A rapid colorimetric assay for the extracellular lipase ofPseudomonas fluorescensB52 using β-naphthyl caprylate

Abstract

SUMMARYConditions necessary for the determination of crude extracellular lipase activity fromPseudomonas fluorescensB52 using β-naphthyl caprylate (β-NC), an 8-carbon ester, as the substrate were examined. Maximum enzyme synthesis occurred at 20 °C in pyruvate mineral salts medium containing 1 mM-CaCl2. Bile salts were necessary for enzyme activity; 6 mM-Na taurocholate or Na deoxycholate gave maximum activity but the latter compound was inhibitory at higher concentrations. Activity was optimal in N-Tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid buffer pH 8·0 at 40 °C. A comparison of β-NC with β-naphthyl butyrate (a 4-carbon ester) and β-naphthyl myristate (a 14-carbon ester) showed that β-NC was the best substrate;Kmvalues of 0·0415, 0·141 and 0·200 mM andVmaxvalues of 67·2, 20·1 and 5·28 μmol ml-1h-1were obtained for those substrates respectively. The enzyme was inhibited 50% in its activity against β-NC by 0·009 mM-EDTA, 0–0007% (w/v) mixed alkyltrimethylammonium bromide and 0·00275% (w/v) Triton X-100. The biochemical properties determined using β-NC as substrate are consistent with those reported for the lipases of other strains ofPs. fluorescensusing natural substrates.