Purification and biochemical characterization of an extracellular lipase from Pseudomonas fluorescens MTCC 2421

Article abstract of DOI:10.1021/jf803797m

An extracellular lipase produced by Pseudomonas fluorescens MTCC 2421 was purified 184.37-fold with a specific activity of 424.04 LU/mg after anion exchange and gel exclusion chromatography. The enzyme is a homomeric protein with an apparent molecular mass of 65.3 kDa. The lipase exhibited hydrolytic resistance toward triglycerides with longer fatty acyl chain length containing unsaturation as evident from the lower V_max (0.23 mM/mg/min) of the lipase toward glycerol trioleate (C_18:1n9) compared with the fatty acid triglycerides having short to medium carbon chain lengths (C _18:0-12:0, V_max 0.32-0.51 mM/mg/min). This indicates a preferential specificity of the lipase toward cleaving shorter carbon chain length fatty acid triglycerides. The lipase exhibited optimum activity at 40 °C and pH 8.0, respectively. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the thermostability of lipase with a significantly high residual activity (100%) after 30 min at 40 °C, as compared to 90.6% after incubation with Ca²⁺ alone. The lipase activity was inhibited by Cu²⁺ and Fe²⁺ (42 and 48%,respectively) at 10 mM. The enzyme lost 31% of its initial activity by 0.001 mM EDTA and 42% by 0.1 mM EDTA. Significant reduction in lipase activity was apparent by 2-mercaptoethanol and phenylmethanesulfonyl fluoride at diluted concentration (0.001 mM), thereby indicating an important role of sulfhydryl groups in the catalytic mechanism.