Purification and characterization of a mesophilic organic solvent tolerant lipase produced by Acinetobacter sp. K5b4

The extracellular lipase produced by Acinetobacter sp. K5b4 was purified to homogeneity using ultrafiltration (cutoff 30?KDa) followed by gel filtration chromatography on Sephadex G-50. The enzyme was purified to homogeneity with an apparent molecular mass of 133?KDa by SDS-PAGE. This purification resulted on 10.24 fold with 18.3% recovery. The K_m and V_max of purified enzyme when using pNPL hydrolysis were 4.0?mM and 73.53?nmol/ml/min, respectively. The pure enzyme was greatly stimulated in the presence of 20, 40 and 60% (v/v) methanol, DMSO and acetone whereas, ethanol, acetonitrile and propanol decreased the enzyme activity. Maximum enzyme activity was achieved at pH 7.0 and incubation temperature of 27?°C. The enzyme was stable within a pH range of 6.5 to 7 at 27?°C for 1?h. The enzyme activity was enhanced up to 36% by KCl, BaCl₂, MgCl₂ and CaCl₂ while obviously inhibited (10–20%) by CoCl₂, ZnCl₂, MnCl₂ and CuCl₂. No inhibitory effects were observed with 1.0 and 5.0?mM of 2-mercaptoethanol and EDTA. Similarly, SDS at 1.0?mM does not affect the enzyme activity while high reduction (80%) was observed at 5.0?mM SDS concentration. The enzyme was active against p-nitrophenyl esters of C8, C12 and C16 with highest preference to the medium carbon chain p-nitrophenyl caprylate (C8). The fact that the enzyme displays distinct stability in the presence of methanol, DMSO and acetone suggests that this lipase is suitable as biocatalyst in organic synthesis where such hydrophilic organic solvents are used as a reaction media.