Linoleic acid isomerase from Propionibacterium acnes: purification, characterization, molecular cloning, and heterologous expression.
Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors.
Deng
Ming-De
MD
Bio-Technical Resources, 1035 South 7th Street, Manitowoc, WI 54220, USA. mddeng@yahoo.com
Grund
Alan D
AD
Schneider
Kenneth J
KJ
Langley
Kim M
KM
Wassink
Sarah L
SL
Peng
Susan S
SS
Rosson
Reinhardt A
RA
D000818:Animals; D002849:Chromatography, Gas; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D052200:Limosilactobacillus reuteri / Q000201:enzymology; D019787:Linoleic Acid / Q000378:metabolism*; D044243:Linoleic Acids, Conjugated / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011425:Propionibacterium acnes / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D019745:cis-trans-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D044243:Linoleic Acids, Conjugated; D011994:Recombinant Proteins; D019787:Linoleic Acid; D019745:cis-trans-Isomerases