Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle.
Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.
Park
Doo-Sang
DS
Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Oh
Hyun-Woo
HW
Heo
Sun-Yeon
SY
Jeong
Woo-Jin
WJ
Shin
Dong Ha
DH
Bae
Kyung Sook
KS
Park
Ho-Young
HY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019117:Burkholderia / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D002412:Cations / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D001517:Coleoptera / Q000382:microbiology*; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D013816:Thermodynamics
D002412:Cations; D008049:Lipase