Linoleic Isomerase Research Articles

Proteomics and Metabolomics Analysis Reveal the Regulation Mechanism of Linoleate Isomerase Activity and Function in Propionibacterium acnes.

Conjugated linoleic acid (CLA) holds significant application prospects due to its anticancer, anti-atherosclerosis, lipid-lowering, weight-loss, and growth-promoting functions. The key to its efficient production lies in optimizing the biocatalytic performance of linoleic acid isomerase (LAI). Here, we constructed a Propionibacterium acnes mutant library and screened positive mutants with high linoleate isomerase activity. The proteomics and metabolomics were used to explore the mechanism in the regulation of linoleic acid isomerase activity. High-throughput proteomics revealed 104 differentially expressed proteins unique to positive mutant strains of linoleic acid isomerase of which 57 were upregulated and 47 were downregulated. These differentially expressed proteins were primarily involved in galactose metabolism, the phosphotransferase system, starch metabolism, and sucrose metabolism. Differential metabolic pathways were mainly enriched in amino acid biosynthesis, including glutamate metabolism, the Aminoacyl-tRNA biosynthesis pathway, and the ABC transporter pathway. The upregulated metabolites include dl-valine and Acetyl coA, while the downregulated metabolites include Glutamic acid and Phosphoenolpyruvate. Overall, the activity of linoleic acid isomerase in the mutant strain was increased by the regulation of key proteins involved in galactose metabolism, sucrose metabolism, and the phosphotransferase system. This study provides a theoretical basis for the development of high-yield CLA food.

Impact of Lactobacillus-originated metabolites on enterohemorrhagic E. coli in rumen fluid.

Rumen is one of the richest microbial ecosystems naturally harboring many zoonotic pathogens. Controlling the colonization of cattle originated zoonotic pathogens in rumen, particularly enterohemorrhagic Escherichia coli (EHEC), is critical in reducing foodborne enteric diseases in humans. In this study, we aimed to inhibit the growth of EHEC in a simulated rumen system with collected rumen fluids (RFs) using live probiotics, synbiotics, and their metabolites. EHEC inoculated RF was treated with live wild type Lactobacillus casei (LCwt), LCwt with 0.5% peanut flour (LCwt+PF), an engineered LC capable of overexpressing linoleate isomerase (LCCLA), and their metabolites collected in cell-free culture supernatants (CFCSwt, CFCSwt+PF, and CFCSCLA) at various time points. A growth stimulatory effect toward Lactobacillus spp. was exerted by all CFCS, while the EHEC was suppressed. Among other treatments only LCwt+PF reduced EHEC by 2.68 logs after 72h. This observation was also supported by metataxonomic analysis. A reduction in Bacteroidetes and Proteobacteria while increase in Firmicutes was observed at 48h by the presence of CFCSs as compared to the control. Our observation implies probiotic-originated metabolites modulate rumen microbiota positively which can be deployed to control the transmission of cattle-borne pathogens specifically EHEC.

Linoleate Isomerase Complex Contributes to Metabolism and Remission of DSS-Induced Colitis in Mice of Lactobacillus plantarum ZS2058.

A linoleate isomerase complex including myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase, and acetoacetate decarboxylase has been confirmed as the pivotal factor for conjugated linoleic acid (CLA) production in Lactobacillus plantarum. However, its role in the metabolism and health-associated benefits of Lactobacillus remain unclear. In the current study, the mild type, knockout, and complemented mutants of the linoleate isomerase complex of L. plantarum ZS2058 were used to investigate those putative effects. The metabonomic results showed that a linoleate isomerase complex could significantly influence the glycol-metabolism, lipid metabolism, and antioxidant compounds. Especially, with the stress of linoleic acid, linoleate isomerase complex knockout mutants induced the increase of several antioxidant compounds, such as glutamic acid, glycine, l-cysteine, glycerol, and l-sorbosone. Moreover, the linoleate isomerase complex played a pivotal role in ameliorating DSS-induced colitis. The knockout mutants showed effects similar to those in the DSS group, whereas complementation of the corresponding gene in the knockout mutants could restore the anti-inflammatory activity, wherein the integrity of a mucus layer was repaired, the level of pro-inflammatory cytokines decreased, and the amount of anti-inflammatory cytokines increased significantly. All the results indicated that the linoleate isomerase complex plays a key role in CLA production and metabolism as well as the health-associated benefits of L. plantarum ZS2058. These results are conducive to promote clinical trials and product development of probiotics for colitis.

