LARGE-SCALE PURIFICATION, ENZYMATIC CHARACTERIZATION, AND CRYSTALLIZATION OF THE LIPASE FROM GEOTRICHUM-CANDIDUM

Lipases from several strains of the imperfect fungus Geotrichum candidum are renowned for their high, though not absolute, specificity for DELTA-9-unsaturated fatty acids. Starting from a commercially available raw material (GC-4 lipase from Amano), an expeditious two-step isolation method was developed that allowed us to prepare 260 mg of pure enzyme within 2 days. The 61.6-kDa lipase was homogeneous in SDS-PAGE, had a carbohydrate content of 8.8 wt%, and revealed a microheterogeneic pattern between pH 4.4 and 4.8 on isoelectric focusing. Deglycosylation with endoglycosidase H abolished this microheterogeneity, and the lipase focused as a single band at pH 4.63. Over a broad pH range the deglycosylated enzyme had higher specific activities than the native glycosylated form. In hydrolysis as well as in transesterification, a high preference for oleic acid over saturated fatty acids was found for the GC-4 lipase. Crystals were obtained from the native and deglycosylated enzyme as well as from the lipase modified by p-chloromercuribenzoate and iodine. High-quality crystals of native GC-4 lipase grew from media containing polyethylene glycol 20,000. They diffracted to at least 2.5 angstrom, the lattice constants were a = 53.1 angstrom, b = 83.5 angstrom, c = 57.8 angstrom, beta = 100-degrees and the space group was monoclinic P 2(1). A space-filling coefficient of 2.3 angstrom 3 was calculated.