EUROPEAN CONGRESS OF CHEMICAL ENGINEERING – ECCE 6 Characterization of an Extracellular lipase from Yarrowia lipolytica A.I.S. Brígida a, P.F.F. Amaral a, J.A.P. Coutinho b, L.R.B. Gonçalvez c, M.A.Z. Coelho a a Departamento de Eng. Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, , R.J., Brasil b CICECO, Departamento de Química, , Universidade de Aveiro, Aveiro, Portugal c Departamento de Eng. Química, Universidade Federal do Ceará, , CE, Brasil INTRODUCTION Lipase (triacylglycerol eser hydrolases, E.C ) constitute a group of enzymes that catalyse lipids hydrolysis as its biological function. Somes studies have reported the yeast Yarrowia lipolytica, formerly known as Candida, Endomycopsis or Saccharomycopsis lipolytica, as a good lipase producer. This yeast is able to produce extracellular, membrane-bound and intracellular lipases encoded by approximating, eighteen genes. Therefore, the aim of this study was to characterize the lipase activity of Yarrowia lipolytica IMUFRJ (Baía de Guanabara isolated strain, Brazil), produced under submerged fermentation in a multiphase reactor, due to its great importance for different applications as catalysts in chemical processes. The lipase extract produced by Y. lipolytica IMUFRJ under different conditions (Pereira-Meirelles et al., 1997) showed a pH range of 6 – 10 and an optimum pH of 9. Assay of hydrolytic activity with methyl butirate: Methyl butyrate hydrolysis was used to determine the esterasic activity of LYL. The reaction was initiated by the addition of 0.1 mL of extract to 30 mL methyl butyrate solution dissolved in 25 mM phosphate buffer pH 7.0. Experiments were performed using an automatic titrator (pHstat) and 50 mM NaOH as titrating agent. Cell Growth: Cell growth was followed by optical density measurements at 570 nm converted to mg.mL -1 using a factor previously established. Glucose: Extracellular glucose concentration was determined by glucose oxidase method (Enzimatic Colorimetric Glucose Assay Kit, HUMAN GmbH - Germany. Assay of hydrolytic activity with p-nitrophenyl laurate (pNPL): The reaction occurred at 37 ºC by the addition of 0.2 mL of enzyme solution to 1.8 mL of 560 μM pNP-laurate dissolved in 50mM potassium-phospate buffer (pH 7.0), containing 1% (v/v) of dimethyl sulfoxide (DMSO). Protein analysis: Protein was estimated by the Folin-Ciocalteau’s phenol reagent as outlined by Lowry et al. (1951), at 660 nm, using bovine serum albumin (BSA) as standard. RESULTS AND DISCUSSION Lipase Production: The lipase extract produced presebted specific activity of 22.4 pNPLU/g protein, determined by p-nitrophenyl laurate (pNPL) hydrolysis. However, for the hydrolysis of methyl butirate, only 3 MBU/mL of extract was found, which is a low activity value for this method. By these results, it is posible to assume that the amount of esterase in the extract is minimum, or null. Therefore, the activity expressed for pNPL hydrolysis is due to lipase presence. RESULTS AND DISCUSSION Figure 1: Effect of pH reaction on activity (a) and stability (b) of Y. lipolytica lipase. Effect of pH on activity and stability of lipase extract: Figure 5: Effect of chemical compounds on activity of lipase extract produced by Y. lipolytica and type B from C. antarctica. Figure 2: Effect of temperature on activity (a) and stability (b) of lipase from Y. lipolytica. Effect of temperature on activity and stability of lipase extract: Comparing LYL with a commercial enzyme (Lipozyme CALB L, Novozyme), both products have the same optimum temperature: 37ºC. The studied extract revealed to be more stable then purified YlLip2 since it is capable to retain 86% of its initial activity for 5 h at 37ºC while YlLip2 retained 83% for 4 h at 35ºC. Effect of substrate concentration: Effect of some additives: Km = mM Km = mM Storage stability: Figure 3: Initial rate of pNPL hydrolysis by lipase from Y. lipolytica and tye B from C. antarctica. Figure 4: Storage stability of crude extract of lipase from Y. lipolytica at -10ºC. CONCLUSIONS The crude lipase extract produced by Y. lipolytica IMUFRJ showed to have similar characteristics to other purified lipase extracts from Y. lipolytica, mainly for YlLip2 strain producer. Nevertheless LYL produced under conditions reported in this paper possess behaviour hardly similar to that obtained by the same strain in different production conditions. MATERIALS AND METHODS Strain, Media and Culture Conditions: Yarrowia lipolytica IMUFRJ a wild type strain isolated from Baía de Guanabara, Rio de Janeiro – Brazil, was kept at 4ºC on YPD-agar medium. Pre-inoculum: Production: 28ºC 1 mg d.w. cells. mL -1 1,500 mL of YPD (w/v: Yeast extract 1%; Peptone 0.64%; Glucose 2%) 20% (v/v) of perfluorodecalin 28ºC 160 rpm 200 mL of YPD medium (w/v: Yeast extract 1%; Peptone 2%; Glucose 2%)