Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

Enzymatic synthesis of geranyl acetate inn-hexane withCandida antarctica lipases

Geranyl acetate is an important flavor and fragrance compound. Two immobilizedCandida antarctica lipases, SP382 and SP435, were investigated for their use in the synthesis of geranyl acetate by direct esterification. Yields between 95 and 99% molar conversion were obtained with 2 and 15% (w/w reactants) of SP435 and SP382 lipases, respectively. Optimum yields were obtained at 0.1M acetic acid and 0.12M geraniol after 16-h incubation. No inhibitory effect was observed at increasing concentrations of geraniol. Addition of 60% (w/w reactants) water led to 50 and 60% reduction in the esterification activity of SP382 and SP435 lipases, respectively. The best yields were obtained at added water contents between 0–5% (w/w reactants). Solvents with a logP value of 0.85 or more gave reaction yields of more than 80% molar conversion. Higher logP values did not necessarily lead to higher conversion yields. The immobilized lipase SP382 was still active after reusing ten times in the direct esterification reaction.     

Enzymatic synthesis of geranyl acetate by transesterification with acetic anhydride as acyl donor

Pseudomonas sp. lipase (PS) was immobilized by adsorption technique onto glass beads and tested for its ability to synthesize geranyl acetate by transesterification with acetic anhydride as the acyl donor. Reactions were carried out inn-hexane containing 0.1 M geraniol, 0.1 M acetic anhydride, and 200 units of lipase PS. Enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse were studied. Yields of up to 96% were obtained with 200 units (approximately 11% w/w of reactants) of enzyme. Increasing amounts of geraniol inhibited lipase activity, while excess acyl donor concentration enhanced ester production. Yields as high as 97% were obtained at 50°C, 24 h incubation, with no added water. Solvents with logP values ≥3.0 showed the highest conversion yields. Solvent-free samples also performed well. The pH range of 4–9 gave good yields (92–98.4%). Enzyme reuse studies showed the lipase remained active after 15 runs.     

Synthesis of monoterpene esters by alcoholysis reaction with Mucor miehei lipase in a solvent-free system

The syntheses of geranyl acetate and citronellyl acetate by alcoholysis reaction catalyzed by immobilized lipase from Mucor miehei was studied for the first time in a solvent-free system. Reactions were carried out at a terpene alcohol/acyl donor molar ratio of 1:5 with Lipozyme at 10% of the total weight of the reactants in a solvent-free system. Incubations were carried out at 55 to 60°C for ethyl and butyl acetates as acyl donors, whereas for methyl acetate the incubation temperature was 40 to 45°C. Excess concentration of acyl donor increases the percentage of geranyl acetate and citronellyl acetate, while excess of terpene alcohol concentration decreases the same. Yields from 75 to 77% molar conversion (90 to 98% conversion, w/w) were obtained after 8 to 28 h of reaction time.     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.     

Partial purification of <Emphasis Type="Italic">nigella sativa</Emphasis> L. Seed lipase and its application in hydrolytic reactions. Enrichment of γ-linolenic acid from borage oil

Selective hydrolysis of borage (Borago officinalis L.) oil was catalyzed by two lipase preparations of Nigella sativa L. seeds at 40°C in a mixture of borage oil, water, and hexane. Ammonium sulfate-precipitated lipase (Nigella PL) and lipase partially purified by DEAE-ion exchange chromatography (Nigella CPL) exhibited a negative specificity toward γ-linolenic acid (GLA). Best results were obtained in the experiments conducted with 330 U/g oil of Nigella PL and 200 U/g oil of nigella CPL. When 330 U/g oil of Nigella PL was used, after 8 h the GLA level rose from 21.9% in the starting oil to 29.6 and 41.8% in TAG and DAG fractions of the product mixtures, respectively (1.5-fold enrichment of GLA in the total unhydrolyzed acylglycerol fraction). At 200 U/g oil enzyme concentration of Nigella CPL, after 77 h maximum GLA enrichment was observed in the DAG fraction. The GLA content of the DAG increased to 34.6%, corresponding to almost 1.6-fold enrichment. The relative inability of Nigella sativa lipase(s) to hydrolyze γ-linolenoyl moieties of TAG can be used for the enrichment of this acid in the unhydrolyzed acylglycerol fractions of GLA-containing oils.     

