Chain Fatty Alcohols Research Articles

Synthesis of Anionic Surface Active Agents Containing Heterocyclic Moiety From Long Chain Fatty Alcohols

A series of novel groups of anionic surface active agent were synthesized. Synthesis of these surfactants via the reaction of long chain fatty alcohols (octyl, decyl and dodecyl) with maleic anhydride to give monoester. The monoester chloride reacted with amino derivatives of heterocyclic rings followed by addition of NaHSO3 .The surface tension, interfacial tension; Kraft point, emulsifying and wetting power were evaluated. Stability to hydrolysis, biodegradability and biological activities were measured. A comparison studies between the chemical structures and the results were done.

Effects of alkyl polyglycoside (APG) on Bombyx mori silk degumming and the mechanical properties of silk fibroin fibre

Alkyl polyglycoside (APG), a nonionic surfactant, is often considered to be a green surfactant and is synthesized using glucose and long chain fatty alcohols. It is used as a degumming agent of Bombyx mori silk fibre in this study for the first time. We studied APG systematically in comparison to the traditional degumming methods, such as aqueous solutions of sodium carbonate (Na2CO3) and neutral soap (NS). After repeatedly boiling silk fibres in an aqueous solution of 0.25% APG three times for 30min and using a bath ratio of 1:90–120 (g/mL), sericin was completely removed from the fibre. SDS-PAGE showed that the degumming in APG did not induce an evident breakage of the silk fibroin peptide chains, including the light chain and P25 protein. The tensile properties, thermal analysis, and scanning electron microscopic (SEM) observation of the degummed fibroin fibre all show that APG is a degumming agent similar to NS and far superior to Na2CO3. These results indicate that APG is an environment-friendly silk degumming/refining agent in the silk textile industry and in the manufacture of silk floss quilts.

Preparation and properties of microemulsion detergent with linear medium chain fatty alcohols as oil phase

The formation of promising oil-in-water microemulsion as detergent has been achieved by using linear medium chain fatty alcohols (C5-C8) as the oil phase, sodium dodecyl sulfate (SDS) and isomeric alcohol ethoxylate (IC13EO6) as a mixed surfactant, and 1-butanol as cosurfactant. Pseudoternary phase diagrams were constructed by addition of the water. The effect of the SDS to IC13EO6 mixing ratio on single phase microemulsion area size was systematically investigated. The structure transition of the microemulsion was measured by conductivity. The results show that with addition of the water, the structure of microemulsion transformed from water-in-oil, bicontinuous to oil-in-water. After diluted 500 times, the equilibrium contact angle and dynamic surface tension of the microemulsion were much lower than this for standard liquid detergent under the same condition indicating that the dilute microemulsion exhibited excellent spreading and wetting properties on hydrophobic surfaces. The detergency of the as-prepared microemulsions with different oil types was tested by Terg-O-Tometer in order to select optimum formulations from the infinite dilution zone. With optimum formulations, the detergency of microemulsion was higher than that of standard liquid detergents, because microemulsions exhibit strong solubilization ability on oil stain.

Synthesis of Fatty Acetoacetates Under Microwave Irradiation Catalysed by Sulfamic Acid in a Solvent‐Free System

AbstractThe 1,3‐dicarbonyl compounds are important building blocks to obtain products with various biological activities and technological applications. In this work, we used a simple transesterification method to develop fatty acetoacetates in a solvent‐free medium using a green catalyst, sulfamic acid (NH2SO3H), under microwave irradiation. The experimental results demonstrate good yields in a short reaction time (13 min), which makes this method an efficient approach to synthesize fatty acetoacetates from a wide range of saturated, unsaturated, and polyunsaturated long chain fatty alcohols, and ricinoleic derivatives. Experiments of recycling of the catalyst were performed and no decrease in catalytic activity of sulfamic acid was observed.

Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6–C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 & C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP+ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.

The study of binding agents used to inlay turquoise onto bronze objects in Eastern Zhou Period

Abstract The paper presents the analysis results of the binding agents used on Turquoise-inlayed bronze artifacts in Eastern Zhou Period. The technique applied is pyrolysis-gas chromatography and mass spectrometry with thermal assisted hydrolysis and methylation (THM-Py-GC/MS). Mastic resin was identified as binding agent to inlay turquoise onto the bronze pots, based on the detection of the marker compounds of 3-oxo-olean-18-en-28-oic acid, 3-oxo-olean-12-en-28-oic acid and ursa-2, 12-dien-28-oic acid. Beeswax was determined as the binding agent used on turquoise-inlayed bronze sword according to the detection of a series of alkanes, long chain fatty acids and long chain alcohols. The results clearly demonstrate that different binding agents were used to inlay turquoise onto artifacts respectively during Eastern Zhou Period in China.

