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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: 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.This type of self-immobilised enzyme particle has been named spherezymes.

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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Size distribution of lipase spheres made with surfactant in mineral oil as determined by LLS. Standard formulation method (_____), 50% lipase concentration (.........), 50% lipase concentration and surfactant omitted (-------).
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Figure 4: Size distribution of lipase spheres made with surfactant in mineral oil as determined by LLS. Standard formulation method (_____), 50% lipase concentration (.........), 50% lipase concentration and surfactant omitted (-------).

Mentions: From the SEM images, the size of the individual spherical particles were determined to be between 0.5–10 μm in diameter (Fig. 3b). However from laser light scattering experiments it appears that the particles aggregate with a size distribution between 10 and 100 μm. Using the current standard procedure for spherezyme formation the average particle size diameter was 62.0 μm, (Sauter mean diameter d32 = 62.0 μm, span = 1.96, Fig. 4).


Spherezymes: a novel structured self-immobilisation enzyme technology.

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

Size distribution of lipase spheres made with surfactant in mineral oil as determined by LLS. Standard formulation method (_____), 50% lipase concentration (.........), 50% lipase concentration and surfactant omitted (-------).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Size distribution of lipase spheres made with surfactant in mineral oil as determined by LLS. Standard formulation method (_____), 50% lipase concentration (.........), 50% lipase concentration and surfactant omitted (-------).
Mentions: From the SEM images, the size of the individual spherical particles were determined to be between 0.5–10 μm in diameter (Fig. 3b). However from laser light scattering experiments it appears that the particles aggregate with a size distribution between 10 and 100 μm. Using the current standard procedure for spherezyme formation the average particle size diameter was 62.0 μm, (Sauter mean diameter d32 = 62.0 μm, span = 1.96, Fig. 4).

Bottom Line: 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.This type of self-immobilised enzyme particle has been named spherezymes.

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
Related in: MedlinePlus