Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121–1

The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column. Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates. The optimum temperature was determined to be 52 °C with p-NPB, in the range 50-60 °C with Tween 80 and in the range 50-65 °C with olive-oil emulsion. The optimum pH was determined to be in the pH range 7.2-7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB. The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 °C, pH 7.0-8.2, but was rapidly inactivated at 70-80 °C and at pH 7.0. The lipase was shown to be more thermolabile at 60 °C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after pre-incubating the enzyme for 30 min at 70 °C. The enzyme was found to be pH-tolerant when stored at 20 °C, pH 6.3-10.3 (100 mM Briton-Robson buffer) as the half-life (t 1/2 ) was more than 240 h when p-NPB was used as the substrate. By contrast, the pH-stability range was more narrow (pH 8.0-10.5) with olive-oil emulsion. The effect of various metal ions and EDTA dependedon the nature of the substrate.