved by the immobilised enzyme particles than for free enzyme, presumably because the lipase conformation required for catalysis had been locked in place during immobilisation. The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme. This type of self-immobilised enzyme particle has been named spherezymes.

Show MeSH