Biotransformation of linoleic acid into different metabolites by food derived Lactobacillus plantarum 12-3 and in silico characterization of relevant reactions

Lactic acid bacteria have been reported to be capable of converting polyunsaturated fatty acids, e.g. linoleic acid (LA) into bioactive and other fatty acid metabolites that are not toxic to the bacteria themselves, but the mechanism of this conversion is not clear. Here we reported for the first time that probiotic L. plantarum 12-3 derived from Tibet kefir when supplemented with LA from 1% to 10% in the MRS medium transformed LA to various fatty acid derivatives. These derivatives formed in the medium were identified with gas chromatography and mass spectrometry. In silico studies were done to confirm the enzymatic reactions responsible for this conversion. We found that L. plantarum 12-3 could convert LA at different concentrations to 8 different fatty acid derivatives. Putative candidate enzymes involved in biotransformation of LA into fatty acid derivatives were identified via whole genome of L. plantarum 12-3, including linoleate isomerase, acetoacetate decarboxylase and dehydrogenase. Therefore, the present study provides further understanding of the mechanism of conversion of LA to health-beneficial fatty acid metabolites in probiotic L. plantarum, which can be explored for potential application in functional foods.

Linoleic acid isomerization ability of Lactobacillus spp. isolated from Vietnamese human intestinal origins

Conjugated linoleic acid (CLA) have been shown to exert numerous health benefits, including anti-carcinogenic, anti-atherogenic, anti-diabetic, antiobesity, cholesterol reducing, antioxidant, anti-microbial, immune system modulator and growth-stimulating properties. In human, CLA is produced from Linoleic acid (LA) by gut bacteria. In this study, nineteen Lactobacillus (Lac.) strains isolated from human feces were studied to determine their ability to metabolize LA. The bacteria were grown in the liquid form of anaerobic MRS medium supplemented with 0.5 mg/mL LA. The linoleate isomerase activity in bacteria grown on MRS medium was determined by Gas chromatograpy. The results indicated that 4 out of 19 strains, including strains Lac.02, Lac.05, Lac.14 and Lac.16 are capable of producing about 40-50 μg/mL CLA from LA. Among them, the highest ability to produce CLA from LA is Lac.02 strain. In the production of CLA from LA, enzymes involved in this metabolism in Lactobacillus act as catalysts of hydration/dehydration (CLA-HY), oxidation of hydroxy groups/reduction of oxo groups (CLA-DH), migration of carbon-carbon double bonds (CLA-DC), and saturation of carbon-carbon double bonds (CLA-ER). The cla-dh, cla-dc, cla-hy and cla-er genes that encode enzymes CLA-DH, CLA-DC, and CLA-ER had been found in all Lac.02, Lac.05, Lac.14 and Lac.16 strains. Gas chromatography traces indicated that these strains produced the same compounds, which was subsequently identified as cis-9, trans-11, and trans-10, cis-12 CLA. In the next study, we will optimize the conditions such as substrate concentrations, pH values, temperature and culture time of each strain to obtain the best rerults.

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli.

Enhancing extracellular metabolic byproducts of probiotics is one of the promising strategies to improve overall host health as well as to control enteric infections caused by various foodborne pathogens. However, the underlying mechanism of action of those metabolites and their effective concentrations are yet to be established. In this study, we determined the antibacterial potential of the metabolites in the cell-free culture supernatant (CFCS) collected from wild-type Lactobacillus casei (LCwt) and genetically modified LC to overexpress linoleate isomerase (LCCLA). We also evaluated the mechanism of action of CFCSs collected from the culture of LCwt in the presence or absence of 0.5% peanut flour (CFCSwt and CFCSwt+PF, respectively) and LCCLA alone (CFCSCLA) against enterohemorrhagic Escherichia coli (EHEC). The metabolites present in CFCSwt+PF and CFCSCLA eliminated EHEC within 24 and 48 h, respectively. Whereas CFCSwt failed to eliminate EHEC but reduced their growth by 6.7 logs (p < 0.05) as compared to the control. Significant downregulation of the expression of cell division gene, ftsZ, supported the observed degree of bactericidal and bacteriostatic properties of the collected CFCSs. Upregulation of EHEC genes related to maintaining cell membrane integrity, DNA damage repair, and molecular chaperons indicated an intensive stress condition imposed by the total metabolites present in CFCSs on EHEC growth and cellular structures. A range of deviated morphological features provoked by the metabolites indicated a membrane-targeted action, in general, to compromise the membrane permeability of EHEC. The information obtained from this study may contribute to a more efficient prevention of EHEC related infections.

