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 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.     

Production of geranyl propionate by enzymatic esterification of geraniol and propionic acid in solvent‐free system

Background: This work reports the optimization of geranyl propionate production by esterification of geraniol and propionic acid in a solvent‐free system using a commercial lipase as catalyst. For this purpose, a sequential strategy was performed applying two experimental designs. Results: The operating conditions that optimized geranyl propionate production were determined to be 40 °C, geraniol to propionic acid molar ratio of 3:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the optimum reaction parameters, a kinetic study was carried out evaluating the influence of substrates molar ratio, enzyme concentration and temperature on reaction conversion. Results obtained in this step allow one to conclude that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (5 wt%), temperature of 40 °C and substrates molar ratio of 1:1 afforded nearly complete reaction conversion after 30 min of reaction. Conclusion: New experimental data on enzymatic esterification of geraniol and propionic acid for geranyl propionate production are reported in this work, showing a promising perspective of the technique to overcome the well‐known drawbacks of the chemical‐catalyzed route. Copyright © 2010 Society of Chemical Industry     

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).     

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.     

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.     

Lipase catalyzed synthesis of oleyl oleate: Optimization by response surface methodology

Lipase catalyzed production of oleyl oleate, which is an analogue of jojoba oil, was carried out using oleic acid and oleyl alcohol in the solvent-free system. Novozym 435, immobilized Candida antarctica lipase, was used as a biocatalyst. Response surface methodology (RSM) based on five-level, four-variable central composite rotatable design was used to evaluate the effects of important parameters on the production of oleyl oleate. Acid/alcohol molar ratio (0.5-1.5), enzyme quantity (2-10% w/w of substrates), reaction temperature (40-60°C), and reaction time (30-90 min) were chosen as process variables for the optimization. Among these parameters, enzyme quantity and acid/alcohol molar ratio have significant effects compared with temperature and time on the production of oleyl oleate. Optimum conditions were found to be a acid/alcohol molar ratio of 1, enzyme quantity of 7% (w/w), reaction temperature of 51°C, and reaction time of 75 min. The coefficient of determination (R 2 ) for the model is 0.97. Probability value is 2.9 ‐ 10 m 9 (P-value<0.01). This P-value demonstrates a very high significance for the regression model. The maximum oleyl oleate concentration predicted by the equation (737 g/L) agrees well with the experimentalvalue (734 g/L) obtained from the experimental verification at the optimum values.     

Effects of acetic acid addition methods on butyl acetate enzymatic synthesis

Abstract

Butyl acetate is a versatile chemical due to its excellent solvency for polymers, resins, and oils. In this work, the butyl acetate synthesis was studied by the esterification of n-butanol with acetic acid using the commercial immobilized lipase Novozym^® 435. The acetic acid and n-butanol concentrations have to be limited in the enzymatic synthesis of this ester due to the deleterious effects caused by these reagents on the enzyme. Three methods of acetic acid addition (single, stepwise and continuous addition) were investigated to minimize the acid concentration. A significant drop on acid conversion was observed in the presence of a higher concentration of the reagents when single or stepwise acid addition was used. However, a solvent-free system using a continuous addition of acetic acid favored butyl acetate synthesis, reaching a conversion of 84% at 60?°C, employing 5?wt% of enzyme and n-butanol/acetic acid final molar ratio equal to 1/1.     

Lipase-Catalyzed Synthesis of Saccharide-Fatty Acid Esters Utilizing Solvent-Free Suspensions: Effect of Acyl Donors and Acceptors,and Enzyme Activity Retention

The effect of acyl donor (oleic, caprylic, lauric and myristic acids) and acceptor (fructose, sucrose, glucose and xylose) for synthesis of saccharide-fatty acid esters was conducted using solvent-free (50–200 μm sized) suspensions of saccharide crystals in a mixture of fatty acid/fructose oleate (90 wt% monoester and 10 wt% diester) at a ratio of 75/25 w/w initially, and a bioreactor system containing a packed bed bioreactor filled with immobilized Rhizomucor miehei lipase (Lipozyme^®IM, Novozymes, Franklinton, NC, USA) at 53 °C or a stirred tank bioreactor (STBR) at 65 °C. A nearly linear relationship between initial saccharide concentration and initial rate of reaction and final ester concentration was achieved which was independent of acyl donor or acceptor type. Slightly lower reaction rate and yield were obtained for operation in STBRs. Suspensions containing the highest saccharide concentration coincided with saccharide crystals of the smallest average size, since large-sized crystals sedimented out during the workup for formation of the suspensions. The best performance was achieved using fructose and oleic acid as substrates (92.3 wt% ester, consisting of 92 wt% monoester) in a packed-bed bioreactor (PBBR). The activity of RML did not decrease appreciably after four successive runs for solvent-free fructose-oleate esterification, or equivalently, a 22 day reaction period.     

