Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Biotechnological production of biodiesel fuel using biocatalysed transesterification: A review

Biotechnological production of biodiesel has attracted considerable attention during the past decade compared to chemical-catalysed production since biocatalysis-mediated transesterification has many advantages. Currently, there are extensive reports on enzyme-catalysed transesterification for biodiesel production; the related research can be classified into immobilised-extracellular and immobilised-intracellular biocatalysis and this review focusses on these forms of biocatalyst for biodiesel production. The optimisation of the most important operating conditions affecting lipase-catalysed transesterification and the yield of alkyl esters, such as the type and form of lipase, the type of alcohol, the presence of organic solvents, the content of water in the oil, temperature and the presence of glycerol, are discussed. However, there is still a need to optimise lipase-catalysed transesterification and reduce the cost of lipase production before it is applied commercially. Optimisation research of lipase-catalysed transesterification could include development of new reactor systems with immobilised biocatalysts, the use of lipases tolerant to organic solvents, intracellular lipases (whole microbial cells) and genetically modified microorganisms (intelligent yeasts). Biodiesel fuel is expensive in comparison with petroleum-based fuel and 60–70% of the cost is associated with feedstock oil and enzyme. Therefore ways of reducing the cost of biodiesel with respect to enzyme and substrate oils reported in literature are also presented.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Enzymatic Preparation of Mono- and Di-Stearin by Glycerolysis of Ethyl Stearate and Direct Esterification of Glycerol in the Presence of a Lipase from Candida Antarctica (Novozym 435)

Mixtures of 1(3)-monostearin and distearin were prepared by direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym 435) using a variety of solvents of differing polarity. In all cases, the transesterification resulted in higher product yields. In n-heptane as reaction medium the addition of water (3%) was essential for high product yields, with mono- and distearin being produced in almost equal amounts. Using more polar solvents as reaction media, such as acetonitrile or acetone, again the highest yields were obtained in the transesterification mode; employing these solvents the reactions were much more selective towards the formation of monostearin.     

Pseudomonas cepacia lipase catalyzed esterification and transesterification of 3-(furan-2-yl) propanoic acid/ethyl ester: A comparison in ionic liquids vs hexane

A comparison of the Pseudomonas cepacia lipase (lipase PS) catalyzed esterification of 3-(furan-2-yl) propanoic acid and transesterification of ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols (propanol to octanol) in ionic liquids and hexane was carried out. The ionic liquids selected, [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisted of an identical cation and different anions. This is the first report on the biocatalyzed synthesis of these esters. In all the media, lipase PS catalyzed esterification of 3-(furan-2-yl) propanoic acid resulted in high yields of the esters compared to the transesterification of ethyl 3-(furan-2-yl) propanoate. [Bmim]Tf₂N proved to be the best; yielding 98–67% of the product by lipase PS catalyzed esterification. The lipase PS–[Bmim]Tf₂N and lipase PS–[Bmim]PF₆ mixture was recycled five times without any decrease in the yields of the products and was found to be operationally stable up to 10 months at room temperature.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system

The monoethyl esters of the long chain fatty acids (biodiesel) were prepared by alcoholysis of Jatropha oil, a non-edible oil, by a lipase. The process optimization consisted of (a) screening of various commercial lipase preparations, (b) pH tuning, (c) immobilization, (d) varying water content in the reaction media, (e) varying amount of enzyme used, and (f) varying temperature of the reaction. The best yield 98% (w/w) was obtained by using Pseudomonas cepacia lipase immobilized on celite at 50 °C in the presence of 4–5% (w/w) water in 8 h. It was found that yields were not affected if analytical grade alcohol was replaced by commercial grade alcohol. This biocatalyst could be used four times without loss of any activity.     

Production of biodiesel by Burkholderia cepacia lipase as a function of process parameters

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME.     

Alcoholysis of olive oil for producing wax esters by intracellular lipase in immobilized fungus cells

Summary Alcoholysis of olive oil was carried out with lauryl and palmityl alcohols for producing wax esters. The reaction can be catalyzed efficiently by cell-bound lipase of Rhizopus niveous fungus cells immobilized within cellulose biomass support particles. Influences of reaction conditions such as water content, temperature, and substrate concentrations on reaction rates and yields were investigated.     