Synthesis of geranyl and citronellyl esters of coconut oil fatty acids through alcoholysis by Rhizomucor miehei lipase catalysis

Synthesis of geranyl and citronellyl esters of mixed fatty acids has been investigated by alcoholysis of coconut oil (CNO) using Rhizomucor miehei lipase. CNO fatty acid esters of geraniol and citronellol have unique mild flavors that can be used in food materials. Both geraniolysis and citronellolysis of CNO produce flavor esters in good yield. Depending on substrate concentration the molar yield is more than 50%. The optimized reaction conditions were: pressure, atmospheric; temperature, 50°C; incubation period, 5 h; and Lipozyme, 10% (w/w).     

Characterization of Cross-Linked Lipase Aggregates

Commercially available microbial lipases from different sources were immobilized as cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linkers. These CLEAs were assayed based on esterification between lauric acid and n-propanol in solvent-free systems. Precipitants were found to have a profound influence on both specific activities and total activity recovery of CLEAs, as exemplified by Candida antarctica lipase B (CALB). Among the CLEAs of CALB studied, those obtained using PEG600, ammonium sulfate, PEG200 and acetone as precipitants were observed to attain over 200% total activity recovery in comparison with acetone powder directly precipitated from the liquid solution by acetone. PEG200 precipitated CLEA gave the best specific activity (139% relative to acetone powder). The results of kinetic studies showed that V _max/K _m does not significantly change upon CLEA formation. This work presents a characterization of CLEAs based on an esterification activity assay, which is useful for exploring the synthetic application potential of CLEA technology with favorable perspectives.     

Partial purification and properties of lipase from germinating seeds of Jatropha curcas L.

Lipase present in the seeds of Jatropha curcas L. was isolated and some of its properties studied. Lipase activity was detected in both dormant and germinating seeds. The lipase was partially purified using a combination of ammonium sulfate precipitation and ultrafiltration, which increased the relative activity of the lipase by 28- and 80-fold, respectively. The lipase hydrolyzed palm kernel, coconut, and olive oils at comparable rates (approximately 5 μg FFA/μg protein/min); palm—Raphia hookeri and Jatropha curcas L.—oils at about twice the rate of the first group of oils; and palm and fish oils at a higher rate than all other oils. The lipase, however, had the highest activity with monoolein. Optimal pH and temperature for maximal lipase activity were 7.5 and 37°C, respectively. The addition of ferric ion (15 mM) to the lipase assay medium caused 90% inhibition of lipase activity, whereas calcium and magnesium ions enhanced lipase activity by 130 and 30%, respectively.     

Synthesis of geranyl acetate by lipase entrap-immobilized in cellulose acetate-TiO2 gel fiber

Lipase from Candida antarctica was entrap-immobilized in cellulose acetate-TiO₂ gel fiber (fiber-immobilized lipase) by the sol-gel method. Syntheses of geranyl acetate and citronellyl acetate catalyzed by the fiber-immobilized lipase were studied in heptane solution. Conversions reached 85% for geranyl acetate after 100 h, and 75% for citronellyl acetate after 80 h, and these values were almost identical to those for syntheses catalyzed by nonimmobilized lipase, although the reaction rate was decreased by immobilization. Compared to those of the non-immobilized lipase and commercially available immobilized lipase (Novozyme 435), the activity of the fiber-immobilized lipase was not particularly affected by changes in reaction conditions, such as bulk water content or substrate concentration. The fiber-immobilized lipase retained a high level of activity after six repeated uses, and almost no enzyme leakage from fiber was observed. However, the reactivity of the fiber-immobilized lipase was depressed at higher temperature, presumably due to dehydration by thermal contraction of the gel fiber.     

Synthesis of geranyl butyrate with the poly(acrylic acid‐co‐hydroxy propyl methacrylate‐cl‐ethylene glycol dimethacrylate) hydrogel immobilized lipase of Pseudomonas aeruginosa MTCC‐4713