Pattern recognition analysis of chromatographic fingerprints of Crocus sativus L. secondary metabolites towards source identification and quality control.

Chromatographic fingerprinting is an effective methodology for authentication and quality control of herbal products. In the presented study, a chemometric strategy based on multivariate curve resolution-alternating least squares (MCR-ALS) and multivariate pattern recognition methods was used to establish a gas chromatography-mass spectrometry (GC-MS) fingerprint of saffron. For this purpose, the volatile metabolites of 17 Iranian saffron samples, collected from different geographical regions, were determined using the combined method of ultrasound-assisted solvent extraction (UASE) and dispersive liquid-liquid microextraction (DLLME), coupled with GC-MS. The resolved elution profiles and the related mass spectra obtained by an extended MCR-ALS algorithm were then used to estimate the relative concentrations and to identify the saffron volatile metabolites, respectively. Consequently, 77 compounds with high reversed match factors (RMFs > 850) were successfully determined. The relative concentrations of these compounds were used to generate a new data set which was analyzed by multivariate data analysis methods including principal component analysis (PCA) and k-means. Accordingly, the saffron samples were categorized into five classes using these techniques. The results revealed that 11 compounds, as biomarkers of saffron, contributed to the class discrimination and characterization. Eleven biomarkers including nine secondary metabolites of saffron (safranal, α- and β-isophorone, phenylethyl alcohol, ketoisophorone, 2,2,6-trimethyl-1,4-cyclohexanedione, 2,6,6-trimethyl-4-oxo-2-cyclohexen-1-carbaldehyde, 2,4,4-trimethyl-3-carboxaldehyde-5-hydroxy-2,5-cyclohexadien-1-one, and 2,6,6-trimethyl-4-hydroxy-1-cyclohexene-1-carboxaldehyde (HTCC)), a primary metabolite (linoleic acid), and a long chain fatty alcohol (nanocosanol) were distinguished as the saffron fingerprint. Finally, the individual contribution of each biomarker to the classes was determined by the counter propagation artificial neural network (CPANN) method.

In vitro Inhibition of Zika Virus by Metadichol®, A Novel Nano Emulsion Lipid

Objective: To show the antiviral activity of Metadichol® against Zika virus by an in-vitro assay. Methods: The primary Anti-Zika virus assay was performed using real-time RT-qPCR (TaqMan) to measure extracellular Zika virus RNA copy number associated with virions released from vero cells. The ‘vero’ cell line (kidney epithelial cells extracted from an African green monkey) treated with antiviral test articles is infected with Zika virus followed by Zika virus associated RNA measurement in the cell culture supernatant. Antiviral compounds blocking any step of viral replication such as transcription, translation, encapsidation, the particle assembly and release were identified and characterized using this sensitive assay system. Findings: Metadichol® [1,2] in vitro assays, inhibited the Zika Virus with a EC50 of 1.48 µg/ml. Conclusion: Metadichol is a safe and effective inhibitor for enveloped viruses in humans. Since it is known to bind to the vitamin D receptor (VDR), its action mechanism likely involves the competitive displacement of virus particles from VDR’s on host cell membranes. Metabolism studies of long chain alcohol in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly interconverted in a fatty alcohol cycle [3]. Metadichol consists of natural components of common foods (classified as GRAS), Metadichol has no known negative side effects. The inhibition of Zika virus by Metadichol is not surprising, given that we have recently published the results of Metadichol which showed broad-spectrum antiviral activity against Dengue, Ebola, H1N1, SARS, Chikungunya and other enveloped viruses [4].

