Lipase-catalyzed Esterification Reactions Research Articles

A review of synthesis of esters with aromatic, emulsifying, and lubricant properties by biotransformation using lipases.

The esterification reactions catalyzed by lipases are among the most significant biochemical processes of industrial relevance. The lipases have the function of versatility by catalyzing a diversity of reactions with extreme ease, obtaining quality products with high yield. Therefore, enzyme-catalyzed esterification has gained increasing attention in many applications, due to the importance of derived products. More specifically, lipase-catalyzed esterification reactions have attracted interest in research over the past decade, due to the increased use of organic esters in the chemical and biotechnology industry. These esters can be obtained by three techniques: extraction from natural sources, chemical and enzymatic syntheses. Biotechnological processes have offered several advantages and are shown as a competitive alternative to chemical methods due to high catalytic efficiency, mild operating conditions, and selectivity of natural catalysts. These an industrial point of view, reactions catalyzed by enzymes are the most economical approach to achieve green products with no toxicity and no harm to human health. Thus, this review presents a descriptive evaluation of the trends and perspectives applied to enzymatic esterification, mainly for the synthesis of esters with different properties, such as aromatics, emulsifiers, and lubricants using the esterification process. An emphasis is given to essential factors, which affect the lipase-catalyzed esterification reaction. In which, the parameters are dependent on the lipase source, a form of the biocatalyst (free or immobilized), the polarity of the reaction medium, the molar ratio between alcohol and acid, among other variables, are also discussed.

One-pot production of butyl butyrate from glucose using a cognate \u201cdiamond-shaped\u201d E. coli consortium

Esters are widely used in plastics, textile fibers, and general petrochemicals. Usually, esters are produced via chemical synthesis or enzymatic processes from the corresponding alcohols and acids. However, the fermentative production of esters from alcohols and/or acids has recently also become feasible. Here we report a cognate microbial consortium capable of producing butyl butyrate. This microbial consortium consists of two engineered butyrate- and butanol-producing E. coli strains with nearly identical genetic background. The pathways for the synthesis of butyrate and butanol from butyryl-CoA in the respective E. coli strains, together with a lipase-catalyzed esterification reaction, created a “diamond-shaped” consortium. The concentration of butyrate and butanol in the fermentation vessel could be altered by adjusting the inoculation ratios of each E. coli strain in the consortium. After optimization, the consortium produced 7.2 g/L butyl butyrate with a yield of 0.12 g/g glucose without the exogenous addition of butanol or butyrate. To our best knowledge, this is the highest titer and yield of butyl butyrate produced by E. coli reported to date. This study thus provides a new way for the biotechnological production of esters.

Lipase-catalysed synthesis of mono- and di-acyl esters of glyceryl caffeate in propylene carbonate and their antioxidant properties in tuna oil

Development of new non-toxic antioxidants with diverse hydrophobic properties is important due to growing concerns about the toxicity of artificial oil-soluble antioxidants, the comparatively low effectiveness of natural options, and the complex role hydrophobicity plays in antioxidant effectiveness. Using caffeic acid, a naturally occurring phenolic acid with potent antioxidant activity, a range of glyceryl caffeate esters (decanoate and palmitate) were prepared using lipase-catalysed esterification reactions. Glyceryl-1-caffeate (GC) was prepared from ethyl caffeate and glycerol (acting as both the solvent and the substrate), catalysed by immobilised Candida Antarctica lipase B (Novozym-435) at 80 °C under vacuum. Esterification of GC with decanoic acid using immobilised Thermomyces lanuginosus lipase (TLIM) or Novozym-435 was found to be selective towards mono-acylated or di-acylated products, respectively. The reaction was performed in an unconventional solvent, propylene carbonate (PC), which has many of the attributes of a green solvent. Product conversions in PC were comparable to the best performing conventional solvents. In contrast to conventional volatile solvents, the low volatility of PC allowed the reaction to be performed under vacuum, without the need for molecular sieves for removal of water produced during the reaction. Diisopropyl ether was effective at extracting the more lipophilic products from PC. Both the lipase (Novozym-435) and PC were reused four times with only a small loss in conversion efficiency. Glyceryl caffeate esters performed much better than α-tocopherol at protecting bulk tuna oil from oxidation (analysed using Rancimat). A comparison of glyceryl caffeate esters (decanoate/palmitate and mono-/di-acylated) showed that their antioxidant effectiveness in bulk tuna oil was not affected by chain-length, but compounds containing only one fatty ester were slightly more effective than those containing two fatty esters.

