Determination of the extracellular lipases of Pseudomonas fluorescens spp. in skim milk with the beta-naphthyl caprylate assay.
A method based on the hydrolysis of beta-naphthyl caprylate (beta-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0.02 ml raw SM in a 2.0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 micrograms/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of beta-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 degrees C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 degrees C when the cell density exceeded 10(8) colony forming units/ml. The presence of butterfat (3.5%) in the SM assay inhibited B52 lipase by 97%. The beta-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the beta-NC assay may be useful for determining lipase activity in raw SM.
D000818:Animals; D002210:Caprylates / Q000378:metabolism; D002417:Cattle; D005260:Female; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D008049:Lipase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D008892:Milk / Q000201:enzymology / Q000382:microbiology*; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods
D002210:Caprylates; D007202:Indicators and Reagents; C048448:2-naphthyl caprylate; D008049:Lipase