Immobilized Candida Antarctica Research Articles

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Enzymatic Synthesis of Glucose Fatty Acid Esters Using SCOs as Acyl Group-Donors and Their Biological Activities

Sugar fatty acid esters, especially glucose fatty acid esters (GEs), have broad applications in food, cosmetic and pharmaceutical industries. In this research, the fatty acid moieties derived from polyunsaturated fatty acids containing single-cell oils (SCOs) (i.e., those produced from Cunninghamella echinulata, Umbelopsis isabellina and Nannochloropsis gaditana, as well as from olive oil and an eicosapentaenoic acid (EPA) concentrate) were converted into GEs by enzymatic synthesis, using lipases as biocatalysts. The GE synthesis was monitored using thin-layer chromatography, FTIR and in situ NMR. It was found that GE synthesis carried out using immobilized Candida antarctica B lipase was very effective, reaching total conversion of reactants. It was shown that EPA-GEs were very effective against several pathogenic bacteria and their activity can be attributed to their high EPA content. Furthermore, C. echinulata-GEs were more effective against pathogens compared with U. isabellina-GEs, probably due to the presence of gamma linolenic acid (GLA) in the lipids of C. echinulata, which is known for its antimicrobial activity, in higher concentrations. C. echinulata-GEs also showed strong insecticidal activity against Aedes aegypti larvae, followed by EPA-GEs, olive oil-GEs and N. gaditana-GEs. All synthesized GEs induced apoptosis of the SKOV-3 ovarian cancer cell line, with the apoptotic rate increasing significantly after 48 h. A higher percentage of apoptosis was observed in the cells treated with EPA-GEs, followed by C. echinulata-GEs, U. isabellina-GEs and olive oil-GEs. We conclude that SCOs can be used in the synthesis of GEs with interesting biological properties.

Production of Glyceryl Monostearate by Immobilized Candida Antarctica B Lipase in Organic Media

Production of Glyceryl Monostearate by Immobilized Candida Antarctica B Lipase in Organic Media

Ferulic acid-based renewable esters and amides-containing epoxy thermosets from wheat bran and beetroot pulp: Chemo-enzymatic synthesis and thermo-mechanical properties characterization

In this study, ferulic acid – a natural phenolic acid present in bagasse, wheat and rice brans, and beetroot pulp – was used as renewable starting material to prepare new biobased epoxy precursors. This biobased building block was first esterified then reduced to provide ethyl dihydroferulate which was then trans-esterified/amidified with biobased diols, triol and diamines. Immobilized Candida antarctica (aka CAL-B or Novozyme 435®) was used to perform the solvent-free-transesterification/amidation under mild conditions. The phenolic architectures synthesized were then glycidylated using a TEBAC-mediated procedure to afford four biobased epoxy precursors – with various linker structures between the ferulic units – which were then cured with conventionnal and biobased diamine curing agents. The thermal and thermomechanical properties of the thermosets prepared were assessed by TGA, DSC and DMA to highlight the influence of the linker configuration (aliphatic vs cycloaliphatic), that of the epoxy functionality (di vs tri), and that of the nature of the function used for coupling ferulic units (ester vs amide) on the thermosets properties. Analyses of these thermosets revealed glass transition temperatures ranging from 32 to 85°C and high thermal stabilities around 300°C. The mechanical behavior of the biobased thermosets proved equivalent to that of the DGEBA-IPDA reference sytem at low temperatures (up to 40–100°C depending on the epoxy precursor and diamine).

Lipase-catalyzed biodiesel production and quality with Jatropha curcas oil: exploring its potential for Central America.

BackgroundExtensive native Jatropha curcas L. (Jatropha) crop areas have been planted in Central America marginal lands since 2008 as a non-edible prospective feedstock alternative to high-value, edible palm oil. Jatropha biodiesel is currently exclusively produced in the region at commercial scale utilizing alkaline catalysts. Recently, a free, soluble Thermomyces lanuginosus (TL) 1,3 specific lipase has shown promise as biocatalyst, reportedly yielding up to 96 % ASTM D6751 compliant biodiesel after 24 h transesterification of soybean, canola oils and other feedstocks. Biodiesel conversion rate and quality of enzymatically catalyzed transesterification of Jatropha oil was evaluated. Two lipases: free, soluble TL and immobilized Candida antarctica (CA) catalyzed methanolic transesterification of crude Jatropha and refined palm oil.ResultsJatropha yields were similar to palm biodiesel with NaOH as catalyst. After 24 h transesterification, Jatropha (81 %) and palm oil (86 %) biodiesel yields with TL as catalyst were significantly higher than CA (<70 %) but inferior to NaOH (>90 %). Enzymatic catalysts (TL and CA) produced Jatropha biodiesel with optimum flow properties but did not complied with ASTM D6751 stability parameters (free fatty acid content and oil stability index).ConclusionsBiodiesel production with filtered, degummed, low FFA Jatropha oil using a free liquid lipase (TL) as catalyst showed higher yielding potential than immobilized CA lipase as substitute of RBD palm oil with alkaline catalyst. However, Jatropha enzymatic biodiesel yield and stability were inferior to alkaline catalyzed biodiesel and not in compliance with international quality standards. Lower quality due to incomplete alcoholysis and esterification, potential added costs due to need of more than 24 h to achieve comparable biodiesel yields and extra post-transesterification refining reactions are among the remaining drawbacks for the environmentally friendlier enzymatic catalysis of crude Jatropha oil to become an economically viable alternative to chemical catalysis.

