Polyethylene glycol-modified subtilisin forms microparticulate suspensions in organic solvents

Khan, Shahnaz A.; Halling, Peter J.; Bosley, John A.; Clark, Alan H.; Peilow, Alan D.; Pelan, Eddie G.; Rowlands, David W.

doi:10.1016/0141-0229(92)90164-j

Cited by 30 publications

(9 citation statements)

References 21 publications

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“…Enzymes covalently linked to amphipatic polymers such as PEG give transparent solutions in aromatic and chlorinated solvents and retain catalytic activity (Inada et al, 1986) and native conformation (Pasta et al, 1988). Even though the true solubility of these derivatives has been questioned by Khan et al (1992), who showed that PEG-subtilisin forms aggregates in organic solvents, the dispersion of PEG-enzymes should be much greater than that of suspended powder or immobilized enzymes. Table II Effects of water activity (a, ) on V,, and Kr,,, for n-octanol of PEG-lipase in different organic solvents I.…”

Section: Resultsanddiscussionmentioning

confidence: 99%

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Bovara¹,

Carrea²,

Ottolina³

et al. 1993

Biotechnol Lett

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The V,, and KM of various forms of lipase from Pseudomonas cepacia ( powder, adsorbed onto Celite or covalently linked to polyethylene glycol ) were determined in organic solvents preequilibrated to water activities ( a, ) from ~0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that Ki,., for the nucleophile increased with increasing a, for all three lipase forms. V,, increased with increasing a, for polyethylene glycol-lipase, whereas there was an optimum at intermediate a, values ( 0.11 -0.38 ) for lipase powder and Celite-immobilized lipase.

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Section: Resultsanddiscussionmentioning

confidence: 99%

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Bovara¹,

Carrea²,

Ottolina³

et al. 1993

Biotechnol Lett

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“…98 However, it has been shown that, at least in some cases, the PEG-modified enzymes formed relatively large aggregates in organic solvents. 99 Recently, a comb-shaped PEG was used to covalently modify CRL to make it soluble in ionic liquids. 100 Interestingly, the activity in a transesterification reaction with vinyl acetate was much higher in ionic liquids than in other organic solvents.…”

Section: Covalent Couplingmentioning

confidence: 99%

Immobilisation and application of lipases in organic media

2013

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Different methods of preparing lipases for use in organic media are critically reviewed. Solid lipase preparations can be made by typical immobilisation methods such as adsorption, entrapment, covalent coupling or cross-linking. Immobilisation is especially attractive for lipases because, in addition to the normal benefits of enzyme immobilisation, it can also lead to a considerable increase in catalytic activity, probably caused by conformational changes in the lipase molecules. Activation can be achieved, for example, using hydrophobic support materials or surfactants during the immobilisation procedure. Surfactants can also be used to solubilise lipases in organic media via the formation of hydrophobic ion pairs, surfactant-coated lipase or reversed micelles. Lipase preparation methods are discussed with regard to potential lipase inactivation and activation effects, mass transfer limitations, lipase stability and other features important for applications. The practical applications of lipases in organic media reviewed include ester synthesis, modification of triacylglycerols and phospholipids, fatty acid enrichment, enantiomer resolution, biodiesel production and acylation of carbohydrates and bioactive compounds.

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“…While each method has its own advantages, none has been shown to yield both high solubilization efficiencies and high activities across a broad range of solvents. For example, PEGylation is timeconsuming (Altreuter et al, 2003), leads to modified proteins having limited solubility in several commonly used organic solvents (Kwon et al, 1999), and frequently results in solubilized enzyme with little or no improvement in activity over the suspended enzyme (Khan et al, 1992;Kwon et al, 1999). Furthermore, for RM-encapsulated enzymes, the complete encapsulation of the protein by surfactant and its containment within a pool of water can restrict enzyme activity in organic biotransformations, despite high solubilization efficiencies (Castro and Knubovets, 2003).…”

Section: Introductionmentioning

confidence: 97%

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

Akbar

Aschenbrenner

Harper

et al. 2006

Biotech & Bioengineering

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A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.

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Polyethylene glycol-modified subtilisin forms microparticulate suspensions in organic solvents

Cited by 30 publications

References 21 publications

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Immobilisation and application of lipases in organic media

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

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