We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.