α-Tocopherol을 함유한 Nanostructured Lipid Carriers의 특성과 안정화 효과

Loading of hydrophobic α-tocopherol into nanostructured lipid carrier (NLC) was performed for improving its oxidative stability. First, various NLCs with different constituents and mixing ratios were prepared and their characteristics were investigated. While the stable NLCs were made when cetyl palmitate (CP) or glyceryl monosterate (GMS) was used as a solid lipid, the phase separation occurred in the NLCs consisting of stearic acid. Particle sizes of the NLCs were several hundreds of nanometers and the size decreased with increasing the ratio of solvent to lipid. It was examined from DSC thermogram and anisotropy test that the degree of crystallinity of the lipid phase decreased and the lipid matrix became less ordered when octyldodecanol, a long chain fatty alcohol, was added into the solid lipid. The oxidative stability of α -tocopherol in NLC was remarkably improved compared to that in solution or emulsion under high temperature (45 °C) and UV radiation, which was verified through DPPH test and peroxide value measurement.

GC-MS ANALYSIS OF CHANGE IN FATTY ACID COMPOSITION OF HALOBACTERIUM BACILLUS LICHENIFORMIS HSW-16 UNDER VARYING SALINITY CONDITION

The salt concentration have a pronounced effect on fatty acid composition, therefore the adaption of fatty acid composition of a halophilic bacterium Bacillus licheniformis HSW-16, grown at different concentration of NaCl (0 mM, 150 mM, 175 mM, 200 mM) was studied. The common fatty acid observed at various salt concentration were tetratriacontane, triacontane, tetracosane and pentacosane etc..As the salinity increases from 150 mM to 200 mM, the bacterium started to accumulating the long chain alkanes and fatty alcohol, suggesting the adaption of bacterium through accumulation varying fatty acids for counteracting the increased salinity and maintaining its osmotic balance in the changing environment.

Low-Cost Synthesis and Characterizations of Metal-Organic Framework (MOF-199) Materials by Nonsurfactant Templating Method

In this study, a three-dimensional (3D) copper-based metal-organic framework (MOF-199) is a microporous materials with structural formula Cu3(BTC)2 (1,3,5-benzene tricarboxylate) were successfully synthesized using nonsurfactant templating method. The preparations of MOF-199 utilized different length of straight carbon chain (oleochemical) fatty alcohols derived from palm oil were similar to those reported earlier by our group with some modifications [1]. This new method led to unique structure and properties of as-synthesized MOF-199. The addition of fatty alcohols such as octyl (C8) and decyl (C10) alcohol act as renewable template onto MOF-199 generating the required carbon template for microporous crystalline structure [2]. The MOF-199 had been synthesized using conventional hydrothermal method with the present of fatty alcohols to analyse the resulting MOF-199 in terms of structure, morphology, surface area and adsorption isotherm. MOF-199 synthesized in this study exhibited single crystal orthorhombic morphology at ca. 30 micron. The crystallinity of MOF-199 materials was improved by the addition of fatty alcohols as observed in the X-Ray Diffraction patterns.

Hydrothermal Synthesis and Characterizations of MOF-199 Using Renewable Template

A three-dimensional (3D) copper-based metal-organic frameworks or MOF-199 were successfully synthesized by utilizing palm oil based fatty alcohols that acted as renewable template. The synthesis of MOF-199 employed different length of straight chain of fatty alcohols namely octyl, decyl, lauryl, myristyl and cetyl alcohols derived from palm oil using similar method to those reported earlier with some modifications. The microstructure of MOF-199 exhibited single particles with fine octahedral morphology at various particle sizes in the range of ca. 15-60 μm. The nitrogen adsorption isotherms were conducted to estimate the surface area and pore volume of the materials and were at ca. 400 – 1100 m2g-1 and 0.17 – 0.43 mLg-1 respectively. The surface area and pore volume of the materials decreased with longer straight chain fatty alcohol possibility due to mobility of the long carbon chain.

Lipid profile of mice fed a high‐fat diet supplemented with a wax ester‐rich marine oil

