10-hydroxystearic Acid Research Articles

Continuous Flow Microreactors for the High-efficiency Enzymatic Synthesis of 10-Hydroxystearic Acid from Oleic Acid.

Converting fatty acids into specialty chemicals is sustainable but hindered by the low efficiency and thermal instability of current oleic acid hydratases, along with mass transfer limitations in emulsion reactions. This study introduces an optimized continuous flow micro-reactor (CFMR) that efficiently transforms oleic acid at low (15 g L-1) and high (50 g L-1) concentrations, improving reaction efficiency and overcoming key conversion barriers. The first CFMR model showed reaction speeds surpassing traditional batch stirred tank reactors (BSTR). Optimizations were performed on three key components: liquid storage, mixer, and reaction section of the CFMR, with each round's best conditions carried into the next. This achieved a space-time yield of 597 g L-1 d-1 at a 15 g L-1 oleic acid load. To further enhance the yield, we optimized the emulsifier system to solve incomplete emulsification and developed a two-component feed microreactor (TCFMR) that addressed mass transfer limitations caused by the product at high substrate loads, reaching a 91 % conversion of 50 g L-1 oleic acid in 30 minutes, with a space-time yield of 2312 g L-1 d-1. These advancements represent significant progress in utilizing fatty acids and advancing sustainable chemical synthesis.

Immobilization study of a monomeric oleate hydratase from Rhodococcus erythropolis

AbstractThe chemical, pharmaceutical, and cosmetic industries are currently confronted with the challenge of transitioning from traditional chemical processes to more sustainable biocatalytic methods. To support that aim, we developed various heterogeneous biocatalysts for an industrially relevant enzyme called oleate hydratase that converts oleic acid to 10-hydroxystearic acid, a fatty emollient substance useful for various technical applications. We used cheap support matrices such as silica, chitosan, cellulose, and agarose for further scale-up and economic feasibility at the industrial level alongside more sophisticated supports like metal–organic frameworks. Different physical and chemical binding approaches were employed. Particularly, by immobilizing oleate hydrates on a 3-aminopropyltriethoxysilane surface-functionalized cellulose matrix, we developed an enzyme immobilizate with almost 80% activity of the free enzyme. The long-term goal of this work was to be able to use the developed heterogeneous biocatalyst for multiple reuse cycles enabling profitable biocatalysis. Despite high initial conversion rate by the developed cellulose-based immobilizate, a depletion in enzyme activity of immobilized oleate hydratase was observed over time. Therefore, further enzyme modification is required to impart stability, the optimization of operational conditions, and the development of carrier materials that enable economical and sustainable enzymatic conversion of oleic acid to meet the commercial demand. Graphical abstract

Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

Hydroxylation of Fatty Acids by Lactic Acid Bacteria.

Hydroxy fatty acids (HFAs) are fatty acids with hydroxyl functional groups attached to the main chain. HFAs are used in widely diverse industrial applications, healthy functional foods, artificial food flavorings, and alcoholic beverages. A lactic acid bacterium (LAB), Lactobacillus sakei, hydroxylates oleic acid. Furthermore, the hydroxyl fatty acid was identified by GC-MS as 10-hydroxystearic acid. The Lactobacillus sakei hydroxylated more than 90% of the oleic acid in the medium at 15°C after 30-48h. The hydroxyl enzyme needs a coenzyme for an electron donor as NADPH. The enzyme is useful for assay with monitoring NADPH concentration used an A340 device. The hydroxylate fatty acids are converted by LAB lactonize aroma lactone from commercial yeast strains, which can be detected directly by scent. Commercial beer brewing yeast T-58 produced the highest concentration of aroma lactone from hydroxyl fatty acids. Furthermore, the aroma lactone is identified by GC-MS as gamma-dodecalactone. The ratio of conversion is 87%. These results suggest that the lactonization conversion system is useful to hydroxylate fatty acids for alcoholic beverages.

Ultrastable and Responsive Foams Based on 10-Hydroxystearic Acid Soap for Spore Decontamination.

Currently, there is renewed interest in using fatty acid soaps as surfactants. Hydroxylated fatty acids are specific fatty acids with a hydroxyl group in the alkyl chain, giving rise to chirality and specific surfactant properties. The most famous hydroxylated fatty acid is 12-hydroxystearic acid (12-HSA), which is widely used in industry and comes from castor oil. A very similar and new hydroxylated fatty acid, 10-hydroxystearic acid (10-HSA), can be easily obtained from oleic acid by using microorganisms. Here, we studied for the first time the self-assembly and foaming properties of R-10-HSA soap in an aqueous solution. A multiscale approach was used by combining microscopy techniques, small-angle neutron scattering, wide-angle X-ray scattering, rheology experiments, and surface tension measurements as a function of temperature. The behavior of R-10-HSA was systematically compared with that of 12-HSA soap. Although multilamellar micron-sized tubes were observed for both R-10-HSA and 12-HSA, the structure of the self-assemblies at the nanoscale was different, which is probably due to the fact that the 12-HSA solutions were racemic mixtures, while the 10-HSA solutions were obtained from a pure R enantiomer. We also demonstrated that stable foams based on R-10-HSA soap can be used for cleaning applications, by studying spore removal on model surfaces in static conditions via foam imbibition.

Regio- and stereoselective biocatalytic hydration of fatty acids from waste cooking oils en route to hydroxy fatty acids and bio-based polyesters

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefinery feedstocks, which contribute to a reduced impact on the environment. Frequently, this waste is wrongly disposed of into municipal sewage systems, thereby creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs, which were intended for the production of biofuels, were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)− 10-hydroxystearic acid methyl ester that was further purified with a high purity (> 90%), at gram scale. The purified (R)− 10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction to produce the corresponding polyester. This work highlights the potential of waste products to obtain bio-based hydroxy fatty acids and polyesters through a biorefinery approach.

Targeting intestinal flora and its metabolism to explore the laxative effects of rhubarb.

Rhubarb, a traditional herb, has been used in clinical practice for hundreds of years to cure constipation, but its mechanism is still not clear enough. Currently, growing evidence suggests that intestinal flora might be a potential target for the treatment of constipation. Thus, the aim of this study was to clarify the laxative effect of rhubarb via systematically analyzing the metagenome and metabolome of the gut microbiota. In this study, the laxative effects of rhubarb were investigated by loperamide-induced constipation in rats. The gut microbiota was determined by high-throughput sequencing of 16S rRNA gene. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used for fecal metabolomics analysis. The data showed that rhubarb could significantly shorten gastrointestinal transit time, increase fecal water content and defecation frequency, improve gastrointestinal hormone disruption, and protect the colon mucus layer. Analysis of 16S rRNA gene sequencing indicated that rhubarb could improve the disorder of intestinal microbiota in constipated rats. For example, beneficial bacteria such as Ligilactobacillus, Limosilalactobacillus, and Prevotellaceae UCG-001 were remarkably increased, and pathogens such as Escherichia-Shigella were significantly decreased after rhubarb treatment. Additionally, the fecal metabolic profiles of constipated rats were improved by rhubarb. After rhubarb treatment, metabolites such as chenodeoxycholic acid, cholic acid, prostaglandin F2α, and α-linolenic acid were markedly increased in constipation rats; in contrast, the metabolites such as lithocholic acid, calcidiol, and 10-hydroxystearic acid were notably reduced in constipation rats. Moreover, correlation analysis indicated a close relationship between intestinal flora, fecal metabolites, and biochemical indices associated with constipation. In conclusion, the amelioration of rhubarb in constipation might modulate the intestinal microflora and its metabolism. Moreover, the application of fecal metabolomics could provide a new strategy to uncover the mechanism of herbal medicines.Key points• Rhubarb could significantly improve gut microbiota disorder in constipation rats.• Rhubarb could markedly modulate the fecal metabolite profile of constipated rats.

Identification of New Microbial Isolates Capable of Transforming Oleic Acid to Solely 10‐Hydroxystearic Acid

Hydroxy fatty acids have a wide range of industrial applications including production of lubricants, plastics, waxes, cosmetics, and antimicrobial agents. Many plant oils contain a small amount of hydroxy fatty acids but are rich in oleic acid (OA) and linoleic acids. These fatty acids can be converted to hydroxy fatty acids by microorganisms with low to moderate yields. In some cases, OA can be converted to a mixture of 10-hydroxystearic acid (10-HSA) and 10-ketostearic acid (10-KSA). Oleate hydratase and secondary alcohol dehydrogenase (2o-ADH) are responsible for converting OA to 10-HSA and then 10-KSA, respectively. We are interested in obtaining microorganisms that can convert OA to solely 10-HSA without 10-KSA to eliminate downstream separation and purification of 10-HSA. While using CRISPR/Cas9 genome editing technology to knockout the 2o-ADH gene of Nocardia cholesterolicum NRRL5767 to obtain mutants that convert OA solely to 10-HSA (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230915), we also isolated three new microorganisms from soil of corn and soybean fields that convert OA solely to 10-HSA in high yield. Here we report isolation and identification of three new Isolates capable of transforming OA to solely 10-HSA. These microbial isolates were identified by Gram Stain and 16S rRNA gene sequencing. We have successfully amplified a ~1500 bp region in the 16S rRNA using 27F and 1492R primer pair from the genome of these isolates by PCR. The sequence of the entire fragment was determined on both strands by Sanger's Chain Termination method. The resulting nucleotide sequence was then used to search similar sequences in rRNA/ITS databases using nucleotide BLAST. We were able to identify the genus of these three microbes confidently.

