Wax Ester Synthesis Research Articles

Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

Induction of meibocyte differentiation by three-dimensional, matrigel culture of immortalized human meibomian gland epithelial cells to form acinar organoids

PurposeRecent studies have shown that two-dimensional (2D) culture of primary rabbit and immortalized human meibomian gland epithelial cells (iHMGEC) do not recapitulate normal meibocyte differentiation and fail to express critical enzymes necessary for synthesis of meibum lipids. The purpose of this study was to test the hypothesis that 3D-spheroid culture of iHMGEC can facilitate meibocyte differentiation and induce the expression of acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), shown to be required for synthesis of meibum wax esters. MethodsiHMGEC were suspended in matrigel/basement membrane matrix and grown in proliferation media to form distinct cell clusters or spheroids. Cells were then treated with serum-free, differentiation media (advanced DMEM/F12) with and without FGF10 and synthetic agonists for the nuclear lipid receptor, peroxisome proliferator activator receptor gamma (PPARγ). Cells were then evaluated for differentiation markers using western blotting, immunocytochemistry (ICC) and real-time PCR. Control cells were grown in standard 2D culture systems. ResultsUnder proliferative conditions, 3D culture induced the formation of KRT5+ spheroids that contained a Ki67+/P63+ undifferentiated, basal cell population. When spheroids were switched to differentiation media containing PPARγ agonists, two different organoid populations were detected, a KRT6low population that was AWAT2+/PPARγ+ and a KRT6high population that was AWAT2-/PPARγ-, suggesting that iHMGEC exhibit a dual differentiation potential toward either a ductal or meibocyte organoid phenotype. ConclusionThe 3D culturing of iHMGEC can induce the formation of both meibocyte and ductal organoids and may thus serve as a better in vitro model system for studying the regulatory mechanisms controlling meibomian gland function.

Genome-wide analysis of the WSD family in sunflower and functional identification of HaWSD9 involvement in wax ester biosynthesis and osmotic stress.

The wax esters are important cuticular wax composition that cover the outer surface of plant organs and play a critical role in protection and energy metabolism. Wax ester synthesis in plant is catalyzed by a bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase (WSD). Sunflower (Helianthus annuus L.) is an important oil crop in the world; however, little is known about WSD in sunflower. In this study, we identified and performed a functional analysis of twelve HaWSD genes from sunflower genome. Tissue-specific expression revealed that 12 HaWSD genes were differentially expressed in various organs and tissues of sunflower, except seeds. HaWSD genes were highly induced by salinity, drought, cold, and abscisic acid (ABA) in sunflower. To ascertain their function, HaWSD9, with highly expressed levels in stems and leaves, was cloned and expressed in a yeast mutant defective in triacylglycerol (TAG) biosynthesis. HaWSD9 complemented the phenotype by producing wax ester but not TAG in vivo, indicating that it functions as a wax ester synthase. Subcellular localization analysis indicated that HaWSD9 was located in the endoplasmic reticulum (ER). Heterologous introduction of HaWSD9 into Arabidopsis wsd1 mutant exhibited increased epicuticular wax crystals and cuticular wax contents on the stems. As compared with the wsd1 mutant, HaWSD9 overexpressing transgenic Arabidopsis showed less cuticle permeability, chlorophyll leaching and water loss rate. Further analysis showed that the HaWSD9 transgenics enhanced tolerance to ABA, mannitol, drought and salinity, and maintained higher leaf relative water content (RWC) than the wsd1 mutant under drought stress, suggesting that HaWSD9 play an important physiological role in stress response as well as wax synthase. These results contribute to understanding the function of HaWSD genes in wax ester synthesis and stress tolerance in sunflower.

