Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03.
A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.
Ogino
Hiroyasu
H
Department of Chemical Engineering, Osaka Prefecture University, Osaka, Japan. ogino@chemeng.osakafu-u.ac.jp
Mimitsuka
Takashi
T
Muto
Toshiaki
T
Matsumura
Miharu
M
Yasuda
Masahiro
M
Ishimi
Kosaku
K
Ishikawa
Haruo
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005232:Fatty Acids, Volatile / Q000378:metabolism; D005798:Genes, Bacterial; D005990:Glycerol / Q000494:pharmacology; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005227:Fatty Acids; D005232:Fatty Acids, Volatile; D011994:Recombinant Proteins; D014280:Triglycerides; D008049:Lipase; D005990:Glycerol