Lipase Activity Assay: Improvements to the Classical p-Nitrophenol Palmitate Spectrophotometrical Method Rafael Picazo Espinosa, University of Granada, Spain rafaelpicazo@hotmail.com Abstract Gupta's lipase activity assay is widely used for checking lipase activity of isolates from different sources (olive oil mill wastewaters, sludge from waste water treatment plants, dairy industry effluents, oil contaminated soils, etc) when seeking for biotechnological applications for lipolytic strains in biofuels production, cosmetics, food and even pharmaceutical industry. However, this assay shows several limitations when the study of lipase activity requires the use of different pH, organic solvents or temperature conditions for one single strain or the comparison of the activity levels of several strains. In the present study, several improvements have been realized so the lipase assay can be applied to the simultaneous study of lipase activity of several bacterial strains under different conditions of pH, temperature and presence of organic solvents. Studied strains' lipases showed different performances at different pH, solvents and temperature conditions, and the most interesting conditions for using the isolates for methanolysis and ethanolysis of fatty acids were determined. Particularly, Microbacterium sp. S18 and Proteus sp. S53 showed a better activity level at pH 5 in presence of methanol, while their performance was quite similar both in ethanol and methanol at pH 8. Besides, feasibility of studied lipases for their application in different biotechnological fields could be sugested from the activity levels in presence of most used organic solvents. Gupta's lipase activity assay limitations Lipase assay developed by Gupta and colaborators is based in an spectrophotometrical method to determine the lipase activity of a bacterial culture throw the quantification of the p-nitrophenol released by lipase enzymes from the substrate p-nitrophenol palmitate (pNPP) (Gupta, et al., 2002). In the normal assay, 1 ml of cell-free medium obtained by centrifugation of an overnight culture is mixed with 9 ml of substrate solution. This reaction mix is incubated at 30ºC for 30 minutes. The reaction is finished by incubation at 100ºC for 4 minutes before the spectrophotometrical determination. Improvements to the protocol Nevertheless, this lipase assay requires relatively long volumes of reagents and cultures, and the prolongated incubation time for each sample creates a bottleneck that reduces the applicability of the method to the study of multiple samples or multiple parameters. Besides, the assay has a low replicability because of p-NPP absorbance changes; as well as Triton X100, p-NPP and arabic gum emulsification problems when pH values are far from standard pH 8. Briefly, the changes made to the original protocol have been: 1.The buffer system includes citrate buffer to adjust pH 5-6, Sorensens' phosphate buffer to adjust pH 7-8, glycine-NaOH buffer to pH 9-10.4. Thus, pH of the reaction mix can be modified according to the needs of the study and the performance of the lipases can be analysed under pH conditions far from the standard and it is possible to make a more realistic prediction of the behaviour of the system where the enzyme will be deployed. 2.The emulsification problems have been fixed by directly disolving Triton X100 in isopropanol (200 µl/ml), resuspending the p-NPP directly with the obtained emulsifying solution (6.6 mg of p-NPP per ml of detergent solution) and suplementing the buffer with arabic gum (11 mg of arabic gum in 20 ml of buffer) prior to mixing it with the p-NPP-isopropanol-Triton solution. After incubating at the desired temperature for 45 minutes, absorbance at 410nm is readed in a microtiter reader. 3.The proportions of the different components of the reaction mix have been modified to improve the replicability and adapt the assay to 96 wells plates. Thus, 1 ml of TritonX100-isopropanol-p-NPP is added t