Solvent-free synthesis of oleic acid-based wax esters using recyclable acidic deep eutectic solvent

Wax esters have been widely used in cosmetics and pharmaceutical products. Oleic acid wax esters can be used to replace spermaceti oil or jojoba oil. In this work, the acidic deep eutectic solvent (DES) composed of choline chloride and p-toluenesulfonic acid (1:4, mol/mol) was used as an efficient recyclable catalyst for the synthesis of oleic acid-based liquid wax esters through an esterification reaction. The esterification conversion of cetyl alcohol reached 99.1% under the following optimal reaction conditions: 5% DES as catalyst, molar ratio of fatty acid to alcohol of 1.3:1 and reaction temperature of 70 oC for 3h. The catalyst recovery experiments showed that this low-price acidic DES catalyst could be reused five times with uniform activity. Moreover, DES-catalyzed solvent-free esterification could be applied in the preparation of other oleic acid-based wax esters and excellent conversions (> 96%) could be obtained under such mild conditions.

Stabilization of water‐in‐oil emulsions using a wax ester synthesized by a new homemade heterogeneous biocatalyst

AbstractBACKGROUNDCetyl palmitate is a wax ester naturally found in sperm whale spermaceti, and it is of importance in the cosmetics industry. International regulations prohibit the sale of sperm whale spermaceti, so it is now being replaced by pure cetyl palmitate obtained by a chemical route or from mixtures based on jojoba oil. Another alternative is to use an enzymatic route for wax ester synthesis. For such a purpose, a new heterogeneous biocatalyst was prepared by our research group via physical adsorption of Burkholderia cepacia lipase on aerogel modified with a protic ionic liquid (IB‐IL‐OPT). Its catalytic activity was compared with that of a biocatalyst prepared using a non‐chemically modified silica aerogel (IB‐Control). The objective of this work was to apply this homemade heterogeneous biocatalyst to cetyl palmitate synthesis via a direct esterification reaction in a solvent system and to test the final product as a stabilizing agent in water‐in‐oil emulsions.RESULTSIB‐IL‐OPT achieved maximum acid conversion of 95–100% under optimal experimental conditions established using a central composite rotational design: biocatalyst concentration of 14.8% (w/w), molar ratio of 1:1.3, 41 °C for 15 h of reaction under continuous mechanical agitation (200 rpm). The IB‐Control gave 58.8% acid conversion in similar conditions. IB‐IL‐OPT was successively reused for four cycles. The wax ester showed potential as a good emulsifier for virgin coconut oil.CONCLUSIONSThe cetyl palmitate obtained via an enzymatic route without further purification showed good emulsifying potential, forming Pickering emulsions. This opens new perspectives for study and applications in the cosmetics industry. © 2022 Society of Chemical Industry (SCI).

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes

PurposePreviously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. MethodsRabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70–80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. ResultsRabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a −16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. ConclusionsAWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C36, C34 and C32, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.

The Application of a Rotating Packed Bed Reactor for the Synthesis of High Viscosity Wax Ester

Wax esters are high value-added products and widely used in a broad range of commercial fields. The enzymatic synthesis of wax ester from plant oil is more attractive than the traditional chemical method due to being environmental-friendly and limiting the use of hazardous chemicals. However, the high mass transfer resistance from the high viscous substrate leads to a low conversion and long reaction time in the continues stirred tank reactor (CSTR). The rotating packed bed reactor (RPBR) offers high mass transfer and can be used to enhance the enzymatic wax ester synthesis process. From the results, it could be concluded that the mass transfer was enhanced with the optimization of the centrifugal factor of the RPBR. Under the optimal process conditions, the wax ester yield of 96.4% was obtained after 4 hours reaction, and the half-life of the catalyst corresponded with 64 hours, while nine batches achieved of yields above 90%. The investigation proved that the RPBR is an attractive and effective reactor for heterogeneous bio-catalysis in high viscosity of 7.39~12.27 Cst at 40 °C.

Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds

Utilization of lignin, an abundant renewable resource, is limited by its heterogenous composition and complex structure. Biological valorization of lignin provides advantages over traditional chemical processing as it occurs at ambient temperature and pressure and does not use harsh chemicals. Furthermore, the ability to biologically funnel heterogenous substrates to products eliminates the need for costly downstream processing and separation of feedstocks. However, lack of relevant metabolic networks and low tolerance to degradation products of lignin limits the application of traditional engineered model organisms. To circumvent this obstacle, we employed Acinetobacter baylyi ADP1, which natively catabolizes lignin-derived aromatic substrates through the β-ketoadipate pathway, to produce mevalonate from lignin-derived compounds. We enabled expression of the mevalonate pathway in ADP1 and validated activity in the presence of multiple lignin-derived aromatic substrates. Furthermore, by knocking out wax ester synthesis and utilizing fed-batch cultivation, we improved mevalonate titers 7.5-fold to 1014 mg/L (6.8 mM). This work establishes a foundation and provides groundwork for future efforts to engineer improved production of mevalonate and derivatives from lignin-derived aromatics using ADP1.

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa.

To ensure global food security under the changing climate, there is a strong need for developing ‘climate resilient crops’ that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate.

A simplified kinetic model to describe the solvent‐free enzymatic synthesis of wax esters

AbstractBACKGROUNDThe use of biotechnological processes at industrial scale is a promising tool to replace conventional synthesis as an efficient and eco‐friendly technology. For that purpose, the kinetic modelling of an in‐lab optimized enzymatic process prior to scaling‐up is of great utility.RESULTSIn this work, a kinetic model for the solvent‐free synthesis of cetyl laurate, myristate, palmitate and stearate using different commercial immobilized lipases has been developed. In order to describe the esterification process of the cetyl esters separately and as a mixture similar to natural spermaceti, a pseudo‐first‐order kinetic has been proposed and tested. A relation between the inverse values of the kinetic constant and the amount of biocatalyst has been observed. The effect of temperature on the reaction rate can be accurately described by the Arrhenius equation except for immobilized Thermomyces lanuginosus, which appears to be less resistant to temperatures above 70 °C.CONCLUSIONLow deviations between experimental and predicted values (R2 ≥ 0.99) indicate that this pseudo‐first‐order kinetic model can be considered valid for the data range studied. In addition, the reaction rate of spermaceti can be successfully predicted through a weighted average of the kinetic constants obtained during the synthesis of each cetyl ester. This simple but accurate kinetic model for describing the solvent‐free enzymatic biosynthesis of wax esters from spermaceti may contribute to extending the application of lipases as industrial catalysts. © 2021 Society of Chemical Industry (SCI).

Synthesis, Characterization and Optimization of Oleyl Oleate Wax Ester Using Ionic Liquid Catalysts

In this work, the synthesis of oleyl oleate wax ester using Brønsted acidic ionic liquid catalysts was carried out. Confirmation of oleyl oleate molecular structure has been performed using FTIR, NMR, and ESI-MS spectroscopies. The ability of ionic liquid catalysts for catalyzing the esterification reaction of oleic acid and oleyl alcohol to produce oleyl oleate was optimized. The ionic liquid catalyst ([NMP][CH3SO3]) was found to be the best catalyst for the esterification reaction of oleic acid and oleyl alcohol compared with the other acidic ionic liquids studied. The optimal reaction conditions were determined at a reaction time of 8 h; oleic acid to oleyl alcohol mole ratio of 1:1; ([NMP][CH3SO3]) with 9.9 wt.%; and reaction temperature of 90 °C. Under these conditions, the percentage yield of oleyl oleate wax ester was 86%.

Enzymatic synthesis of coconut oil based wax esters by immobilized lipase EQ3 and commercial lipozyme RMIM

