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.     

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.     

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     

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     

Properties and application of poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetate

A range of fatty acid esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presence of a crosslinker, N,N‐methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a poly(methacrylic acid‐co‐dodecyl methacrylate‐cl‐N,N‐methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed a 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydrolytic potential toward various p‐nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p‐nitrophenyl palmitate at a pH of 8.5 and a temperature of 55°C. The hydrolytic activity of the hydrogel‐bound lipase was enhanced by Hg²⁺, Fe³⁺, and NH ions at a concentration of 1 mM. The hydrogel‐bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n‐heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n‐pentane, n‐hexane, n‐heptane, n‐octane, and n‐nonane), reaction time, temperature, molecular sieve (3 Å × 1.5 mm) and scale up (at 50‐mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n‐alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55°C in n‐heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume (2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel‐bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

固定化Candida sp. 99-125脂肪酶催化合成蜡酯(英文)

Wax esters were synthesized in a solvent free system catalyzed by immobilized lipase from Candida sp. 99-125, with oleic acid and cetyl alcohol. The effects of substrate molar ratio, lipase dosage and water removal were investigated in a 50 ml flask incubated in a thermostatic cultivation cabinet. The optimized conditions were: temperature 40 ℃, shaking at 170 r·min-1, acid/alcohol molar ratio 1:0.9, lipase dosage in 10% (by mass) of oleic acid, and open reaction for water removal. As a result, the conversion rate reached 98% for reaction of 8 h. The volume of reactor was scaled up to 1 L three-neck flask. The optimized parameters were: 200 r·min-1 agitation speed, 2.5% (by mass) lipase dosage, others were the same as the parameters described above. The conversion rate reached 95% for reaction of 24 h. The lipase retained 46% conversion rate after reuse for 6, 7 batches. The products were purified by removing remained cetyl alcohol and fatty acids with ethanol and saturated sodium carbonate so-lution, respectively. The purity of the wax ester, cetyl oleate, was 96%. The physical and chemical properties of cetyl oleate were tested and compared with those of jojoba oil. The results show that the product cetyl oleate has great potential to use as the substitute of natural jojoba oil.     

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.     

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.     

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 esterification of cinnamic acid and oleyl alcohol in organic solvent media

The esterification of cinnamic acid (CA) and oleyl alcohol (OA) in organic solvent media by immobilized lipase Novozym 435 was optimized in terms of selected parameters, including the logarithm of the 1‐octanol/water partition coefficient of the organic solvent (log P, 0.29–4.5), initial water activity (a_w, 0.05–0.75), agitation speed (0–200 rpm), temperature (35–65 °C) and ratio of substrates (CA/OA, 1.0:0.5–1.0:6.0). The results showed that the more hydrophobic solvent mixtures and lower initial a_w values resulted in a higher enzymatic activity and bioconversion yield. The most appropriate solvent medium and initial a_w value was the mixture of iso‐octane/2‐butanone (85:15, v/v) and 0.05, respectively. The results also showed that an agitation speed of 150 rpm and a reaction temperature of 55 °C were optimal for the reaction system. The activation energy (E_a) of the esterification reaction was calculated as 43.6 kJ mol^?1. The optimal ratio of CA to OA was 1.0:6.0, with the absence of any inhibition by OA. Using the optimized conditions, the maximum enzymatic activity was 390.3 nmol g^?1 min^?1, with a bioconversion yield of 100% after 12 days of reaction. In addition, the electrospray ionization‐mass spectroscopy analysis confirmed that the major end product of the esterification reaction was oleyl cinnamate. Copyright © 2005 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.     

Synthesis of Oleic Acid Esters with Immobilized Lipase

Esterification of oleic acid and alcohols with immobilized lipase from fungus Mucor miehei in a stirred batch reactor is presented in this article. The degree of conversion of oleic acid to esters was determined by measurement of acid values. The dependence of the synthesis of n-butyl oleate on the amount of enzyme, the influence of temperature on the equilibrium conversion for this reaction and the influence of temperature on initial velocities is presented. The influence of water added to reaction mixture at various reaction conditions on the activity of the enzyme preparation is also presented.     

Synthesis of geranyl acetate by esterification with lipase entrapped in hybrid sol-gel formed within nonwoven fabric

Candida cylindracea lipase was entrapped in organic-inorganic hybrid sol-gel polymers made from tetramethoxysilane (TMOS) and alkyltrimethoxysilanes. By forming the gels within the pores of a nonwoven polyester fabric, a novel immobilized biocatalyst in sheet configuration based on sol-gel en-trapment of the enzyme was obtained. Lipases immobilized in sol-gel matrices efficiently catalyzed the direct esterification reaction of geraniol and acetic acid in anhydrous hexane to produce geranyl acetate. The optimal formulation of the sol-gel solution for enzyme immobilization was at a 20∶1 molar ratio of water to total silane; a 4∶1 molar ratio of propyltrimethoxysilane to TMOS; hydrolysis time at 30 min; and enzyme loading of 200 mg lipase/g gel. Under these conditions, protein immobilization efficiency was 91%, and the specific activity of the immobilized enzyme was 2.6 times that of the free enzyme. Excellent thermal stability was found for the immobilized enzyme in dry form or in hexane solution in the presence of acetic acid, in which case severe inactivation of free enzyme was observed. The immobilized enzyme retained its activity after heating at 70°C for 2 h, whereas the free enzyme lost 80% of its activity.     