Enzymatic synthesis of alpha-butylglucoside linoleate in a packed bed reactor for future pilot scale-up

The enzymatic synthesis of a mixture of unsaturated fatty acid alpha-butylglucoside esters, containing more than 60% alpha-butylglucoside linoleate, was achieved through lipase-catalyzed esterification. The continuous evaporation under reduced pressure of the water produced enabled substrate conversions greater than 95% to be reached. Two immobilized lipases from Candida antarctica (Chirazyme L2, c.-f., C2) and Rhizomucor miehei (Chirazyme L9, c.-f.) were compared in stirred batch and packed bed configurations. When the synthesis was carried out in stirred batch mode, C. antarctica lipase appeared to be of greater interest than the R. miehei enzyme in terms of stability and regioselectivity. Surprisingly, a change in the process design to a packed bed configuration enabled the stability of R. miehei lipase to be significantly improved, while the C. antarctica lipase efficiency to synthesize unsaturated fatty acid alpha-butylglucoside esters was slightly decreased. Water content in the microenvironment of the biocatalyst was assumed to be responsible for such changes. When the process is run in stirred batch mode, the conditions used promote the evaporation of the essential water surrounding the enzyme, which probably leads to R. miehei lipase dehydration. In contrast, the packed bed design enabled such water evaporation in the microenvironment of the biocatalyt to be avoided, which resulted in a tremendous improvement of R. miehei lipase stability. However, C. antarctica lipase led to the formation of 3% diesters, whereas the final percentage of diesters reached 21% when R. miehei enzyme was used as biocatalyst. A low content of diesters is of greater interest in terms of alpha-butylglucoside linoleate application as linoleic acid carrier, and therefore the enzyme choice will have to be made depending on the properties expected for the final product.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor miehei lipase

Partial acylation of only one primary hydroxyl group of glycerol generates a chiral center at position 2. Rhizomucor miehei lipase (RML) catalyzes the kinetically controlled transesterification of different aromatic carboxylic acids methyl esters with glycerol. High synthetic yields of glyceryl esters (around 70-80%) were obtained even in the presence of significant concentrations of water (from 5% to 20%). After a long incubation of the reaction mixture in the presence of the biocatalyst only pure free acid was obtained. Other lipases (from Geobacillus thermocatenulatus and from Thermomyces lanuginose) also catalyzed similar kinetically controlled transesterifications although less efficiently. RML immobilized on Sepharose-Q showed a high activity and specificity, compared to the immobilization by other techniques, only producing monoglyceryl esters with all substrates. In particular, monoglyceryl-phenylmalonate product was synthesized in 82% overall yield and >99% diastereomeric excess at pH 7.0 and 37 °C and 90% glycerol.     

Resolution of rac-ketoprofen esters by enzymatic reactions in organic media

Enzyme-catalyzed reactions in organic media of rac-ketoprofen esters with different nucleophiles such as alcohols, amines, and water have been studied. Among the parameters optimized are the enzyme, the activated substrate, and the solvent. With the enzymes used in this study the preferred substrate was the trifluoroethyl ester of rac-ketoprofen (rac- 2 ), whose (R)-enantiomer reacted preferentially. The enzyme of choice was the lipase M-AP-10 from Mucor miehei and best results were obtained with diisopropyl ether as solvent. Three different methods have been scaled-up for the resolution of 75–150 g of substrate: transesterification with 1-butanol (90% yield of (S)-ketoprofen, 88% ee), transesterification with 2-(2-pyridyl)ethanol (94% yield, 92% ee), and hydrolysis in wet organic solvent (93% yield, 97% ee). Despite the comparable chemical and optical yields obtained with these three methods, the use of 2-(2-pyridyl)ethanol and the hydrolysis allowed a much easier work-up and isolation of the desired (+)-(S)-ketoprofen. © 1993 Wiley-Liss, Inc.     

Resolution of racemic carboxylic acids via the lipase‐catalyzed irreversible transesterification of vinyl esters

The lipase‐catalyzed irreversible transesterification procedure using vinyl esters was applied to the resolution of racemic 2‐phenoxypropanoic acids. Aspergillus niger lipase showed high enantioselectivities and reasonable reaction rates. The enantioselectivity was found to be affected profoundly by several variables, e.g., the alcohol as nucleophile, the organic solvent used, and the reaction temperature. A gram‐scale resolution of (RS)‐2‐phenoxypropanoic acid was achieved after optimization of the reaction conditions. Then this irreversible transesterification procedure was applied to the resolution of some related 2‐substituted carboxylic acids. Thus, racemic 2‐methoxy‐2‐phenylacetic acid was resolved via the A. niger lipase‐catalyzed transesterification of the corresponding vinyl ester. 2‐Phenylpropanoic acid and 2‐phenylbutanoic acid were resolved using Pseudomonas sp. lipase. A gram‐scale resolution of 2‐phenylbutanoic acid was achieved by this procedure coupled with the porcine liver esterase‐catalyzed hydrolysis of the resulting methyl ester. Chirality 11:554–560, 1999. © 1999 Wiley‐Liss, Inc.     

