PREPARATION OF ORGANIC-SOLVENT-SOLUBLE ENZYME (LIPASE-B) AND CHARACTERIZATION BY GEL-PERMEATION CHROMATOGRAPHY

To analyse lipase-catalysed reactions in organic media; a product was developed to aid both solubilization of lipase B and the preservation of its activity in organic solvents using a synthetic detergent, didodecyl glucosylglutamate, according to Okahata's modification method. By this improved method, > 50% of the lipase B could be converted into a solvent-soluble enzyme, which was called organic-solvent-soluble lipase. The organic-solvent-soluble lipase was successfully separated by gel permeation chromatography (GPC) from the excess of free detergent. The lipase activity was eluted at the fraction corresponding to a molecular weight of 130 kD. The composition of the purified solvent-soluble lipase was also investigated. It was estimated that 153 +/- 25 molecules of the synthetic detergent were attached to one enzyme molecule, and the molecular weight of the complex was calculated to be 131 kD +/- 16 kD based on the composition ratio. This value agreed with the molecular weight determined by GPC. These results confirmed that the organic-solvent-soluble lipase could be chromatographed by the GPC method and that its activity was preserved, suggesting that the GPC might be a very useful technique for the purification of organic-solvent-soluble enzymes.