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C10-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s−1 mM−1 to 119 s−1 mM−1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L−1 day−1 and 160 g L−1 day−1, respectively.

Recombinant Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: Enzyme Expression and Design of a Reliable Experimental Procedure for the Stereoselective Hydration of Oleic Acid

Different microbial strains are able to transform oleic acid (OA) into 10-hydroxystearic acid (10-HSA) by means of the catalytic activity of the enzymes oleate hydratase (EC 4.2.1.53). Lactobacillus rhamnosus ATCC 53103 performs this biotransformation with very high stereoselectivity, affording enantiopure (R)-10-HSA. In this work, we cloned, in Escherichia coli, the oleate hydratase present in the above-mentioned probiotic strain. Our study demonstrated that the obtained recombinant hydratase retains the catalytic properties of the Lactobacillus strain but that its activity was greatly affected by the expression procedure. According to our findings, we devised a reliable procedure for the hydration of oleic acid using a recombinant E. coli whole-cell catalyst. We established that the optimal reaction conditions were pH 6.6 at 28 °C in phosphate buffer, using glycerol and ethanol as co-solvents. According to our experimental protocol, the biocatalyst does not show significant substrate inhibition as the hydration reaction can be performed at high oleic acid concentration (up to 50 g/L).

A Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of Free Hydroxy Fatty Acids in Cow and Goat Milk.

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further β-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers’ point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of β-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

Exploring the abundance of oleate hydratases in the genus Rhodococcus\u2014discovery of novel enzymes with complementary substrate scope

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicumNRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5’ coding region and the 5’ upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Urban sewage scum and primary sludge as profitable sources of biodiesel and biolubricants of new generation

Lipids of sewage scum and primary sludge taken from several wastewater treatment plants were quantified and characterised. In sewage scum, lipids represented 36–50% of total solids and were primarily composed of free fatty acids (45–60%) and calcium soaps (27–35%). In primary sludge, total lipids were 20–24% of total solids, and 71–82% of these were calcium soaps. Estolides and 10-hydroxystearic acid (prevalently present as R enantiomer, with an enantiomeric excess >92%) were also identified and quantified. A scheme of valorisation was then specifically designed and positively tested for both the sludge. Lipids were first recovered (92–99%), activated and finally reacted with methanol and AlCl3∙6H2O (343 K, 2 h, yield >96%). Besides biodiesel, methyl estolides and methyl 10-hydroxystearate were efficiently isolated and purely separated in different fractions. A preliminary feasibility study was finally conducted and a possible integration of processes into a wastewater treatment plant was proposed and positively evaluated.

Sustainable Biotransformation of Oleic Acid to 10-Hydroxystearic Acid by a Recombinant Oleate Hydratase from Lactococcus garvieae

Enzymatic hydration of oleic acid into 10-hydroxystearic acid (10-HSA) represents a theme of substantial scientific and practical interest. In this study, a fatty acid hydratase (OHase) from Lactococcus garvieae was cloned and expressed in Escherichia coli. The recombinantly expressed enzyme was identified as oleate hydratase (EC 4.2.1.53) confirming its highest hydration activity for oleic acid. The optimally yielded enzyme fraction was purified and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A solitary band on SDS-PAGE confirmed the molecular weight of 65 kDa. Gas chromatography-mass spectrometry (GC-MS) analysis scrutinized the silylated hydroxy fatty acid products acquired from the hydration of oleic acid by the oleate hydratase from L. garvieae. Optimal reaction conditions for the enzymatic production of 10-HSA from oleic acid using the purified oleate hydratase were pH 7.5, 30 °C, 105.49 U/mL enzyme solution and 30 g/L oleic acid. In the presence of activity stimulators, that is, magnesium (II) (Mg2+), the oleate hydratase activity was found to be greatly improved at 30 °C. In conclusion, the results revealed the potential efficacy of recombinant enzyme for the biotechnological conversion of oleic acid to 10-HSA acid with high efficiency. The results would be useful for the improved industrial-scale biosynthesis of 10-HSA via an economical and environmentally friendly bioprocess approach.