Optimization of lipase-catalyzed biodiesel by response surface methodology

Biodiesel prepared by catalyzed mild transesterification has become of much current interest for bioenergy. The ability of a commercial immobilized lipase (Novo Industries--Bagsvaerd, Denmark) from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of soybean oil and methanol was investigated in this study. Response surface methodology and 5-level-5-factor central composite rotatable design were employed to evaluate the effects on reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. Based on ridge max analysis, the optimum synthesis conditions giving 92.2% weight conversion were: reaction time 6.3 h, temperature 36.5 degrees C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.     

Biodiesel production through lipase catalyzed transesterification: An overview

Recently, with the global shortage of fossil fuels, excessive increase in the price of crude oil and increased environmental concerns have resulted in the rapid growth in biodiesel production. The central reaction in the biodiesel production is the transesterification reaction which could be catalyzed either chemically or enzymatically. Enzymatic transesterification has certain advantages over the chemical catalysis of transesterification, as it is less energy intensive, allows easy recovery of glycerol and the transesterification of glycerides with high free fatty acid contents. Limitations of the enzyme catalyzed reactions include high cost of enzyme, low yield, high reaction time and the amount of water and organic solvents in the reaction mixture. Researchers have been trying to overcome these limitations in the enzyme catalyzed transesterification reaction. This paper is meant to review the latest development in the field of lipase catalyzed transesterification of biologically derived oil to produce biodiesel.     

Enzymatic synthesis of short‐chain esters in n‐hexane and supercritical carbon dioxide: Effect of the acid chain length

Enzymatic synthesis is the preferred way to produce so‐called “natural products.” Hydrolases have been used for short‐chain ester synthesis. These esters present a pleasant flavor and they have a lot of applications in different industries. Novozym 435 from Candida antarctica (EC 3.1.1.3, triacylglycerol lipase) was used for hexyl ester synthesis in n‐hexane and supercritical carbon dioxide (SCCO₂). Direct esterification provided higher yields than transesterification for the synthesis of esters. Several carboxylic acids of different chain lengths were tested for the esterification reactions: acetic, propionic, butyric, caproic and caprylic acids. The reactions were carried out at 40°C and the amount of enzyme used was 13.8 g/mol alcohol. Substrates were added at equimolar concentrations, with sufficient stirring to avoid external diffusion control. Different substrate concentrations up to 1.5 M were used. The working pressure was 14 MPa in the case of SCCO₂ and atmospheric pressure in the case of organic solvent. The results in both solvents show that the reaction rate increases with the chain length of the acid, but the final yields were similar. However, some of the reactions prove to be faster in SCCO₂, except for hexyl acetate and propionate synthesis, in which acetic and propionic acid presented a lower solubility in SCCO₂ due to its high polarity. Moreover, an acetic acid concentration of 1.5 M brought about a strong inhibition of the enzyme activity.     

Synthesis of the immunosuppressive agent 2-morpholinoethyl mycophenolate by a lipase-catalyzed transesterification

The synthesis of 2-morpholinoethyl mycophenolate was realized by an enzymatic transesterification of simple esters of mycophenolic acid with 2-morpholinoethanol. Best results were achieved by a Candida antarctica lipase B (CAL B) catalyzed transesterification of ethyl mycophenolate in toluene. CAL B showed to selectively transform only the ethyl ester function leaving unreacted the other functional groups present on the substrate. By this way 2-morpholinoethyl mycophenolate was obtained in satisfactory yields from mycophenolic acid (84%).     

Enantiomeric resolution of asymmetric glycol by a lipase‐catalyzed transesterification reaction

The lipase catalyzed enantiomeric resolution of syn‐glycol was carried out to confirm the sector method, which can determine the absolute configuration of anti‐ and syn‐glycol from the ¹H‐NMR spectra of bis‐2‐methoxy‐2‐trifluoromethyl‐2‐phenylacetic acid (MTPA) esters. The lipase catalyzed transesterification reaction was most reactive at the C2 position (C2–OH) of (2R;3R)‐2,3‐octanediol. Both (2S;3S)‐ and (2R;3R)‐2,3‐octanediol were prepared using lipase. The ¹H‐NMR spectra of their bis‐(R)‐MTPA esters agreed well with those prepared previously via mono‐(R)‐MTPA esters. The result suggests the retention of the Mosher plane in MTPA esters possessing a hydroxyl group at the β position. The reaction rate and the stereoselectivity decreased at C2–OH with the addition of 18‐crown‐6. Chirality 11:338–342, 1999. © 1999 Wiley‐Liss, Inc.     

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica

The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 micromol min(-1) g(-1) by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45 degrees C to 60 degrees C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL(-1). Myristic acid esters of fructose (22.3 micromol min(-1) g(-1)), alpha-D-methyl-glucopyranoside (26.9 micromol min(-1) g(-1)) and maltose (1.9 micromol min(-1) g(-1)) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate.