Purification and gene sequencing of conjugated linoleic acid reductase from a gastrointestinal bacterium, Butyrivibrio fibrisolvens.
OBJECTIVE
To characterize the cause for the lack of conjugated linoleic acid (CLA) reductase (CLA-R) activity in the Butyrivibrio fibrisolvens MDT-5 strain that rapidly isomerizes linoleic acid (LA) to CLA without hydrogenation, the CLA-R was purified and its gene (cla-r) sequence was determined.


RESULTS
CLA-R was purified to near homogeneity as a 53-kDa monomeric protein from the high CLA-R activity-expressing strain MDT-10. The purified CLA-R recognized conjugated double bonds. Unsaturated fatty acids containing 18 carbons markedly increased the CLA-R expression at the transcriptional level. Complete sequencing of the cla-r gene revealed that the CLA-R is a novel protein. Sequence analysis of the cla-r gene from the MDT-5 strain revealed that the MDT-5 CLA-R protein sequence differed from that of the MDT-10 at four consecutive amino acids. Northern and Western blotting analyses confirmed that the cla-r mRNA and protein are expressed normally in MDT-5.


CONCLUSIONS
Strain MDT-5 expresses the CLA-R protein that lacks enzyme activity because of mutation, which explains why MDT-5 exclusively produces CLA from LA.


CONCLUSIONS
The cla-r gene was sequenced for the first time. Exogenous fatty acids affected the cla-r transcription. These results will provide additional knowledge on biohydrogenation, and may also augment the CLA production in the gastrointestinal tract.
Fukuda
S
S
Department of Life Science, Meiji University, Kawasaki, Japan.
Suzuki
Y
Y
Komori
T
T
Kawamura
K
K
Asanuma
N
N
Hino
T
T
D000595:Amino Acid Sequence / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D045856:Butyrivibrio / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media; D005231:Fatty Acids, Unsaturated / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D044243:Linoleic Acids, Conjugated / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D005231:Fatty Acids, Unsaturated; D044243:Linoleic Acids, Conjugated; D012329:RNA, Bacterial; D012333:RNA, Messenger; C526119:conjugated linoleic acid reductase, Butyrivibrio fibrisolvens; D044925:Oxidoreductases Acting on CH-CH Group Donors