A rapid colorimetric assay for the extracellular lipase of Pseudomonas fluorescens B52 using beta-naphthyl caprylate.
Conditions necessary for the determination of crude extracellular lipase activity from Pseudomonas fluorescens B52 using beta-naphthyl caprylate (beta-NC), an 8-carbon ester, as the substrate were examined. Maximum enzyme synthesis occurred at 20 degrees 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 degrees C. A comparison of beta-NC with beta-naphthyl butyrate (a 4-carbon ester) and beta-naphthyl myristate (a 14-carbon ester) showed that beta-NC was the best substrate; Km values of 0.0415, 0.141 and 0.200 mM and Vmax values of 67.2, 20.1 and 5.28 mumol ml-1 h-1 were obtained for those substrates respectively. The enzyme was inhibited 50% in its activity against beta-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 beta-NC as substrate are consistent with those reported for the lipases of other strains of Ps. fluorescens using natural substrates.
McKellar
R C
RC
D001647:Bile Acids and Salts / Q000494:pharmacology; D002210:Caprylates; D003124:Colorimetry / Q000379:methods; D003470:Culture Media; D007700:Kinetics; D008049:Lipase / Q000032:analysis* / Q000096:biosynthesis; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000254:growth & development; D013696:Temperature
D001647:Bile Acids and Salts; D002210:Caprylates; D003470:Culture Media; C048448:2-naphthyl caprylate; D008049:Lipase