Purification and characterization of tert-butyl ester-hydrolyzing lipase from Burkholderia sp. YY62

An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) w as purified to homogeneity from crude cell-free extracts of Burkholderia (f ormerly Pseudomonas) sp, YY62 with 9% overall yield. Seventy-four-fold puri fication was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filt ration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G20 00SW(XL) gel-filtration and phenyl 5PW hydrophobic interaction columns). En zymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO- hydrolyzing activity. The peak preparation showed a single band of 40 kDa o n SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denat ured conditions, indicating the monomeric nature of the TBO-hydrolyzing lip ase. The lipase showed maximum activity at pH 7.0 and 28 degrees C. The N-t erminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-A sn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known prote ins, suggesting that the purified enzyme may belong to a novel class of hyd rolase.