Kinetics of lipase catalyzedenantioselective esterification of racemic ibuprofen in isooctane

The kinetics of Candida rugosa lipase catalyzed esteritlcation of racemic ibuprofen with n-butanol in isooctane was studied. The kinetic study was carried out with the addition of 0.1% and 2% (by volume) of water for enzyme activation respectively when celite was added into isooctane for enzyme dispersion. The specific initial rate for S-ibuprofen can be fitted with the Ping Pong Bi Bi mechanism with dead-end competitive inhibition by the alcohol. The time courses of the enantioselective esteriflcation of the two ibuprofen enantiomers with different initial substrate concentrations and water contents were simulated with a model in which both effects of enzyme inactivation by long term reaction and reversed hydrolytic reaction under high water content were taken into consideration.     

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 alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99‐125

A lipase preparation developed from Candida sp. 99‐125 was used for fatty acid alkyl ester synthesis by both enzymatic esterification of fatty acids, and transesterification of oils and fats. The lipase preparation was chosen based on screening of lipases from commercial sources as well as those produced in the laboratory. The effects of enzyme dosage, solvent types, water absorbent additions, inhibition of short‐chain alcohols, alcohol and acid types, molar ratio of substrates, and reusability of the lipase preparation in esterification were studied. Degree of esterification between oleic acid and methanol under optimal conditions reached 92%. Purity of the methyl ester after washing with water and distillation was 98%. Half‐life of the lipase preparation was calculated to be approximately 340 h. For transesterification of rapeseed oil with the same lipase preparation, the amount of methanol and mode of methanol addition to the reaction were also conducted. Transesterification of the oil with stepwise methanol addition reached 83% after 36 h reaction time.     

葡萄糖硬脂酸酯的非水相酶法制备

考察了脂肪酶催化合成葡萄糖硬脂酸酯(GSAE)的非水相转化条件及其效果。从7种商品化脂肪酶中筛选适于非水相酶法制备GSAE的酶,分别探讨脂肪酶种类及用量、助溶剂种类及用量、底物摩尔比、水分、分子筛用量、摇床速率、反应温度、反应时间等因素对GSAE合成反应产率的影响。结果表明,丙酮是适宜的助溶剂,在丙酮反应体系中固定化脂肪酶Novozyme 435是适宜GSAE酯化反应的最好催化剂,水分明显降低脂肪酶活性和酯化反应产率,底物、助溶剂和反应过程的除水对于该酯化反应是十分必要的。正交实验设计优化的酯化反应条件为:Novozyme 435酶用量0.08 g、分子筛用量0.8 g、n(硬脂酸)∶n(葡萄糖)=3∶1、反应温度45℃、丙酮用量10 mL、摇床转速150 r/min、反应时间5 h。该条件下最大反应速率Vm=0.749μmol/(min.g)和Km=0.020 3mmol/L,GSAE产率达64.11%。     

玉米油酶法合成甘油二酯工艺优化研究

以玉米油、甘油为原料,在无溶剂体系中用固定化脂肪酶Lipozyme TL IM催化合成甘油二酯,通过单因素试验和响应面试验研究底物摩尔比(亚油酸/甘油)、反应温度、反应时间、酶质量分数和初始水质量分数等因素对亚油酸转化率的影响。得出:反应温度、底物摩尔比、反应时间、酶质量分数对亚油酸转化率影响较大;影响酯化反应中亚油酸转化率的主次因素依次为反应温度、底物摩尔比、反应时间、酶质量分数;最佳工艺条件为亚油酸和甘油的摩尔比为2.10∶1,反应温度为61.16℃,反应时间为12.17 h,酶质量分数为20.09%,亚油酸转化率达到75.69%。Lipozyme TL IM连续反应3批次,其相对酶活仍有65.7%。     

The effect of immobilization on the hydrolytic/interesterification activity of lipase from Rhizopus niveus

Lipase from Rhizopus niveus was immobilized by physical adsorption on Celite 545 and glass beads. The results showed that the highest immobilization efficiency and specific hydrolytic activity of 96% and 9.2 meq/mg protein/min, respectively, were obtained with Celite as the carrier. However, the specific hydrolytic activity of lipase adsorbed on glass beads by acetone precipitation was similar to that obtained by the Celite carrier, although the protein loading capacity was relatively low. The results showed that lipase immobilized on glass beads exhibited similar activity profiles with respect to reaction time, different enzyme concentrations, and water content, using trimyristin and tripalmitin as substrates, to those obtained with the free enzyme. In contrast, the immobilized lipase on Celite exhibited a considerably lower hydrolytic activity. However, the results also showed that the lipase activities of the free enzyme and the immobilized Celite enzyme were similar when the more hydrophilic triolein was used as the substrate. The interesterification of a mixture of tripalmitin and trimyristin or triolein was carried out using both the free and immobilized enzymes. The results indicated that the hydrolytic activity of lipase was similar in both cases for the first 24 h, after which it decreased dramatically. These findings suggest that at this late stage an equilibrium between the hydrolytic and interesterification reactions was reached.