Oil extracted from the marine copepod Calanus finmarchicus contains the long chain omega‐3 fatty acids eicosapentaenoic acid and DHA in addition to stearidonic acid (18:4n‐3). Unlike other marine lipids, the fatty acids in this oil are esterified with long chain fatty alcohols as wax esters. The aim of this study was to examine the fate of the wax esters in oil from C. finmarchicus when given as a 2% supplement in a high fat diet to C57BL/6J mice for 11 weeks. The study confirmed that feeding mice a high fat diet supplemented with a small amount of oil containing wax esters reduced the body weight gain. During digestion, wax esters were hydrolyzed and the fatty acids absorbed since the fatty acid composition of the adipose tissue and liver reflected the enrichment with the Calanus oil. The composition of the liver lipids demonstrated elongation and desaturation of the C18 omega‐3 fatty acids from the feed and accumulation of longer chained omega‐3 fatty acids. Elevated levels of FFA and FAOH in the feces suggest that the absorption process, not the hydrolysis, could be a rate limiting step in utilization of small amounts of wax esters included in high fat diets in mice.Practical applications: The limited amount of available fish oil has led to extensive search for alternative sources of long‐chain PUFA. One suggestion is to harvest at lower trophic levels, like small crustaceans, which may be abundantly present in the oceans. In this investigation, we have studied the effects of including the astaxanthin‐rich oil from the marine copepod C. finmarchicus in a high fat diet in mice. This oil is different from other marine oils since most of the fatty acids are esterified to long‐chain fatty alcohols, not in TAG, and that stearidonic acid is the major omega‐3 fatty acid present. The results provide knowledge and understanding of aspects related to digestion and possible physiological effects of including small amounts of marine wax esters in diets.The lipid class composition in the feces from mice fed a high fat diet or a high fat diet supplemented with a wax ester rich oil were similar except for the pronounced amount of fatty alcohols in feces of the latter.

Solubility limits and phase diagrams for fatty alcohols in anionic (SLES) and zwitterionic (CAPB) micellar surfactant solutions

By analysis of experimental data, a quantitative theoretical interpretation of the solubility limit of medium- and long-chain fatty alcohols in micellar solutions of water-soluble surfactants is presented. A general picture of the phase behavior of the investigated systems is given in the form of phase diagrams. The limited solubility of the fatty alcohols in the micelles of conventional surfactants is explained with the precipitation of their monomers in the bulk, rather than with micelle phase separation. The long chain fatty alcohols (with n=14, 16 and 18 carbon atoms) exhibit an ideal mixing in the micelles of the anionic surfactant sodium laurylethersulfate (SLES) and the zwitterionic surfactant cocamidopropyl betaine (CAPB) at temperatures of 25, 30, 35 and 40°C. Deviations from ideality are observed for the alcohols of shorter chain (n=10 and 12), which can be explained by a mismatch with the longer chains of the surfactant molecules. Using the determined thermodynamic parameters of the systems, their phase diagrams are constructed. Such a diagram consists of four domains, viz. mixed micelles; coexistent micelles and precipitate (dispersed crystallites or droplets); precipitate without micelles, and molecular solution. The four boundary lines intersect in a quadruple point, Q. For ionic surfactants (like SLES), a detailed theory for calculating the boundary lines of the phase diagrams is developed and verified against data for the positions of the kinks in surface tension isotherms. The theory takes into account the electrostatic interactions in the micellar solutions and the effect of counterion binding. The results can be useful for a quantitative interpretation and prediction of the phase behavior of mixed solutions of two (or more) surfactants, one of them being water soluble and forming micelles, whereas the other one has a limited water solubility, but readily forms mixed micelles with the former surfactant.

The Leaf, Wood and Bark Oils of three Species of Myodocarpus (Myodocarpaceae) Endemic to New Caledonia

The leaf, wood and bark oils of three species of Myodocarpus, M. viellardii Brongn. & Gris, M. fraxinifolius Brongn. & Gris and M. lanceolatus Dubard & R. Viguier have been examined. From the wood oil of M. viellardii the major components were α-pinene (22.4%) and a monoterpene methyl ester, methyl myodocarpate (methyl 3,7-dimethylbicyclo [4,1,0]hept-3-ene-7-carboxylate) (61%), based on the δ-3-carene skeleton. Also present in lesser amounts was the corresponding acid, myodocarpic acid (3,7-dimethylbicyclo[4,1,0]hept-3-ene-7-carboxilic acid) and the corresponding alcohol, myodocarpol (3,7- dimethylbicyclo[4,1,0]hept-3-en-7-yl) methanol), in lesser (< 3%) amounts. The bark oil contained β-caryophyllene (13.8%) and a series of long chain fatty alcohols, dodecanol, tetradecanol, hexadecanol, octadecanol and octadec-9-en-1-ol in amounts of 1.4-15% (all but octadecanol > 11%). The leaf oil of this species produced a sesquiterpenic oil with the principal components being β-caryophyllene (36%), α-humulene (11.1%) and bicyclogermacrene (10.6%). In M. fraxinifolius, both the wood and bark oils contained a series of long chain alcohols, tetradecanol (30-38%), hexadecanol (23-29%) and octadec-9-en-1-ol (12%) as principal components. In the leaf oil of this species the principal component was β-caryophyllene (63%), with lesser amounts of humulene oxide II (2.9%), isocomene (2.8%) and viridiflorol (1.4%). In M. lanceolatus, the leaf oil was dominated by the monoterpene hydrocarbons α-pinene (22.9%) and δ-3-carene (32.6%). In the wood oil of this species the principal components were geraniol (7.4%), citronellol (4.7%), germacrene-B (7.1%), zingiberine (6.8%) and linalool (6.7%), while in the bark oil they were geraniol (12.4%), citronellol (2.6%), germacrene-B (5.8%), and linalool (6.5%).