Novel Lactobacillus reuteri HI120 Affects Lipid Metabolism in C57BL/6 Obese Mice.

Intestinal probiotics are a primary focus area of current medical research. Probiotics such as bifidobacteria and lactobacilli can positively impact obesity and other metabolic diseases by directly or indirectly affecting lipid metabolism. However, the precise mechanisms of these effects remain unclear. In our previous work, the novel strain Lactobacillus reuteri HI120 was isolated and identified. HI120 expresses high levels of linoleic isomerase, resulting in the production of large amounts of conjugated linoleic acid (CLA) when mixed with linoleic acid (LA). As HI120 can efficiently transform LA into CLA, the effect of HI120 on the lipid metabolism in C57BL/6 obese mice was studied and the underlying molecular mechanism was explored in vitro. The results revealed no significant change in the diet, body weight, and serum triglyceride levels in mice. However, serum cholesterol levels were significantly decreased. The underlying mechanism may involve a CLA-mediated reduction in the gene expression levels of NPC1L1, SREBP-2, and HMG-CR, resulting in reduced cholesterol synthesis and absorption. Thus, HI120 can be developed as a potential probiotic formulation. After oral administration, LA from certain food sources can be converted into CLA in the human intestine to contribute to the prevention and treatment of obesity and hyperlipidemia.

CONVERSION OF LINOLEIC ACID TO DIFFERENT FATTY ACID METABOLITES BY Lactobacillus Plantarum 13-3 AND IN SILICO CHARACTERIZATION OF THE PROMINENT REACTIONS

Lactobacillus plantarum strains have been extensively used in food processing and preservation. L. plantarum also has the potential to convert polyunsaturated fatty acids, e.g. linoleic acid (LA) into bioactive and less toxic fatty acid metabolites. The objective of this study was to assess the capability of probiotic L. plantarum 13-3 to convert Linoleic Acid (LA) to different fatty acid metabolites in the medium supplemented with differential concentrations of LA, and the relevant reactions were characterized by in silico calculation. L. plantarum 13-3 was grown in MRS medium containing LA from 1% to 10%, and the fatty acid metabolites formed in the medium were identified and quantitated by GC-MS and in silico studies were done to confirm the enzymatic reactions involved in its conversion. The results showed that L. plantarum 13-3 could convert LA at different concentrations to 5 different fatty acid metabolites i.e, (Z)-Ethyl heptadec-9-enoate, 9,12-Octadecadienoic acid (Z, Z), methyl ester, Octadec-9-enoic acid, cis-11,14-Eicosadienoic acid, methyl ester and (Z)-18-Octadec-9-enolide. Among these metabolites, the formation of an long chain fatty acid Octadec-9-enoic Acid, also known as 18:1, N-9 or Delta(9)-Octadecenoic acid, is classified as a member of the Long-chain fatty acids in media supplemented with 4% to 10% LA, is being reported for the first time. Putative candidate enzymes involved in biotransformation of LA into fatty acid metabolites were identified in whole genome of L. plantarum 13-3, sequenced previously. In silico studies confirmed that several enzymes including the linoleate isomerase, acetoacetate decarboxylase and oxidoreductase may be involved in biotransformation of LA into fatty acid metabolites. These enzymes could effectively bind the LA molecule mainly by forming hydrogen bonding between the acidic groups of LA and the proline residues at the active sites of the enzymes validating the putative reacting partners.