Liquid wax esters are widely used in cosmetic as well as pharmaceutical and other industries. The demand of organic and natural products is increasing nowadays. Coconut oil contains benefit fatty acids and has been mainly used for oil-based and moisturizer products. Liquid wax esters from coconut oil and unsaturated fatty alcohol can be synthesized by enzymatic reaction; and it is interesting for using as an alternative natural ingredient in these industries. Optimal condition for coconut oil based wax ester synthesis by immobilized lipase EQ3 was 10 U of enzyme, temperature at 30°C and molar ratio of coconut oil to oleyl alcohol at 1:3 (mol/mol) (0.33X) dissolved in isooctane for 12 h, while for Lipozyme RM IM optimal condition was 10 U of enzyme, temperature at 45°C and oil/alcohol molar ratio at 1:3 (0.33X) dissolved in isooctane for 3 h. Percentage of wax esters synthesized by both lipases reached more than 88%. Both immobilized lipases catalyzed high yield of wax esters within the 2nd batch; after that, the immobilized lipases showed reduced activity and synthesized < 60% of wax esters from the 3rd to 5th batch. The main composition of wax esters was ~ 48% oleyl laurate with 10% degradation at ~ 250°C. The liquid wax ester synthesis by commercial Lipozyme RM IM had higher effect than immobilized lipase EQ3, but both catalysts were stable within 2 batches in the optimum condition. The characteristic properties of wax esters showed potential for use as components in cosmetics and skin care products. Ungcharoenwiwat P, H-Kittikun A. Enzymatic synthesis of coconut oil based wax esters by immobilized lipase EQ3 and commercial lipozyme RMIM. Electron J Biotechnol 2020;47. https://doi.org/10.1016/j.ejbt.2020.06.005

Uncovering the role of a positive selection site of wax ester synthase/diacylglycerol acyltransferase in two closely related Stipa species in wax ester synthesis under drought stress.

Natural selection drives local adaptations of species to biotic or abiotic environmental stresses. As a result, adaptive phenotypic divergence can evolve among related species living in different habitats. However, the genetic foundation of this divergence process remains largely unknown. Two closely related alpine grass species, Stipa capillacea and Stipa purpurea, are distributed in different rainfall regions of northern Tibet. Here, we analyzed the drought tolerance of these two closely related Stipa species, and found that S. purpurea was more resistance to drought stress than S. capillacea. To further understand the genetic diversity behind their adaptation to drought environments, a comprehensive gene repertoire was generated using PacBio isoform and Illumina RNA sequencing technologies. Bioinformatics analyses revealed that differential transcripts were mainly enriched in the wax synthetic pathway, and a threonine residue at position 239 of WSD1 was identified as having undergone positive selection in S. purpurea. Using heterologous expression in the Saccharomyces cerevisiae mutant H1246, site-directed mutagenesis studies demonstrated that a positive selection site results in changes to the wax esters profile. This difference may play an important role in S. purpurea in response to drought conditions, indicating that S. purpurea has evolved specific strategies involving its wax biosynthetic pathway as part of its long-term adaptation to the Qinghai-Tibet Plateau.

Waste Cooking Oil for Wax Esters Synthesis Using Candida Rugosa Lipase Immobilized on Fe3o4@oa Nanoparticles

Wax ester is an important skin conditioning agent derived from the esterification of fatty acids with alcohols. It is widely used in cosmetic as it has a composition similar to that of natural skin lipids. This study presents the synthesis of wax esters from hydrolyzed WCO in presence of cetyl alcohol and Candida rugosa lipase immobilized on Fe3O4@OA. The effects of the reaction time and cycles of reuse were investigated. The wax ester conversion has been monitored for 24 h. High conversion of ~ 89.3 % was observed already at 12 hours of synthesis. No significant reduction, during four cycles of use, of the lipase activity occurs.

Triacylglycerol and phytyl ester synthesis in Synechocystis sp. PCC6803

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

G_Fe3O4/Ag supporting Candida rugosa lipase for the “green” synthesis of pomegranate seed oil derived liquid wax esters

Wax esters were obtained through hydrolysis/esterification of pomegranate seed oil (PSO) with cetyl alcohol in the presence of Candida rugosa lipase (CRL) immobilized on G_Fe3O4/Ag@OA (oleic acid-coated magnetic nano-hybrid) catalyst. Lipase was immobilized successfully on the as-produced nanoparticles with an efficiency, depending on the operating conditions, up to ∼94%. The immobilized CRL shows very high activity on the respect of the free counterpart, which can be attributed to the lipase conformational structure modification related to interfacial activation. The immobilized lipase was used for the wax esters synthesis from PSO in a solvent-free system. A conversion of ∼83% was observed after 12 h alcohol/FFA molar ratio 1:1, it increases up to ∼95% with an alcohol/FFA molar ratio of 2:1. After the first three cycles of use, no significant reduction of the lipase activity was observed. Analyses of wax esters properties compared with jojoba oil evidence the feasibility of the produced wax ester for different applications. Iodine number of ∼209 (gI2/100 g oil) was obtained indicating a high degree of anti-oxidant and moisturizing properties.

