Large-scale purification, enzymic characterization, and crystallization of the lipase from Geotrichum candidum
1991; Elsevier BV; Volume: 13; Issue: 10 Linguagem: Inglês
10.1016/0141-0229(91)90069-m
ISSN1879-0909
AutoresHans Christian Hedrich, Friedrich Spener, Ulrich Menge, Hans‐Jürgen Hecht, Rolf D. Schmid,
Tópico(s)Biofuel production and bioconversion
ResumoLipases from several strains of the imperfect fungus Geotrichum candidum are renowned for their high, though not absolute, specificity for Δ9-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 Å, the lattice constants were a = 53.1 Å, b = 83.5 Å, c = 57.8 Å, β = 100° and the space group was monoclinic P 21. A space-filling coefficient of 2.3 Å3 was calculated.
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