Purification and characterization of tert-butyl ester-hydrolyzing lipase from Burkholderia sp. YY62.
An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp. YY62 with 9% overall yield. Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SWXL gel-filtration and phenyl 5PW hydrophobic interaction columns). Enzymes 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 on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase. The lipase showed maximum activity at pH 7.0 and 28 degrees C. The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase.
Yeo
S H
SH
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Nihira
T
T
Yamada
Y
Y
D000595:Amino Acid Sequence; D019117:Burkholderia / Q000737:chemistry / Q000201:enzymology*; D002210:Caprylates / Q000032:analysis / Q000378:metabolism*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009596:Nitrophenols / Q000032:analysis / Q000737:chemistry; D017421:Sequence Analysis