Dose-dependent production of linoleic acid analogues in food derived Lactobacillus plantarum K25 and in silico characterization of relevant reactions

The objective of this study was to assess and scrutinize the competency of probiotic L. plantarum K25 to produce linoleic acid analogues in the medium supplemented with different concentrations of linoleic acid, ranging from 1% to 10%, in a dose dependent manner. The analogues produced were identified and quantitated by GC-MS and in silico studies were done to confirm enzymatic reactions involved in its conversion. The results showed that L. plantarum K25 could convert linoleic acid at different concentrations to 9 different fatty acid analogues at concentrations ranging from 0.01 to 17.24 mg/L. Among these metabolites, formation of an essential fatty acid, the linolenic acid, in media supplemented with 9% linoleic acid, is being reported for the first time. Putative candidate enzymes involved in biotransformation of linoleic acid into linoleic acid analogues were identified in the whole genome of L. plantarum K25, which was sequenced previously. In silico studies confirmed that many enzymes, including linoleate isomerase and dehydrogenase, may be involved in biotransformation of linoleic acid into linoleic acid analogues. Both enzymes could effectively bind the linoleic acid molecule, mainly by forming hydrogen bonding between the acidic groups of linoleic acid and the proline residues at the active sites of the enzymes, validating putative reaction partners.

In silico characterization of linoleic acid biotransformation to rumenic acid in food derived Lactobacillus plantarum YW11.

Lactobacillus plantarum YW11 capability to convert linoleic acid into conjugated linoleic acid and other metabolites was studied in a dose-dependent manner by supplementing LA at different concentrations. L. plantarum YW11 displayed a uniform distinctive growth curve of CLA and other metabolites at concentrations of LA ranging from 1% (w/v) to 10% (w/v), with slightly increased growth at higher LA concentrations. The biotransformation capability of L. plantarum YW11 evaluated by GC-MS revealed a total of one CLA isomer, i.e. 9-cis,11-trans-octadecadienoic acid, also known as the rumenic acid (RA), one linoleic acid isomer (linoelaidic acid), and LA metabolites: (E)-9-octadecenoic acid ethyl ester, trans, trans-9,12-octadecadienoic acid, propyl ester and stearic acid. All the metabolites of linoleic acid were produced from 1 to 10% LA supplemented MRS media, while surprisingly the only conjugated linoleic acid compound was produced at 10% LA. To assess the presence of putative enzymes, responsible for conversion of LA into CLA, in silico characterization was carried out. The in silico characterization revealed presence of four enzymes (10-linoleic acid hydratase, linoleate isomerase, acetoacetate decarboxylase and dehydrogenase) that may be involved in the production of CLA (rumenic acid) and LA isomers. The biotransformation ability of L. plantarum YW11 to convert LA into RA has great prospects for biotechnological and industrial implications that could be exploited in the future scale-up experiments.

Conjugated linoleic acid producing potential of lactobacilli isolated from goat (AXB) rumen fluid samples.

ObjectiveThe present investigation was aimed to explore the potential of lactobacilli for conjugated linoleic acid (CLA) production, isolated from rumen fluid samples of lactating goats.MethodsA total of 64 isolates of lactobacilli were obtained using deMan-Rogosa-Sharpe (MRS) agar from rumen fluid of goats and further subjected to morphological and biochemical characterizations. Isolates found as gram-positive, catalase negative rods were presumptively identified as Lactobacillus species and further confirmed by genus specific polymerase chain reaction (PCR). The phylogenetic tree was constructed from the nucleotide sequences using MEGA6.ResultsOut of the 64 isolates, 23 isolates were observed positive for CLA production by linoleate isomerase gene-based amplification and quantitatively by UV-spectrophotometric assay for the conversion of linoleic acid to CLA as well as gas chromatography-based assay. In all Lactobacillus species cis9, trans11 isomer was observed as the most predominant CLA isomer. These positive isolates were identified by 16S rRNA gene-based PCR sequencing and identified to be different species of L. ingluviei (2), L.salivarius (2), L. curvatus (15), and L. sakei (4).ConclusionThe findings of the present study concluded that lactic acid bacteria isolated from ruminal fluid samples of goat have the potential to produce bioactive CLA and may be applied as a direct fed microbial to enhance the nutraceutical value of animal food products.