Enzymatic preparation of structured triacylglycerides containing γ-linolenic acid

Abstract Structured triacylglycerides (STAG) rich in γ-linolenic acid (GLA) were prepared using a three-step process. First, with the addition of molecular sieves (MS) at different addition times (3, 9, 12 and 15 h) and loads (2.5, 5, 10 and 15%, based on the total weight of substrates), the enrichment of GLA from evening primrose oil (EPO) and 1-butanol (BtOH) was improved via Candida rugosa lipase-catalysed esterification reactions. Secondly, the GLA-enriched fraction was separated by thin-layer chromatography (TLC) to be further set to react during the third step in the presence of glycerol and Candida antarctica fraction B (CALB), under different enzyme loadings (5, 10, 15 and 20%, based on the total weight of substrates), temperatures (30, 40, 50 and 60 °C) and substrates molar ratios (1:1, 2:1, 3:1 and 4:1, GLA:glycerol). 60% of STAG containing 49 wt% of GLA were produced by using 15% of CALB at 60 °C and a 3:1 molar ratio.

Performance Evaluation and Biodegradation Study of Polyvinyl Chloride Films with Castor Oil‐based Plasticizer

AbstractIn the present study, a renewable resource‐based plasticizer was synthesized by the lipase‐catalyzed esterification reaction of furfuryl alcohol (FA) and castor oil fatty acid (COFA). The resultant ester (FA‐COFA ester) was used as secondary plasticizers to the polyvinyl chloride (PVC) films. The PVC films were formulated using the combination of a conventional plasticizer di‐butyl phthalate (DBP) and FA‐COFA ester as a secondary plasticizer at different concentrations. Films were characterized by X‐ray diffraction analysis, scanning electron microscopy, thermal analysis, mechanical performance, and migration stability. A biodegradability study of the PVC films showed increased degradability with increasing concentration of the FA‐COFA ester in the PVC film. The study showed that ester of FA and COFA could be a substitute of DBP by as much as 80% of the total plasticizer with improved elongation and tensile properties, and such a kind of sustainable resource‐based PVC blend films could be used as a good packaging material with biodegradable property.

Lipase-Catalyzed Esterification: An Inquiry-Based Laboratory Activity To Promote High School Students’ Understanding and Positive Perceptions of Green Chemistry

An inquiry-based laboratory learning unit was designed to enhance high school students’ understanding of the application of lipase as a catalyst to synthesize isoamyl and geranyl ester derivatives as well as to promote their understanding and positive perceptions of green chemistry. The learning unit consisted of three laboratories and a final presentation. The first laboratory activity allowed students to learn how to adjust macroscale titration techniques to microscale titration techniques so that the method could be used for quantitative determination of ester products in the next two laboratory activities. The second laboratory activity introduced students to a greener way to synthesize ester compounds by replacing a harmful strong acid catalyst with a more environmentally friendly catalyst, an enzyme lipase. The third laboratory activity challenged students to design their own experiments to synthesize other esters using lipase-catalyzed esterification reactions. At the end of the learning unit, students in each group presented the results and conclusions of the third laboratory activity to the whole class and explained how the 12 principles of green chemistry were applied in the laboratory. The results from the Learning Unit Conceptual Test and Learning Unit Perceptions Questionnaire indicate that the learning units can promote students conceptual understanding. They also have positive perceptions toward green chemistry and learning units.

