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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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Spherezyme particles consisting of various percentage combinations of P. fluorescens lipase and BSA. Lipase specific activity (on p-nitrophenyl palmitate) is represented as a function of lipase mass as opposed to total protein mass. Data was generated from particles that were formed using 40 μl glutaraldehyde-EDA mix as a cross-linker and nonoxynol-4 as the surfactant.
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Figure 6: Spherezyme particles consisting of various percentage combinations of P. fluorescens lipase and BSA. Lipase specific activity (on p-nitrophenyl palmitate) is represented as a function of lipase mass as opposed to total protein mass. Data was generated from particles that were formed using 40 μl glutaraldehyde-EDA mix as a cross-linker and nonoxynol-4 as the surfactant.

Mentions: The immobilisation of multiple proteins in single spherezymes was investigated. The results of including BSA in combination with P. fluorescens lipase provided a strong indication of the predicted selective migration of lipase to the phase interface. The difference in molecular mass of the two p-nitrophenyl esters used to assay activity permitted evaluation of the mass transfer properties of the spheres based on substrate size. An important observation was that although absolute specific activity decreased with increasing percentage BSA inclusion in the particles (as would be expected), the lipase specific activity (calculated solely on lipase mass) increased for p-nitrophenyl palmitate (Fig. 6). This was not observed in the case of p-nitrophenyl-butyrate (data not shown), indicating that it was specifically a surface related phenomenon.


Spherezymes: a novel structured self-immobilisation enzyme technology.

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

Spherezyme particles consisting of various percentage combinations of P. fluorescens lipase and BSA. Lipase specific activity (on p-nitrophenyl palmitate) is represented as a function of lipase mass as opposed to total protein mass. Data was generated from particles that were formed using 40 μl glutaraldehyde-EDA mix as a cross-linker and nonoxynol-4 as the surfactant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Spherezyme particles consisting of various percentage combinations of P. fluorescens lipase and BSA. Lipase specific activity (on p-nitrophenyl palmitate) is represented as a function of lipase mass as opposed to total protein mass. Data was generated from particles that were formed using 40 μl glutaraldehyde-EDA mix as a cross-linker and nonoxynol-4 as the surfactant.
Mentions: The immobilisation of multiple proteins in single spherezymes was investigated. The results of including BSA in combination with P. fluorescens lipase provided a strong indication of the predicted selective migration of lipase to the phase interface. The difference in molecular mass of the two p-nitrophenyl esters used to assay activity permitted evaluation of the mass transfer properties of the spheres based on substrate size. An important observation was that although absolute specific activity decreased with increasing percentage BSA inclusion in the particles (as would be expected), the lipase specific activity (calculated solely on lipase mass) increased for p-nitrophenyl palmitate (Fig. 6). This was not observed in the case of p-nitrophenyl-butyrate (data not shown), indicating that it was specifically a surface related phenomenon.

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