Parameters affecting the synthesis of geranyl butyrate by esterase 30,000 from Mucor miehei

The factors affecting the synthesis of geranyl butyrate by esterase 30,000 of Mucor miehei were studied in a solvent-free system. The effects of substrate molar ratio, temperature, agitation speed, and initial addition of water were investigated. The equimolar ratio was most interesting for ester production in batch. There were no diffusion limitations, and the reaction could be realized at low agitation. The catalytic activity of the enzyme was irreversibly deactivated at 60°C, and the initial addition of water decreased the rate of conversion after 75 h of reaction. The enzyme activity increased with increased linear chainlength of the acid and was also affected by the alcohol structure. Esterase 30,000 gave the highest conversion of butyric acid with hexanol and terpenic alcohols (citronellol, nerol) and the lowest with the secondary alcohol (2-hexanol). Finally, five other industrial enzymatic preparations were investigated for their ability to synthesize geranyl butyrate and to hydrolyze olive oil. We observed, for the lipase from Rhizopus javanicua, that there is no relationship between hydrolytic and synthetic activities; this example shows that the hydrolytic lipase activity data cannot predict the capability of lipases in esterification reactions.     

Parameters affecting the synthesis of geranyl butyrate by esterase 30,000 from Mucor miehei

The factors affecting the synthesis of geranyl butyrate by esterase 30,000 of Mucor miehei were studied in a solvent-free system. The effects of substrate molar ratio, temperature, agitation speed, and initial addition of water were investigated. The equimolar ratio was most interesting for ester production in batch. There were no diffusion limitations, and the reaction could be realized at low agitation. The catalytic activity of the enzyme was irreversibly deactivated at 60°C, and the initial addition of water decreased the rate of conversion after 75 h of reaction. The enzyme activity increased with increased linear chainlength of the acid and was also affected by the alcohol structure. Esterase 30,000 gave the highest conversion of butyric acid with hexanol and terpenic alcohols (citronellol, nerol) and the lowest with the secondary alcohol (2-hexanol). Finally, five other industrial enzymatic preparations were investigated for their ability to synthesize geranyl butyrate and to hydrolyze olive oil. We observed, for the lipase from Rhizopus javanicua, that there is no relationship between hydrolytic and synthetic activities; this example shows that the hydrolytic lipase activity data cannot predict the capability of lipases in esterification reactions.     

An original transesterification route for fatty acid ester production from vegetable oils in a solvent-free system

Wax ester production from a long-chain alcohol and methyl ester has been investigated with an immobilized thermostable lipase (lipozyme IM from Novo Nordisk). The transesterification reaction rate was monitored in solvent-free medium that was exclusively composed of the reactants and the enzyme. The transesterification is performed by simply mixing the two substrates in various stoichiometric amounts at a temperature range from 55 to 65°C under constant stirring in the presence of low concentrations of enzyme preparation (0.12 to 2%, w/w). Long-chain reactants produce waxes of high molecular mass that induce low solubility and high viscosity. On average, high transesterification yields are obtained (around 95%). Thermodynamic parameters involving substrate concentration and temperature have also been investigated. The balance between optimal working temperature and the molar ratio of substrates in such a complex medium appears to be 60°C, with a molar ratio methyl oleate/stearyl alcohol of 1:0.5. Substrate inhibition due to stearyl alcohol has been observed. A study of kinetic parameters has confirmed these results.     

Enzymatic synthesis of oleyl oleate in dense fluids

Esterification between oleic acid and oleyl alcohol, catalyzed by theMucor miehei immobilized lipase in a batch-stirred tank reactor with supercritical carbon dioxide as solvent produced higher reaction rates at supercritical conditions than in the solvent-free system. A continuous fixed-bed reactor was designed based on the results obtained from batch experiments. At 150 bar, 40°C, and with water activity 0.46% w/w, the activity of the enzyme preparation is practically unchanged when CO₂ was used as solvent. The addition of small amounts of water increases the conversion rate. The higher conversion also was observed at longer residence time. Whenn-butane was used as reaction medium, a decrease in conversion was observed.     

Application of lipase immobilized on nylon‐6 for the synthesis of butyl acetate by transesterification reaction in n‐heptane

A purified alkaline thermotolerant bacterial lipase from Bacillus coagulans BTS‐3 was immobilized on nylon‐6 matrix activated by glutaraldehyde. The matrix showed ～ 70% binding efficiency for lipase. The bound lipase was used to perform transesterification in n‐heptane. The reaction studied was conversion of vinyl acetate and butanol to butyl acetate and vinyl alcohol. Synthesis of butyl acetate was used as a parameter to study the transesterification reaction. The immobilized enzyme achieved ～ 75% conversion of vinyl acetate and butanol (100 mmol/L each) into butyl acetate in n‐heptane at 55°C in 12 h. When alkane of C‐chain lower or higher than n‐heptane was used as an organic solvent, the conversion of vinyl acetate and butanol to butyl acetate decreased. During the repetitive transesterification under optimal conditions, the nylon bound lipase produced 77.6 mmol/L of butyl acetate after third cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Study on synthesis parameters of lipase-catalyzed hexyl acetate in supercritical CO<Subscript>2</Subscript> by response surface methodology

