Triacylglycerol lipase has been purified to homogeneity from pancreas of a number of species, with the porcine enzyme the most thoroughly investigated. The procedure originally developed by Verger et al. (117)
essentially includes an aqueous extraction of a defatted pancreas powder, followed by DEAE-cellu-lose and Sephadex G-100 chromatographic steps. Various modifications of the purification have been described (10, 118, 119). The procedure, according to Brockman (119), consists of (a) delipidation of fresh porcine pancreas tissue by chloroform-ra-butanol; (b) extraction of lipase with potassium phosphate buffer containing NaCl and diisopropylfluorophosphate; (c) affinity chromatography using hydrophobic glass beads; and (d) DEAE-cellulose chromatography (Table 1). Colipase contamination can be removed by additional chromatography on concanavalin A-Sepaharose or hydroxyapatite (120). The use of fresh pancreas is critical, as most commercial pancreatic powders may contain degraded lipase, and so is the addition of di-isopropylfluorophosphate to the buffer for inhibition of proteolytic enzymes present in the extract.
Purification of extracellular lipases from microbial sources is less cumbersome than from animal tissues. Rhizomucor miehei lipase is purified by passing the culture supernatant onto an anion exchange column, following by affinity chromatography on Con A-Sepharose, hydrophobic interaction chromatography (Phenyl-Sepharose), and gel filtration (12, 13). The Geotrichum candidum lipases I and II have been obtained by ethanol precipitation, gel filtration (Sephacryl-200 HR), anion exchange (Mono Q), and chemofocusing (Polybuffer exchanger 94) (14). A similar scheme has been used to purify multiple isoforms of Candida rugosa lipase (25).
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