ChemInform Abstract: Immobilized Candida Antarctica B as Efficient Catalyst for the Synthesis of Local Anaesthetic Intermediates.

AbstractNovel reaction conditions are reported for the synthesis of benzoic esters using candida antarctica lipase B as the promotor.

Immobilised Candida antarctica B as efficient catalyst for the synthesis of local anaesthetic intermediates

Abstract We hereby present the development of new reaction conditions for the CALB catalysed esterification of substituted benzoic acids. Using cyclohexane as the reaction media a number of heptyl benzoates have been easily isolated in good to excellent yields (up to 100% at 80 °C, 20–24 h). Moreover, the catalytic system has been successfully applied to the synthesis of local anaesthetics intermediates also showing good productivity in recycling experiments.

Enzymatic synthesis of 6- and 6′-O-linoleyl-α-d-maltose: From solvent-free to binary ionic liquid reaction media

Abstract The eco-efficient lipase catalyzed synthesis of sugar fatty acid ester surfactants from renewable commodities is a stimulating challenge in biotransformation. The biocatalytic investigations went from solvent-free and minimal ionic liquid use to bulk organic solvents, and binary solvents thereof. Disaccharide maltose was acylated with linoleic acid (C18:2) on the primary O-6,6′ hydroxyl functions. The ionic liquid [emim][MeSO3] and the potentially renewable acetone enabled best conversions. Binary solvents like acetone/DMF and [emim][MeSO3]/[bmpyr][PF6] tended to double conversions. The lipase selection is also crucial and the enzymes Pseudomonas cepacia and immobilized Candida antarctica allowed highest yields in a screening with 10 different lipases. Also other non-solvent parameters such as reaction time and molecular sieve content improved maltose transformation further up to 82%. Analysis by HPLC, ESI-MS, and NMR indicated the formation of mono-6 or 6′-O-linoleyl-α- d -maltose as a mixture of two regioisomers in a 1.4:1 ratio. From an applied point of view, the best solvent is acetone. Unlike ionic liquids and binary mixtures, it is easily removable from reaction mixtures. Moreover, acetone is to some degree a green solvent as it can be produced directly from renewable feedstock.

ChemInform Abstract: Enzymatic Synthesis of N‐Acylethanolamines: Direct Method for the Aminolysis of Esters.

Abstract Immobilized Candida antarctica (Novozyme 435) catalyzed synthesis of N -acylethanolamines is described. Treatment of methyl esters with lipase and amines yielded the desired amides within 2–24 h with yields ranging from 41% to 98%.

ChemInform Abstract: Application of Chemoenzymatic Hydrolysis in the Synthesis of 2‐Monoacylglycerols.

Abstract The selective biocatalyzed synthesis of 2-monoacylglycerols (2-MAGs) through the use of commercially available immobilized Candida antarctica (Novozym435) and Rhizomucor miehei is explored. Reactions at room temperature result in the formation of a 2-MAG and a corresponding ethyl ester of the fatty acid with immobilized C. antarctica within 2 h with yields ranging from 36% to 83%. Similar reaction conditions with immobilized R. miehei yielded exclusively the 2-MAG after 24 h with yields ranging from 37% to 88%. Yields vary on the acyl group at the sn -2 position and choice of enzyme involved.

Enzymatic synthesis of N-acylethanolamines: direct method for the aminolysis of esters

Immobilized Candida antarctica (Novozyme 435) catalyzed synthesis of N-acylethanolamines is described. Treatment of methyl esters with lipase and amines yielded the desired amides within 2–24h with yields ranging from 41% to 98%.

Application of chemoenzymatic hydrolysis in the synthesis of 2-monoacylglycerols

The selective biocatalyzed synthesis of 2-monoacylglycerols (2-MAGs) through the use of commercially available immobilized Candida antarctica (Novozym435) and Rhizomucor miehei is explored. Reactions at room temperature result in the formation of a 2-MAG and a corresponding ethyl ester of the fatty acid with immobilized C. antarctica within 2h with yields ranging from 36% to 83%. Similar reaction conditions with immobilized R. miehei yielded exclusively the 2-MAG after 24h with yields ranging from 37% to 88%. Yields vary on the acyl group at the sn-2 position and choice of enzyme involved.