Hexyl acetate, a short-chain ester with fruity odor, is a significant green note flavor compound that is widely used in the food industry. The ability of immobilized lipase from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of hexanol with triacetin in supercritical carbon dioxide was investigated in this study. Response surface methodology and a 3-level-3-factor fractional factorial design were adopted to evaluate the effects of synthesis variables, such as reaction time (30 to 90 min), temperature (35 to 55°C), and pressure (1500 to 3500 psi), on percent molar conversion of hexyl acetate. The results showed that reaction time and pressure were the most important parameters and temperature had less effect on percent molar conversion. Based on canonical analysis, optimal synthesis conditions were as follows: reaction time 69.0 min, synthesis temperature 46.7°C, pressure 2640 psi. The predicted value was 75.6% and the actual value was 77.3% molar conversion.     

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     

Optimization and kinetic modeling of lipase catalyzed enantioselective N‐acetylation of ( ± )‐1‐phenylethylamine under microwave irradiation

BACKGROUND: Optically pure amines are used in the fine chemical industry as resolving agents, chiral auxiliaries, and chiral synthetic building blocks for pharmaceuticals as well as agrochemicals. Lipase‐catalyzed kinetic resolution of ( ± )‐1‐phenylethylamine with ethyl acetate as an acyl donor was achieved using immobilized lipase (Novozyme 435) as a biocatalyst under microwave irradiation. RESULTS: Response surface methodology was employed with a four‐factor‐three‐level Box‐Behnken design to evaluate the effect of synthesis parameters (speed of agitation, enzyme loading, temperature and acyl donor:amine molar ratio) on conversion, enantiomeric excess, enantioselectivity and initial rate. The optimum reaction conditions obtained were mole ratio of acyl donor:amine 1:1, temperature 49.86 °C, 0.03 g of catalyst loading and 345 rpm speed of agitation, giving 49.12% conversion, 78.83% enantiomeric excess and an enantioselectivity of 38.21. R‐stereopreference of lipase was analyzed in detail from the aspects of enzymatic kinetic mechanism and reaction activation energy of both enantiomers. CONCLUSION: Novozyme 435 was found to be the most active chiral catalyst for resolution of ( ± )‐1‐phenylethylamine under microwave irradiation. Statistical analysis was satisfactorily used to determine the optimum reaction conditions. It was found that lipase has R‐stereopreference and the reaction matches the Ping Pong Bi Bi mechanism with dead‐end inhibition of 1‐phenylethylamine. Copyright © 2011 Society of Chemical Industry     

Five-factor response surface optimization of the enzymatic synthesis of citronellyl butyrate by lipase IM77 from Mucor miehei

Response surface methodology (RSM) and a five-level-five-factor central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (3 to 27 h), temperature (25 to 65 °C), enzyme amount (10 to 50%), substrate molar ratio of citronellol to butyric acid (1∶1 to 1∶3), and added water amount (0 to 20%) on molar percent yield of citronellyl butyrate by direct esterification, using lipase IM77 from Mucor miehei. Reaction time and temperature were the most important variables. Substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimal synthetic conditions were these: reaction time 24 h, temperature 60°C, enzyme amount 20%, substrate molar ratio 1∶1.5, and added water 0%. The predicted molar conversion value was 100%. An actual experimental value of 98% molar conversion was obtained.     

无溶剂法合成聚酰乙基苯基次膦酰哌嗪的研究

在无溶荆条件下,以羧乙基苯基次膦酸、哌嗪为原料,合成元卤膨胀型阻燃剂聚酰乙基苯基次膦酰哌嗪.其最适宜的工艺条件为羧乙基苯基次膦酸与哌嗪的摩尔比为1:1,在150℃下反应8 h,产品得率为100.4%,用红外、核磁、元素分析仪、热分析等对产品结构进行了表征,并对其阻燃性能进行了研究.     

Optimization of the enzymatic production of isoamyl acetate with novozym 435 from candida antarctica

In this study the production of isoamyl acetate by esterification of isoamyl alcohol and acetic acid was carried out using immobilized C . antarctica lipase (Novozym 435) as a catalyst without any organic solvent. The esterification yield was optimized with response surface methodology. This method was used with four parameters to evaluate the effects of important variables on the esterification yield. The parameters are acid/alcohol mole ratio (0.2-0.8), enzyme amount (4-12%, w/w), temperature (30-50 °C), and reaction time (4-8 hr). It was found that the most effective parameter was acid/alcohol mole ratio. As acid/alcohol mole ratio increased at any given reaction time and amount of enzyme, ester concentration, C p (mmol ester/g mixture), increased up to an acid/alcohol mole ratio of 0.7 and thereafter decreased. The model indicated the optimum conditions for maximum esterification (3.45 mmol ester/g mixture) in the acid/alcohol mole ratio of 0.52 for 8.15% enzyme at 43.2 °C and after 5.27 hr, which were in good agreement with the experimental value (3.5 mmol ester/g mixture).