Distribution of microbial fatty acids and fatty alcohols in soils from an altitude transect of Mt. Jianfengling in Hainan, China: Implication for paleoaltimetry and paleotemperature reconstruction

The temperature gradient along the altitude transect of Mt. Jianfengling provides a good opportunity to establish and evaluate the microbial lipid-based environmental proxies. The soils collected from 14 different altitudes of Mt. Jianfengling contain abundant microbial fatty acids and fatty alcohols, including iso/anteiso fatty acids (i/aC12:0-i/aC19:0), 10-Me-C16:0 fatty acid, iso/anteiso fatty alcohols (i/aC13-iC26), 10-Me-C16:0 fatty alcohol and unsaturated fatty alcohols, which can indicate a strong microbial activity in the Jianfengling soils. The branched and unsaturated fatty alcohols can be only detected when saponification is performed, implying that these lipids are present as the constituents of bacterial wax esters in the soils. The ratio of aC15/iC15 fatty acids is positively correlated with altitude, suggesting that the decrease in temperature can induce the increase in the relative abundance of anteiso C15 fatty acid. In contrast, the ratio of aC15/iC15 fatty alcohols and of aC15/nC15 fatty alcohols both decrease with increased altitude or decreased temperature. Similarly, the ratio of nC18:1/nC18:0 fatty alcohols also decreases with decreased temperature, which is opposite to the previous observation that unsaturation of fatty acids in microorganism increases in response to decreased temperature. Besides, the average chain length (ACL) of long chain fatty alcohols (C22–C30) from leaf waxes and carbon preference index (CPI) of all n-fatty alcohols are also significantly correlated with altitude or mean annual temperature, demonstrating their potential for paleoclimate reconstruction. The correlation of microbial fatty acids and alcohols as well as ACL and CPI of plant wax-derived fatty alcohols with altitude may provide novel ways to reconstruct paleotemperature and paleoaltimetry.

Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages

Ethnopharmacological relevanceEvening primrose (Oenothera biennis L., Onagraceae) is a wild medicinal plant of Central American origin that is now one of the most widely used herbal medicines in different parts of the world. Oil extracted from it seeds is traditionally used in the treatment of eczema, asthma, rheumatoid arthritis, breast problem, premenstrual and menopausal syndrome, all they have an inflammatory component. The present study demonstrates the in vitro anti-inflammatory effect of long-chain fatty alcohols, minor compounds isolated from Evening primrose oil (EPO). Material and methodsA mixture of long chain fatty alcohols (LCFAs) was isolated from the non-triacylglycerol fraction of the EPO. Hexacosanol (C26OH: 38.65%), tetracosanol (C24OH: 31.59%), docosanol (C22OH: 11.36%) and octocosanol (C28OH: 7.64%), were the major constituents, identified and quantified by GC and GC–MS. LCFA was tested with LPS stimulated murine peritoneal macrophage. This fraction, significantly and dose-dependently decreased nitric oxide production induced by LPS (P<0.001) and the inhibitory effect seems to be consequence of an action at the level of the inducible nitric-oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The release of PLA2 and TXB2 also was significantly inhibited by LCFAs (P<0.001) although LCFAs did not affect to PGE2 generation, however the western blot assay showed that LCFAs reduced cyclooxygenase-2 enzyme gene expression at all doses assayed. In the same way, the secretion of inflammatory cytokines interleukin 1β (IL-1β) and tumour necrosis factor α (TNF-α) from LPS-stimulated murine macrophage, were also significantly reduced (P<0.001). ConclusionThese results demonstrates the anti-inflammatory activity of LCFAs, providing an additional value about the role of bioactive minor compounds in the beneficial effect of EPO and supports its traditional uses in inflammatory processes management.

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.

Detailed characterization of the substrate specificity of mouse wax synthase.

Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.