New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate

The enzyme catalyzed esterification of butyric acid by ethanol under anhydrous conditions in n-hexane, through continuous removal of the formed water, follows a ping pong bi-bi reaction mechanism; immobilized lipase-B from Candida antarctica on acrylic resin (Novozyme 435) was used as biocatalyst. For first time, the kinetic data of an enzymatic bi-substrate reaction generating simultaneous double dead-end substrate inhibitions were processed by surface fitting through multiparametric non-linear equations. Both anhydrous CH3CH2OH and/or CH3CH2OD were employed in an attempt to apply the technique of kinetic isotope effects and validate the selection of the best system among two (ping pong bi-bi and ordered bi-bi), which could describe the aforementioned reaction.

Production of structured lipids from hazelnut oil with conjugated linoleic acid by lipase-catalyzed esterification: Optimization by response surface methodology

The aim of this study was to synthesize structured lipid from hazelnut oil with conjugated linoleic acid (CLA) by lipase-catalyzed esterification reaction. The incorporation of CLA into hazelnut oil was optimized by response surface methodology (RSM). Substrate molar ratio, time, and temperature were independent variables chosen for optimization of the reaction. Substrate molar ratio was the most significant variable on the incorporation of conjugated linoleic acid. Good quadratic model was achieved by multiple regression analysis and backward elimination. Optimal incorporation conditions were determined as follows: reaction temperature, 59 °C; time, 6 hours; substrate molar ratio, 4. Under these optimum conditions, a maximum incorporation ratio of CLA into hazelnut oil of 49.42% was achieved.

Lipase-Catalyzed Acetylation and Esterification of Bile Acids.

In this chapter we describe the application of lipases as catalysts in reactions on a relevant family of steroids: the bile acids. Twenty three monoacetyl, diacetyl, and ester derivatives of deoxycholic, chenodeoxycholic, lithocholic, and cholic acids, 15 of them new compounds, were obtained through lipase-catalyzed acetylation, esterification, and alcoholysis reactions in very good to excellent yield and a highly regioselective way. Among them, acetylated ester products, in which the lipase catalyzed both reactions in one pot, were obtained. The influence of various reaction parameters in the enzymatic reactions, such as enzyme source, nucleophile/substrate ratio, enzyme/substrate ratio, solvent, and temperature, was studied. Some of the reported products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. Due to its singular structure containing three hydroxyl groups, cholic acid showed a different behavior in the enzymatic reactions, from that observed for the other three bile acids studied. In order to shed light to different behaviors of bile acids in the enzymatic reactions, molecular modeling was applied to substrates and some derivatives.

Synthesis of L-Ascorbyl Flurbiprofenate by Lipase-Catalyzed Esterification and Transesterification Reactions.

The synthesis of L-ascorbyl flurbiprofenate was achieved by esterification and transesterification in nonaqueous organic medium with Novozym 435 lipase as biocatalyst. The conversion was greatly influenced by the kinds of organic solvents, speed of agitation, catalyst loading amount, reaction time, and molar ratio of acyl donor to L-ascorbic acid. A series of solvents were investigated, and tert-butanol was found to be the most suitable from the standpoint of the substrate solubility and the conversion for both the esterification and transesterification. When flurbiprofen was used as acyl donor, 61.0% of L-ascorbic acid was converted against 46.4% in the presence of flurbiprofen methyl ester. The optimal conversion of L-ascorbic acid was obtained when the initial molar ratio of acyl donor to ascorbic acid was 5 : 1. kinetics parameters were solved by Lineweaver-Burk equation under nonsubstrate inhibition condition. Since transesterification has lower conversion, from the standpoint of productivity and the amount of steps required, esterification is a better method compared to transesterification.