A spectrophotometric transesterification-based assay for lipases in organic solvent

A new method to evaluate lipase activities in nonaqueous conditions using vinyl ester absorbance at ultraviolet (UV) wavelengths is described. The model reaction is the transesterification between vinyl stearate and pentanol in hexane at 30 °C or in decane at 50 °C. The conversion of vinyl stearate into pentyl stearate is monitored through decreasing UV absorbance at 200 nm. Six commercial lipases were tested with this method, and results were compared with gas chromatography (GC) quantification and a classical spectrophotometric method using p-nitrophenyl palmitate. Results from the new spectrophotometric assay are similar both to results from GC quantification ( R 2 = 0.999) and to results from p-nitrophenyl palmitate ( R 2 = 0.989). The proposed method is able to evaluate both high activity from immobilized lipases such as immobilized Candida antarctica B lipase (3060 ± 350 U g −1) and low activity from crude enzymatic extracts such as Carica papaya dried latex (0.1 ± 0.04 U g −1). The method has also been used to measure kinetic parameters of C. antarctica B lipase for vinyl stearate and the correlation between its synthesis activity and its concentration. The method has also proved to be effective in studying the acyl selectivity of a lipase by comparing its activities with increasing chain lengths of vinyl esters.

Ketoprofen resolution by enzymatic estirification and hydrolysis of the ester product

ImmobilizedCandida antarctica lipase was used to catalyze the separation of ketoprofen into its components by means of esterification followed by the enzymatic hydrolysis of the ester product. In this study, ketoprofen underwent esterification to ethanol in the presence of isooctane. When the reaction was complete, 58.3% of the ketoprofen had been transformed into an ester. The ketoprofen remaining in solution after the reation was complete consisted primarily of itsS-enantiomer (83.0%), while the 59.4% of the ketoprofen component of the ester consisted of itsR-enantiomer. We then subjected the ester product to enzymatic hydrolysis in the presence of the same enzyme and produced a ketoprofen product rich in theR-enantiomer; 77% of this product consisted of theR-enantiomer when 50% of the ester had been hydrolyzed, and 90% of it consisted of theR-enantiomer when 30% of the ester had been hydrolyzed. By contrast, theR-enantiomer levels only reached approximately 42 and 65%, respectively, when 50 and 30% of the racemic ester was hydrolyzed under the same conditions.

Analysis of solvent-free ethyl oleate enzymatic synthesis at equilibrium conditions

This work reports experimental equilibrium data for the esterification of pure oleic acid and a fatty acid mixture with ethanol, using an immobilized Candida antarctica B lipase as catalyst. Reactions are performed in a solvent-free system, containing a mixture of substrates and different amounts of distilled water. According to the initial amount of water and the extent of the reaction, one or two liquid phases are present. Therefore, when the equilibrium is achieved, the liquid–liquid and chemical reaction equilibria have to be simultaneously satisfied. Several reports dealing with enzymatic reactions performed in two-phase systems have found that the value of the reaction equilibrium constant calculated from overall experimental concentrations varies not only with temperature but also with substrate ratio and water content. Although this approach is a valuable way to explore equilibrium shifts in biphasic systems, it is limited to ideal systems with constant partition coefficients. The aim of this work is to consider the biphasic nature of the reactive mixture through a computational procedure that simultaneously takes into account liquid–liquid and reaction equilibria. This approach enables the determination of a classical temperature-dependent thermodynamic equilibrium constant, which accurately fits experimental equilibrium conversions over a wide range of operating conditions.

Enzymatic kinetic resolution of secondary alcohols by esterification with FA under vacuum

AbstractKinetic resolution of some chiral secondary alcohols [2‐octanol, 1‐phenylethanol, and 1‐(2‐naphthyl)ethanol] with high enantioselectivity (E&gt;300) was achieved by direct esterification with FFA catalyzed by immobilized Candida antarctica B lipase. The reaction equilibrium was shifted toward the synthetic side by the removal of the water formed under vacuum. Esterification of rac‐2‐octanol at an alcohol/FFA molar ratio of 2∶1 was used as a model reaction. The chain length of FFA and their structure influenced the reaction rate but did not have a measureable effect on E. The best acyl donor was decanoic acid: &gt;47% conversion at 4 h with virtually perfect E. Temperature did not affect E in the range studied (15–45°C), but temperatures at the higher end afforded improved reaction rates. The reaction rates and E were compared for three different acyl donors. The initial reaction rate increased in the following order: ethyl laurate &lt; lauric acid &lt; vinyl acetate. E was high (E&gt;300) for all acyl donors. Racemic 1‐phenylethanol and 1‐(2‐naphthyl)ethanol were also resolved by direct esterification with decanoic acid in short times (3 and 4 h, respectively) with E&gt;300 and excellent conversions. Preparative‐scale kinetic resolution of 2‐octanol was also performed.