Microbial lipases (E.C. 3.1.1.3) are the preferred biocatalysts for the synthesis of various fragrance compounds, such as linalool acetate, citronellal acetate, and geranyl acetate, in organic solvents over chemical synthesis. In this study, a purified alkaline extracellular lipase of Pseudomonas aeruginosa MTCC‐4713 was efficiently immobilized onto a synthetic poly(AAc‐co‐HPMA‐cl‐EGDMA) hydrogel by surface adsorption, and the bound lipase was evaluated for its hydrolytic potential toward various p‐nitrophenyl acyl esters, which differed in their C‐chain length. Among four series of hydrogels prepared by the variation of the concentrations of monomer and crosslinker, two hydrogels, namely, I_5d and I_20d, that exhibited relatively higher protein (lipase activity) bindings were selected to perform hydrolytic and synthetic (geranyl butyrate) reactions in aqueous and organic solvents. The hydrogel‐bound lipase was highly hydrolytic toward p‐nitrophenyl ester (C: 16; p‐nitrophenyl palmitate). The hydrogel‐immobilized lipase was quite stable and retained approximately 57.6% of its original hydrolytic activity after the fifth cycle of reuse under optimized conditions (pH 8.5, 65°C). The hydrogel‐immobilized lipase when used to perform the esterification of geraniol/butyric acid (400 : 100 mM) in n‐heptane resulted in 98.8 mM geranyl butyrate at 65°C under shaking (120 rpm) after 15 h of reaction time. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 100 mg per reaction volume (1 mL) resulted in the complete conversion of the reactants into geranyl butyrate. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading

Rhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost-effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency-loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.     

Production of MAG of CLA by esterification with dehydration at ordinary temperature using <Emphasis Type="Italic">Penicillium camembertii</Emphasis> lipase

Production of MAG with CLA using Penicillium camembertii mono- and diacylglycerol lipase (referred to as lipase) was attempted for the purpose of expanding the application of CLA. The commercial product of CLA (referred to as FFA-CLA) is a FFA mixture containing almost equal amounts of 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA. Esterification of FFA-CLA with glycerol without dehydration achieved 84% esterification but produced almost equal amounts of MAG and DAG. Esterification with dehydration not only achieved a high degree of esterification but also suppressed the formation of DAG. When a mixture of FFA-CLA/glycerol (1∶2, mol/mol), 1% water, and 200 units/g-mixture of P. camembertii lipase was agitated at 30°C for 72 h with dehydration at 5 mm Hg, the degree of esterification reached 95% and the contents of MAG and DAG were 90 and 6 wt%, respectively. This reaction system may be applied to the industrial production of MAG with unstable CLA.     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

由月桂烯合成香叶基丙酮的研究

以月桂烯为原料,与氯化氢反应生成香叶基氯及其同分异构体,产物与乙酰乙酸乙酯发生取代反应,经水解脱羧制得香叶基丙酮。月桂烯的加成反应是整个反应过程的关键步骤,对合成香叶基丙酮有决定性影响;重点探讨了催化剂种类及用量、反应温度及原料配比对月桂烯与氯化氢加成反应的影响。得到最适宜反应条件:以CuCl和四丁基溴化铵为催化剂,用量为月桂烯摩尔分数的1%,温度为10 ℃,n(月桂烯):n(氯化氢)=1:1。该条件下,氯化物的产率可达92.86%,且香叶基氯和橙花基氯占氯化物总量的75.71%。氯化物经取代反应,水解脱羧后得到香叶基丙酮,产率为76.62%。     

New Sex Attractant Composition for the Click Beetle <Emphasis Type="Italic">Agriotes proximus</Emphasis>: Similarity to the Pheromone of <Emphasis Type="Italic">Agriotes lineatus</Emphasis>

While testing traps baited with a blend of geranyl octanoate and geranyl butanoate (pheromone components previously identified for Agriotes lineatus, Coleoptera, Elateridae) in Portugal and Bulgaria, large numbers of the closely related Agriotes proximus were captured. In the literature, two different compounds, (E,E)-farnesyl acetate and neryl isovalerate had previously been identified as pheromone components of A. proximus. Subsequent field tests, conducted in several European countries, revealed that A. proximus was weakly attracted to geranyl butanoate on its own, while A. lineatus was weakly attracted to geranyl octanoate on its own. However, the largest catches for both species were observed with a blend of both compounds. No A. proximus was caught in traps baited with the blend of (E,E)-farnesyl acetate and neryl isovalerate at any of the test sites. In electroantennographic studies, antennae of male A. proximus and A. lineatus both gave greater responses to geranyl butanoate than to geranyl octanoate, suggesting that the perception of these two compounds was similar for both species. A 1:1 blend of geranyl octanoate and geranyl butanoate can be used as a bait in traps for the detection and monitoring of both A. lineatus and A. proximus in many European countries.     