Lipase Catalyzed Esterification Reactions–A Kinetic Model

Lipase Catalyzed Esterification Reactions–A Kinetic Model

An insight into microbial lipases and their environmental facet

Lipases are serine hydrolases that catalyze the hydrolysis and synthesis of esters formed from glycerol and long-chain fatty acids, by acting at the oil–water interface. Lipases from microbial sources have received heightened attention for an array of industrial applications, and these enzymes have been well exploited in the environmental sector as well. In this article, we present an overview of microbial lipase, including the microorganisms from which it could be produced; the application of recombinant DNA technology tools to produce lipase with enhanced properties, the effective use of waste materials as substrates for lipase production; the usage of statistical tools to efficiently optimize the production medium; lipase purification strategies; and the immobilization of the enzyme on a variety of support materials. The next section of the article provides a gist of its application in diversified spheres and focusses exclusively on the environmentally relevant ones. Lipase-catalyzed esterification, transesterification, and interesterification reactions, an emerging area of green chemistry; lipase-mediated in vitro biopolymer synthesis and degradation; and the application of lipase for remediating fat and oil constituents in wastewater are dealt with in-depth. When its full potential is harnessed, the enzyme could play a pivotal role in environmental management.

Chemical and enzymatic synthesis of a library of 2-phenethyl esters and their sensory attributes

We report a simple enzymatic approach to synthesise phenethyl esters as natural flavouring materials. Chemical and lipase-catalysed esterification reactions between fatty acids of C4–C18 and 2-phenethyl alcohol were studied. Both methods were compared qualitatively and quantitatively by GC–MS/FID. The acid and thermal stabilities of 2-phenethyl esters were excellent and can meet the requirements of food matrices under most processing conditions. Sensory evaluation showed that each 2-phenethyl ester with a different carbon-chain-length fatty acid had unique sensory notes. Moreover, through Lipozyme TL IM-mediated transesterification, valuable 2-phenethyl alcohol-derived esters were synthesised from butter oil and 2-phenethyl alcohol. The influence of several physicochemical parameters (temperature, substrate molar ratio, enzyme loading, shaking speed and time) on the transesterification reaction was investigated to give optimal reaction conditions, leading to a high yield of 80.0%.

Optimization of lipase-catalyzed synthesis of fructose stearate using response surface methodology

In this study, immobilized lipase-catalyzed esterification reaction between fructose and stearic acid was examined for the synthesis of a useful compound, fructose stearate, using response surface methodology. The increase of water content in the reaction medium was the negative effect while the increase in initial stearic acid/fructose molar ratio was the greatest positive effect on the yield. The highest fructose stearate yield was obtained as 65% in tert-butanol. The product yield was enhanced in 1-butyl-3-methylimidazolium trifluoromethanesulfonate obtained as 74% under the optimized conditions. The spectroscopic and elemental analysis methods showed that the esterification reaction is regioselective and the product is fructose monostearate.

Lipase-catalyzed regioselective preparation of fatty acid esters of hydrocortisone

A series of fatty acid derivatives of hydrocortisone has been prepared by an enzymatic methodology. Nine 21-monoacyl products and one 3,11,17-triacetyl derivative, nine of them novel compounds, were obtained in a highly regioselective way through lipase-catalyzed esterification, transesterification and alcoholysis reactions. The influence of various reaction parameters such as acylating agent: substrate ratio, enzyme: substrate ratio, solvent, temperature and nature of acylating agent and alcohol was evaluated. Among the tested lipases, Candida antarctica lipase appeared to be the most appropriate and showed a high efficient behavior especially in a one-pot transesterification. The advantages presented by this methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare acyl derivatives of hydrocortisone. These lipophilic compounds are potential products in the pharmaceutical industry.

