The hydrolytic activity of Pseudomonas mendocina 3121-1 lipase. A kinetic study

The hydrolytic activity of Ps. mendocina 3121-1 (further marked as Ps. mendocina*) lipase was investigated using p-nitrophenyl butyrate (p-NPB) solutions in selected organic solvents (2-propanol, acetone, 1,4-dioxane and acetonitrile) as well as other soluble p-nitrophenyl fatty acid esters of different chain length in 2-propanol as substrates. In acetone, lower K m (app) and higher V max (app) were determined than in the other organic solvents used. The calculated V max (app) /K m app) ratio was found to be higher in the latter case as well. The k cat (app) of p-NPB hydrolysis in 1,4-dioxane was shown to be nearly 4-fold higher than in other organic solvents, indicating the hydrophilicity of that solvent to be favourable for the hydrolysis. The kinetic parameters of hydrolysis of other fatty acid esters in the same organic solvent (2-propanol) were determined. This enzyme was shown to display highest activity when hydrolysing substrates of a moderate chain length (p-nitrophenyl laurate, p-NPL) at the same concentration of all esters (0.1 mM). Kinetic parameters were also determined for the hydrolysis of all fatty acid esters used, and the calculated parameters were compared with the determined ones. K m (app) was found to be higher for short fatty acid esters, and the lowest characteristics were determined for p-nitrophenyl caprylate (p-NPC), indicating Ps. mendocina* lipase to be more specific to soluble fatty acid esters of moderate and long chains. No strong dependence of lipase K m (app) on fatty acid chain length was determined, while the highest V max (app) was determined for p-NPL and p-nitrophenyl myristate (p-NPM), and the lowest one was shown to be for p-nitrophenyl acetate (p-NPA). The k cat (app) was found to be higher for p-NPC and p-NPL, showing the lipase specificity to fatty acid esters of a moderate chain. The V max (app) /K m (app) ratio was different for all substrates used, but for fatty acid esters of moderate and long chains it by far exceeded the ratio for p-NPA and p-NPB. The determined V max (app) was compared with the calculated parameter at the substrate concentration close to K m (app) , and a significant correlation was found. Analysis of kinetic parameters at the substrate concentration close to K m and 5-fold lower than K m (at the same "kinetic profile") showed the enzyme to be the most active upon p-NPL at "K m " profile, and the general dependence of activity on fatty acid chain length was found to be similar to the depen