Enzymatic synthesis of 10-oxostearic acid in high space-time yield via cascade reaction of a new oleate hydratase and an alcohol dehydrogenase

Hydroxy fatty acids and carbonyl fatty acids are important multi-functional materials for the manufacture of fragrances and other fine chemicals. In this study, a novel oleate hydratase (PaOH) was cloned from Paracoccus aminophilus DSM 8538 and solubly expressed in Escherichia coli. The recombinant PaOH efficiently catalyzed the hydration of oleic acid, with a specific activity of 5.21 U mg−1 protein. Enzymatic hydration of oleic acid was optimized for the production of 10-hydroxystearic acid. Under the optimal conditions, a pilot reaction was performed on a 1-L scale, where 90 g L−1 of oleic acid was converted into 10-hydroxystearic acid by 10 g L−1 of lyophilized enzyme within 4 h, achieving a conversion of 96.1% and a space-time yield of up to 552 g L−1 d−1. The resultant 10-hydroxystearic acid was further converted into 10-oxostearic acid, via enzymatic cascade with a secondary alcohol dehydrogenase (MlADH) from Micrococcus luteus WIUJH20, using a lactate dehydrogenase (LdLDH) from Lactobacillus delbrueckii to drive the co-enzyme regeneration. The cascade reaction was carried out stepwise in one pot. Total conversion reached 95.0% after 10 h reaction at a scale of 1 L, with a space time yield of 217 g L−1 d−1. The final yield of 10-oxostearic acid isolated was 52.2% (mol mol−1), with a purity of >99.0%.

Screening the Lactic Acid Bacteria converting Hydroxy Fatty Acid from Unsaturated Fatty Acid.

Hydroxyl fatty acids (HFAs) are used in widely diverse industrial applications, healthy functional foods, artificial food flavorings, and alcoholic beverages. A lactic acid bacterium (LAB), Lactobacillus sakei, hydroxylates oleic acid. Furthermore, the hydroxyl fatty acid was identified by GC-MS as 10-hydroxystearic acid. The Lactobacillus sakei hydroxylated more than 90% of the oleic acid in the medium at 15°C after 30-48h. The hydroxyl enzyme needs a coenzyme for an electron donor as NADPH. The enzyme is useful for assay with monitoring NADPH concentration used an A340 device. The hydroxylate fatty acids converted by LAB lactonize aroma lactone from commercial yeast strains, which can be detected directly by scent. Commercial beer brewing yeast T-58 produced the highest concentration of aroma lactone from hydroxyl fatty acids. Furthermore, the aroma lactone is identified by GC-MS as gamma-dodecalactone. The ratio of conversion is 87%. These results suggest that the lactonization conversion system is useful to hydroxylate fatty acids for alcoholic beverages.

Crystal structure of oleate hydratase from Stenotrophomonas sp. KCTC 12332 reveals conformational plasticity surrounding the FAD binding site

Unsaturated fatty acids are toxic to various bacteria, causing their death or growth inhibition. To prevent this toxicity, unsaturated fatty acids should be converted into saturated fatty acids via hydrogenation reaction, which is the complete reduction of double bonds on the carbon chain. In a recent report, we observed that Stenotrophomonas sp. KCTC 12332 exhibited a high biotransformation activity of oleic acid (OA) in 10-hydroxystearic acid and identified the gene encoding oleate hydratase (OhySt) by complete genomic analysis. In the present study, to further investigate the structural features of OhySt, the recombinant protein was expressed in Escherichia coli, and then purified and crystallized. Biochemical assay showed that OhySt produces 10-hydroxystearic acid in a flavin adenosine dinucleotide (FAD)-dependent manner, indicating that it requires FAD as a cofactor. The OhySt structure, which is determined in its apo state, allows for a structural comparison with the previously reported FAD bound structure of oleate hydratase. The comparison of structures indicates remarkable conformational change of the loop region surrounding the FAD molecule upon binding of FAD. This change forces one of the important catalytic residues into position for catalysis.

Biocatalytic Route for the Synthesis of Oligoesters of Hydroxy-Fatty acids and ϵ-Caprolactone.

Developments of past years placed the bio-based polyesters as competitive substitutes for fossil-based polymers. Moreover, enzymatic polymerization using lipase catalysts has become an important green alternative to chemical polymerization for the synthesis of polyesters with biomedical applications, as several drawbacks related to the presence of traces of metal catalysts, toxicity and higher temperatures could be avoided. Copolymerization of ϵ-caprolactone (CL) with four hydroxy-fatty acids (HFA) from renewable sources, 10-hydroxystearic acid, 12-hydroxystearic acid, ricinoleic acid, and 16-hydroxyhexadecanoic acid, was carried out using commercially available immobilized lipases from Candida antarctica B, Thermomyces lanuginosus, and Pseudomonas stutzeri, as well as a native lipase. MALDI-TOF-MS and 2D-NMR analysis confirmed the formation of linear/branched and cyclic oligomers with average molecular weight around 1200 and polymerization degree up to 15. The appropriate selection of the biocatalyst and reaction temperature allowed the tailoring of the non-cyclic/cyclic copolymer ratio and increase of the total copolymer content in the reaction product above 80%. The catalytic efficiency of the best performing biocatalyst (Lipozyme TL) is evaluated during four reaction cycles, showing excellent operational stability. The thermal stability of the reaction products is assessed based on TG and DSC analysis. This new synthetic route for biobased oligomers with novel functionalities and properties could have promising biomedical applications.