The positional and fatty acid selectivity of oat seed lipase in aqueous emulsions

The positional and fatty acid selectivities of oat (Avena sativa L.) seed lipase (triacylglycerol hydrolase EC 3.1.1.3) were examined. Pure triacylglycerols were used as substrates. The products of lipolysis were examined by thin-layer chromatography and gas-liquid chromatography. Only symmetrical triacylglycerols were used as substrates; thus potential complications arising from stereobias were avoided. Controls were carried out with a lipase specific for primary positions. The lipase from oat seeds catalyzed the hydrolysis of both primary and secondary esters. When the lipase was tested upon mixtures of homoacid triacylglycerols (triacylglycerols composed of the same three fatty acids), the lipase acted most rapidly upon those containing oleate, elaidate, linoleate and linolenate. Strong intermolecular selectivity against homoacid triacylglycerols containing palmitate, petroselinate and stearate was observed. Comparison of assays performed at 26°C with those performed at 45°C showed that selectivity was temperature-independent. When mixed-acid triacylglycerols containing both oleate and stearate were treated with lipase, intramolecular selectivity was observed, with oleate hydrolysis predominating. From this work and earlier work, it can be concluded that the selectivity exhibited by the oat seed lipase is similar to that of the lipase fromGeotrichum candidum, except that the oat seed lipase attacks elaidate, a fatty acyl group with atrans double bond, whereas theG. candidum lipase strongly discriminates against elaidate.     

Enzymatic syntheses of N-lauroyl-β-alanine homologs in organic media

Enyzmatic amidation of the primary amines β-alanine ethyl ester and 3-aminopropionitrile with methyl laurate by means of immobilized lipase (Candida antarctica lipase, CAL) resulted in the formation in good yield of N-lauroyl-β-alanine ethyl ester and 3-(N-lauroylamino)-propionitrile, respectively. When 3-amino-propionitrile was used as substrate, diisopropyl ether was a suitable solvent. Changing the reaction temperature (12–80°C) did not affect the yields, and room temperature was a suitable temperature for this reaction. In the investigation of reaction conditions, the use of equimolar amounts (5 mmol) of substrate and ester, along with 0.5 g of CAL, in diisopropyl ether gave the best yield (99.3%) after 24 h of incubation at 24°C. The enzyme activity in the amidation reaction did not decrease even after six uses. With β-alanine ethyl ester hydrochloride as substrate, diisopropyl ether was unsuited as a solvent owing to the low solubility of the substrate in this solvent. In this reaction, the best yield (82.0%) was attained by using dioxane as solvent. CAL achieved higher extents of amide synthesis with long-chain than with short-chain ester substrates. The enzyme accepted only nonbulky primary amines as substrates.     

Lipase biocatalysis in the production of esters

Lipase biocatalysis was investigated as a tool for the production of butyl oleate and rapeseed oil 2-ethyl-1-hexyl ester by esterification and transesterification, respectively. We screened 25 commercially available lipases and found that butyl oleate was produced at high yields from oleic acid and 1-butanol by lipases fromCandida rugosa, Chromobacterium viscosum, Rhizomucor miehei, and Pseudomonas fluorescens. The initial water content of the system, lipase quantity, and the molar ratio of 1-butanol to oleic acid were important factors in influencing the ester yield. In general, no ester was formed without the addition of water. The exception wasCh. viscosum lipase, which yielded 98% of ester in 12 h with 1-butanol excess without additional water. The addition of 3.2% water increased the initial rate of reaction. With an oleic acid excess and only 0.3% lipase,C. rugosa andR. miehei lipases yielded 94 and 100% esters with initial water contents of 3.2 and 14%, respectively. Lipase-catalyzed alcoholysis of low-erucic acid rapeseed oil and 2-ethyl-1-hexanol without additional organic solvent also was studied in stirred batch reactors. In this case,C. rugosa lipase was the best biocatalyst with an optimal 2-ethyl-1-hexanol to rapeseed oil molar ratio of 2.8, a minimum of 1.0% added water, and 37°C. An increase in temperature up to 55°C increased the rate of reaction but did not affect the final ester yield. The enzyme was inactivated at 60°C. Under optimal conditions, the ester yield increased from 88% in 7 h to nearly complete conversion in 1 h when the lipase content was increased from 0.3 to 14.6%. In a 2-kg small pilot scale, up to 90% conversion (97% of theoretical) was obtained in 8 h at 37°C with 3.4% lipase in the presence of Amberlite XAD-7 resin with 3% added water.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.