The first complete genomic structure of Butyrivibrio fibrisolvens and its chromid

Butyrivibrio fibrisolvens forms part of the gastrointestinal microbiome of ruminants and other mammals, including humans. Indeed, it is one of the most common bacteria found in the rumen and plays an important role in ruminal fermentation of polysaccharides, yet, to date, there is no closed reference genome published for this species in any ruminant animal. We successfully assembled the nearly complete genome sequence of B. fibrisolvens strain INBov1 isolated from cow rumen using Illumina paired-end reads, 454 Roche single-end and mate pair sequencing technology. Additionally, we constructed an optical restriction map of this strain to aid in scaffold ordering and positioning, and completed the first genomic structure of this species. Moreover, we identified and assembled the first chromid of this species (pINBov266). The INBov1 genome encodes a large set of genes involved in the cellulolytic process but lacks key genes. This seems to indicate that B. fibrisolvens plays an important role in ruminal cellulolytic processes, but does not have autonomous cellulolytic capacity. When searching for genes involved in the biohydrogenation of unsaturated fatty acids, no linoleate isomerase gene was found in this strain. INBov1 does encode oleate hydratase genes known to participate in the hydrogenation of oleic acids. Furthermore, INBov1 contains an enolase gene, which has been recently determined to participate in the synthesis of conjugated linoleic acids. This work confirms the presence of a novel chromid in B. fibrisolvens and provides a new potential reference genome sequence for this species, providing new insight into its role in biohydrogenation and carbohydrate degradation.

Bioconversion of conjugated linoleic acid by Lactobacillus plantarum CGMCC8198 supplemented with Acer truncatum bunge seeds oil.

Conjugated linoleic acid (CLA) isomers, c9, t11-CLA and t10, c12-CLA, have been proved to exhibit excellent biomedical properties for potential use in anti-cancer applications and in reducing obesity. Acer truncatum Bunge (ATB), which is rich in unsaturated fatty acids, including oleic acid, linoleic acid, and nervonic acid, is a new resource for edible oil. In the present study, we developed a new method for producing two CLA isomers from ATB-seed oil by fermentation using Lactobacillus plantarum CGMCC8198 (LP8198), a novel probiotics strain. Polymerase chain reaction results showed that there was a conserved linoleate isomerase (LIase) gene in LP8198, and its transcription could be induced by ATB-seed oil. Analyses by gas chromatography-mass spectrometry showed that the concentration of c9, t11-CLA and t10, c12-CLA in ATB-seed oil could be increased by about 9- and 2.25-fold, respectively, after being fermented by LP8198.

Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation.

To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058. CLA has attracted great interests for decades due to its health-associated benefits including anticancer, anti-atherogenic, anti-obesity and modulation of the immune system. A number of microbial CLA producers were widely reported including lactic acid bacteria. Lactobacillus plantarum ZS2058, an isolate from Chinese traditional fermented food, could convert LA to CLA with various intermediates. To characterize the genetic determinants for generating CLA, a cre-lox-based system was utilized to delete the genes encoding myosin cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC) in Lact. plantarum ZS2058, respectively. Neither intermediate was detected in the corresponding gene deletion mutant. Meanwhile all those mutants could recover the ability to convert linoleic acid to CLA when the corresponding gene was completed. The results indicated that CLA production was a multiple-step reaction catalysed by triple-component linoleate isomerase system encoded by mcra, dh and dc. Multicomponent linoleic acid isomerase provided important results for illustration unique mechanism for CLA production in Lact. plantarum ZS2058. Lactobacilli with CLA production ability offer novel opportunities for functional food development.