A Splice Variant of BrrWSD1 in Turnip (Brassica rapa var. rapa) and Its Possible Role in Wax Ester Synthesis under Drought Stress.

Cuticular wax accumulation in plants contributes to drought tolerance. Here, we compared the drought levels on two varieties with different genotypes in turnip (Brassica rapa var. rapa) and found that the drought tolerance was higher in the waxy KTRG-B48a than in the wax-free KTRG-B48b. A combination of PacBio and Illumina sequencing analyses revealed that differential transcripts were mainly enriched in the wax synthesis pathway, and a splice variant (BrrWSD1-X2) was identified in the waxy KTRG-B48a. BrrWSD1-X2 had a stronger ability to synthesize wax esters than BrrWSD1-X1 using heterologous expression in yeast (Saccharomyces cerevisiae) mutant H1246a. Then, we speculated that the T to C transversion of the third intron and the higher number of TA repeats in the third intron of BrrWSD1 DNA in the waxy KTRG-B48a may result in a lower efficiency of splicing recognition of the third intron, resulting in the emergence of BrrWSD1-X2 in waxy varieties.

Enzymatic synthesis of cosmetic grade wax ester in solvent free system: optimization, kinetic and thermodynamic studies

The enzymatic synthesis of cetyl caprate, a cosmetic grade ester was studied by esterification of cetyl alcohol and capric acid using Fermase CALB™10000 under solvent free condition. The process parameters namely, temperature, enzyme loading, molar ratio, agitation speed have been investigated and optimized to attain maximum yield of cetyl caprate. The maximum yield of 91.9% was obtained in 80 min under optimized conditions of 50 °C temperature, 3% enzyme loading, capric acid:cetyl alcohol ratio of 1:3, and 200 rpm stirring speed. The enzyme operational stability study reveals that enzyme can retain its original activity for the synthesis of cetyl caprate and could be reused up to five to six times without any notable loss of catalytic activity. The kinetics of the reaction was derived by employing non-linear regression consideration, and the experimental results and kinetic evaluation showed that reaction obeys ping-pong bi–bi model. The kinetic parameters, Vmax = 21.886 M/min/g catalyst, KA = 1.194 × 10−2 M, KB = 8.4512 M, KiA = 2.02 × 10−2 M, KiB = 3.55 × 10−4 M and SSE = 4.31 × 10−5 measured from initial rate data were used to replicate experimental results with good agreement. The thermodynamic parameters, i.e., change in enthalpy, Gibb’s free energy, change in entropy was evaluated at different operating temperatures. Thus, current research work strikes a significant imprint in the cosmetic industry by synthesizing green and natural product using biocatalysis under solvent free condition.

Enzymatic synthesis of bio-based wax esters from palm and soybean fatty acids using crude lipases produced on agricultural residues

The increased demand for the consumption of vegetable oils has led to the generation of high quantities of by-products, such as palm and soybean fatty acid distillates (PFAD, SFAD), as a result of the refining process. In this study PFAD and SFAD were used as raw materials for the enzymatic production of wax esters in a solvent-free system. A fungal crude lipase was produced by solid state fermentation using babassu cake as substrate and then fermented solids were used as biocatalyst for wax esters synthesis using cetyl and oleyl alcohols. The highest wax esters production of 80% was achieved for the SFAD-cetyl, SFAD-oleyl and PFAD-oleyl esters using 8.8 U/g of biocatalyst at 50 °C. The proposed biocatalysis could be economically feasible since the crude lipase was efficiently reused for up to five repeated batch reactions. The physicochemical properties of the wax esters were also studied. Differential scanning calorimetry and the determination of acid, iodine and saponification values showed that the wax esters have similar properties to natural waxes. This study demonstrated the valorization of by-products from vegetable oil production processes as an alternative feedstock for the synthesis of bio-based wax esters with potential use in food, cosmetics and chemical applications.