Lipases in water-in-ionic liquid microemulsions: Structural and activity studies

Water-in-ionic liquid (w/IL) microemulsions formulated with non-ionic surfactants, (Tween 20 or Triton X-100) in 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF 6), were used as media for lipase-catalyzed esterification reactions. The catalytic behavior and stability of lipases from Candida rugosa, Chromobacterium viscosum and Thermomyces lanuginosa in these novel microemulsions were investigated and compared to other microheterogeneous media used so far for enzyme-catalyzed reactions. The catalytic behavior of the enzymes depends strongly on the surfactant concentration and the water content. The dependence of the esterification activity of lipases on molar ratio of water to surfactant ( w o ) follows a bell-shaped profile, presenting a maximum at w o ≈ 5 . The operational stability of lipases in w/IL microemulsions, especially at high incubation temperature (50 °C), was significantly increased compared to that observed in other microheterogeneous media. The highest half-life times (>100 h) were obtained in w/IL microemulsions with low water content. Conformational studies via Fourier transform-infrared (FT-IR) and circular dichroism (CD) spectroscopy indicated that lipases entrapped in w/IL microemulsions in most cases retain their native structure or adapt a more rigid structure compared to other microheterogeneous media, which correlated well with the stability results. A simple procedure suitable for ester separation and enzyme reuse was developed. T. lanuginosa lipase retained 90% of activity after ten reaction cycles in w/IL microemulsions formulated with Tween 20.

Molecular modeling and experimental verification of lipase-catalyzed enantioselective esterification of racemic naproxen in supercritical carbon dioxide

Experimental and simulation analyses were performed on the lipase-catalyzed esterification reaction of racemic naproxen by CALB (candida antarctica lipase B) enzyme in supercritical carbon dioxide. The reaction pathways were investigated by quantum mechanical analysis, and the enantioselectivity of the products was predicted by molecular dynamics simulation analysis. Calculated results from molecular modeling in supercritical carbon dioxide were qualitatively compared with experimental data by using racemic naproxen as a substrate. All molecular modeling results and experimental data were acquired and compared with those in ambient and supercritical condition. Moreover, to verify the stability of enzymatic reaction in each solvent condition, reaction pathways were investigated in several solvent conditions (vacuum, water, hexane and supercritical carbon dioxide), and the stability of enzymatic reaction in supercritical carbon dioxide was compared with other solvent conditions.

Lipase-catalyzed Esterification of (S)-Naproxen Ethyl Ester in Supercritical Carbon Dioxide

A lipase-catalyzed esterification reaction of (S)-naproxen ethyl ester by CALB (Candida antarctica lipase B) enzyme was performed in supercritical carbon dioxide. Experiments were performed in a high-pressure cell for 10 h at a stirring rate of 150 rpm over a temperature range of 313.15 to 333.15 K and a pressure range of 50 to 175 bar. The productivity of (S)-naproxen ethyl ester was compared with the result in ambient condition. The total reaction time and conversion yields of the catalyzed reaction in supercritical carbon dioxide were compared with those at ambient temperature and pressure. The experimental results show that the conversion and reaction rate were significantly improved at critical condition. The maximum conversion yield was 9.9% (216 h) at ambient condition and 68.9% (3 h) in supercritical state. The effects of varying amounts of enzyme and water were also examined and the optimum condition was found (7 g of enzyme and 2% water content).

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.

Kinetic studies on the Rhizomucor miehei lipase catalyzed esterification reaction of oleic acid with 1-butanol in a biphasic system

The kinetics of the esterification of oleic acid with 1-butanol catalyzed by free Rhizomucor miehei lipase in a biphasic system was studied in a batch reactor. The reaction appeared to proceed via a Ping Pong bi–bi mechanism with 1-butanol inhibition. The kinetic constants of the model were determined from experiments at 30 °C with initial concentrations of 0.1 – 0.95 mol L org − 1 oleic acid and 0.1 – 1 mol L org − 1 1-butanol in the organic phase and 0.05–0.2 g L −1 enzyme in the aqueous phase. The model was used to simulate the batch concentration profiles of the product as well as the initial reaction rates. Agreement of the model with both the batch concentration profiles (average error of 7.2%) and the initial reaction rate per experiment (average error of 16.0%) was good.