Production of conjugated linoleic acid and gamma-aminobutyric acid by autochthonous lactic acid bacteria and detection of the genes involved

In this study, 85 strains of lactic acid bacteria isolated from artisanal cheeses were screened to determine their capacity to synthesise bioactive compounds.Four Lactobacillus plantarum and two Lactobacillus casei subsp. casei strains were capable of synthesizing conjugated linoleic acid (CLA). The highest level of CLA formed in MRS after 48h incubation was 19.26µg/mL. Six Lactobacillus brevis and four Lactococcus lactis subp. lactis strains produced gamma-aminobutyric acid (GABA) and the highest concentration (2524.05µg/mL) was found after 72h incubation.The detection of genes encoding linoleate isomerase or glutamate decarboxylase system could be suitable for use as screening method of CLA or GABA-producer strains, although this should be studied in a larger number of strains.Our findings suggest that the above strains are potential candidates for the design of starter cultures with the capacity to generate bioactive compounds, offering new possibilities for the manufacture of functional dairy products.

Conjugated Linolenic Acid Production by Lactobacilli Isolated from Human Milk, Feces and Fermented Dairy Products

Background: Many investigations have attributed bio-functional properties to a range of conjugated fatty acids (CFAs) of which the conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers have displayed potent health beneficial effects in a large number of in vitro and in vivo studies. CLNA isomers are naturally present in high concentrations in a variety of seed oils but relatively in small amount in meat and dairy products. However, some reports have shown the microbial production of such enzymes by lactic acid bacteria that convert linolenic acid (LNA) into CLNA. Present study aims to isolate CLNA producing lactobacilli of different origins and their potential to produce different CLNA isomers in MRS media. Keywords: Lactobacilli, conjugated fatty acid, conjugated linolenic acid, α-linolenic acid and conjugated linoleic acid, linoleate isomerase gene.

CONJUGATED LINOLEIC ACID IN MILK AND FERMENTED MILKS: VARIATION AND EFFECTS OF THE TECHNOLOGICAL PROCESSES

Background: Conjugated linoleic acid (CLA) is a generic term used to describe a group of geometric and positional isomers of linoleic acid with a conjugated double bond system. CLA-isomers have been widely studied because of their important biological activity and their protective effects against several diseases, such as obesity, atherosclerosis, chronic inflammatory diseases, and cancer. Consequently, these biomolecules have attracted much attention from the dairy industry, since they are naturally found in ruminants’ milk, and because the development of CLA-enriched dairy foods can be a good economic opportunity given the growth of the functional foods market, in which the dairy industry plays an important role. Objectives: This work presents a comprehensive review of the following aspects: (i) The synthesis and concentration of the CLA-isomers in milk, and the main strategies employed to increase their content in a natural manner; (ii) The influence of the main technological treatments applied to milk on the concentration of CLA-isomers; and (iii) The effects of milk fermentation on the content of CLA-isomers, and the challenges of this technological process, which has been though as a promissory alternative to naturally increase the content of CLA in fermented dairy products. Methods: Information available in various databases was reviewed. A total of 103 articles were selected on the basis of their relevance and scientific-technical quality. Results: The CLA concentration in cows’ milk normally ranges between 2 and 37 mg/g fat, and is mainly affected by the dietary regime offered to the animals. From the total CLA-isomers, rumenic acid represents between 75 and 90%. The technological processes normally applied to milk (thermal processing, high pressure processing, and fermentation) might cause slight changes on the CLA concentration, but the mechanisms causing these changes have not been still established. The increase in CLA concentration by milk fermentation is strain-dependent, because of the different linoleate isomerase activity of the species. . Conclusions: Although several studies have reported increases in the concentration of CLA in milk and fermented milks in a natural manner, they are fairly moderate, and the obtained levels of CLA are significantly lower than those recommended to achieve therapeutic effects.

Production of bioactive conjugated linoleic acid by the multifunctional enolase from Lactobacillus plantarum

Lactobacillus plantarum α-enolase, a multifunctional-anchorless-surface protein belonging to the conserved family of enolases with a central role in glycolytic metabolism, was characterized to have a side role in the intricate metabolism of biohydrogenation of linoleic acid, catalyzing the formation of bioactive 9-cis-11-trans-CLA through dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid. The identity of the enolase was confirmed through mass spectrometric analysis that showed the characteristic 442 amino acid sequence with a molecular mass of 48.03kDa. The enolase was not capable of using linoleic acid directly as a substrate but instead uses its hydroxyl derivative 10-hydroxi-12-cis-octadecenoic acid to finally form bioactive conjugated linoleic acid. Biochemical optimization studies were carried out to elucidate the conditions for maximum production of 9-cis-11-trans-CLA and maximum stability of α-enolase when catalyzing this reaction. Furthermore, through structural analysis of the protein, we propose the binding sites of substrate and product molecules that were characterized as two hydrophobic superficial pockets located at opposite ends of the enolase connected through a channel where the catalysis of dehydration and isomerization might occur. These results prove that multifunctional α-enolase also plays a role in cell detoxification from polyunsaturated fatty acids such as linoleic acid, along with the linoleate isomerase complex.

A directed mutational approach demonstrates that a putative linoleate isomerase from Lactobacillus acidophilus does not hydrate or isomerize linoleic acid

Catalytic promiscuity of enzymes is a field of general interest and the application of enzymes, catalyzing more than one chemical reaction has also industrial relevance. Furthermore, comparing the evolutionary relationship in protein families serves as basis for their engineering and adaption in practical applications. Oleate hydratases (EC 4.2.1.53) and linoleate isomerases (EC 5.2.1.5) act on related substrates and share substantial sequence similarity, but catalyze different transformations. The intrinsic cofactor FAD is found in both enzyme classes, but seems to perform different functions: for isomerases a redox‐based mechanism was identified and for hydratases only a structural or charge stabilizing function was suggested. Several fatty acid converting reactions were found in Lactobacillus acidophilus including oleate hydratase and linoleate isomerase activity. Two genes that may encode these two enzymes were identified (GenBank AAV42528.1 and ABB43157.1), cloned, and the corresponding proteins were produced in E. coli, purified, and investigated. This revealed that only the oleate hydratase activity could be confirmed. As both enzymes differ by only three amino acids, a series of mutants was also created and investigated to shed light on their promiscuous behavior.Practical applications: Bacterial isomerization or hydration of cis‐double bonds leading to the formation of trans‐double bonds or hydroxyl groups within fatty acids has been recognized to be useful for biocatalysis, since no additional cofactor recycling is required despite the presence of the cofactor FAD.Lactobacillus acidophilus can convert unsaturated fatty acids to different products through an oleate hydratase and linoleate isomerase activity. The corresponding annotated proteins may differ by only three amino acids. This study, however, confirmed only one as an oleate hydratase, but the identification of the activity of the second protein remains elusive.

Dietary supplementation of Butyrivibrio fibrisolvens alters fatty acids of milk and rumen fluid in lactating goats.

Conjugated linoleic acid (CLA) isomers have high health amelioration potential and hence it is of great interest to increase the CLA content in dairy products. The present study was conducted to investigate the effect of administration of high CLA producing Butyrivibrio fibrisolvens In-1 on fatty acid composition of milk and rumen fluid in lactating goats. Four groups (n = 5) of lactating goats were assigned the following treatments: Control (C) (basal diet); T1 (basal diet + linoleic acid source), T2 (basal diet + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)) and T3 (basal diet + linoleic acid source + suspension of Butyrivibrio fibrisolvens In-1, 10(9) CFU head(-1)). Rumen liquor and milk samples were collected on days 0, 15, 30, 60 and 90 of the experiment and linoleic isomerase enzyme (LA-I) activity and fatty acid profiles were elucidated. Major effects of treatments were seen on day 30 of the experiment. Total CLA content of rumen fluid increased (P < 0.05) by 218.72, 182.26 and 304% whereas total saturated fatty acid (SFA) content was lowered (P < 0.05) by 6.1, 4.44 and 9.55% in T1, T2 and T3, respectively, as compared to control. Vaccenic acid in groups T2 and T3 increased (P < 0.05) by 66.67% and 105.7% as compared to control. In milk, total CLA increased by 2.03, 1.61 and 0.61 folds in T3, T2 and T1, respectively. Total monounsaturated fatty acid and polyunsaturated fatty acid content increased (P < 0.05) in group T3 by 14.15 and 37.44%, respectively. Results of the present study indicated that administration of B. fibrisolvens In-1 along with a linoleic acid (LA) source is a useful strategy to alter the biohydrogenation pattern in the rumen that subsequently decreased SFA content while increased CLA and unsaturated fatty acids in ruminant's milk.