Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media.
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.
Akbar
Umar
U
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
Aschenbrenner
Carl D
CD
Harper
Michael R
MR
Johnson
Harvey R
HR
Dordick
Jonathan S
JS
Clark
Douglas S
DS
D002384:Catalysis; D005591:Chemical Fractionation / Q000379:methods*; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification*; D005656:Fungal Proteins; D008049:Lipase / Q000302:isolation & purification; D009195:Peroxidase / Q000302:isolation & purification; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology; D020860:Subtilisin / Q000302:isolation & purification
D004798:Enzymes; D005656:Fungal Proteins; D012997:Solvents; D009195:Peroxidase; D008049:Lipase; C431349:lipase B, Candida antarctica; D020860:Subtilisin