Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

神经网络优化非水相脂肪酶催化合成抗坏血酸油酸酯的条件研究

抗坏血酸油酸酯具有强抗氧化作用．为了获得脂肪酶催化合成抗坏血酸油酸酯的最适条件,主要研究了反应温度、脂肪酶量、油酸量对抗坏血酸油酸酯合成效果的影响．采用中心组合设计和动量梯度下降神经网络对反应条件网络进行训练仿真,并利用训练好的网络对催化酯化工艺条件进行预测．研究结果表明：经过训练的网络可以很好的模拟反应条件,得到了脂肪酶催化反应的最佳工艺参数．当抗坏血酸0．8g时,反应温度56℃,油酸量0．95g,固定化脂肪酶量0．74g,添加分子筛条件下,抗坏血酸油酸酯的转化率为46.5％．该方法为抗坏血酸酯化催化效果的预测提供了一条可行的途径．     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOT/isooctane). By using n-propanol as the alcohol, an optimal W(0) ([H(2)O]/[AOT] ratio) of 12 was found for the synthesis of n-propyl-ibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short- (propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. (c) 1993 John Wiley & Sons, Inc.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Toward the Synthesis of Pyroglutamate Lauroyl Ester: Biocatalysis Versus Chemical Catalysis

The synthesis of dodecyl pyroglutamate (or pyroglutamate lauroyl ester) was achieved in a two-step process involving a pyroglutamic acid alkyl ester intermediate. The reaction was carried out either by lipase or by chemical catalysis using ion exchange resin. Among the various tested lipases, the one from Candida antarctica B gave the best results allowing 73% formation of the desired ester after 6 h. Comparing the efficiency of this latter lipase with the one of Amberlyst IR120H resin in catalyzing this reaction, the biocatalyst gave a molar yield of pyroglutamate lauroyl ester of 79% compared to 69% when using the ion exchange resin starting with 1.04 mmol substrate in each case.     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Enhancement effect of water activity on enzymatic synthesis of cephalexin

The effect of water activity (a(w)) of the reaction medium on the enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenylglycine methyl ester (PGM) was investigated using the alpha-amino acid ester hydrolase enzyme from Xanthomonas citri. It was found that the synthetic activity of the enzyme and the conversion yield were markedly improved when the a(w) of the reaction medium was lowered to about 0.97. The water activity depressing agents evaluated were glycerol, sucrose, and sorbitol, and the conversion yields were improved up to 170% with 15% glycerol, 230% with 30% sucrose, and 270% with 20% sorbitol, respectively. The extent of favorable effect of a(w) on the conversion yield was not the same among the a(w) depressors, probably due to other unknown interactions between the enzyme and depressors. However, optimal a(w) values corresponding to the maximum conversion yield coincided for all a(w) depressors used. The conversion yield of CEX showed an increasing trend with increasing a(w) up to the optimal a(w) value (0.96-0.97) which corresponds to the maximum conversion yield and a decreasing trend beyond the optimal a(w). There appears to be a delicate balance between the hydrolytic reaction of PGM and synthetic reaction of CEX. The increasing a(w)-[E . PGM] complex and the branched reaction pathway fluxes from [E . PGM] to PG (D-alpha-phenyl glycine) and CEX are balanced in such a way that the maximum CEX conversion yield is obtained at a(w) value of 0.96-0.97. The a(w) depressors stabilized the enzyme somewhat, but this positive effect was considered to be only a minor contribution to the substantial yield enhancement. The a(w) depressor effect on viscosity and in turn the mass transfer rate limitation was ruled out since the change in conversion due to the viscosity change was found to be insignificant. (c) 1993 John Wiley & Sons, Inc.     

Immobilization of steapsin lipase on macroporous immobead-350 for biodiesel production in solvent free system

Commercially available steapsin lipase was immobilized on macroporous polymer beads (IB-350) and further investigated for biodiesel production under solvent free conditions. The fatty acid methyl ester (biodiesel) synthesis was carried out by the methanolysis of fresh and used cooking sunflower oil. The enzymatic reaction for biodiesel synthesis was optimized with various reaction parameters and the obtained reaction conditions were 1: 6 molar ratio (oil: methanol), 50 mg biocatalyst and 20% water content at 45°C for 48 h under solvent free conditions. It was observed that 94% of biodiesel was produced under the optimized reaction conditions. The four step addition of methanol at the interval of 12 h was found to be more effective. Moreover the biocatalyst was effectively reused for four consecutive recycles and was appreciably stable for 90 days. The results obtained highlight potential of immobilized steapsin lipase for biodiesel production.     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Gallic acid‐based alkyl esters synthesis in a water‐free system by celite‐bound lipase of Bacillus licheniformis SCD11501

Gallic acid (3, 4, 5‐ trihydroxybenzoic acid) is an important antioxidant, anti‐inflammatory, and radical scavenging agent. In the present study, a purified thermo‐tolerant extra‐cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross‐linking agent, glutaraldehyde. The celite‐bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite‐bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n‐propyl gallate (72.1%), and n‐butyl gallate (63.8%) at 55^oC in 10 h under shaking (150 g) in a water‐free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and ¹H NMR spectrum analysis. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:715–723, 2015     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6?h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40–60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from Thermomyces lanuginosus

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.     

Isomerase activity of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in the optimized conversion of racemic ibuprofen to (<Emphasis Type="Italic">S</Emphasis>)-ibuprofen

The Candida rugosa lipase catalyzed Dynamic Kinetic Resolution of racemic ibuprofen methyl ester produced (S)-ibuprofen in over 90% yield within 72 h at pH 7.6. The best concentration of various buffers for these reactions ranged from 0.2 to 0.5 M. The commercial lipase was found to be acidic altering the final pH of the reaction mixtures. Dimethylformamide co-solvent maintained the reaction pH better than dimethylsulfoxide. Lower concentrations of ibuprofen methyl ester and higher stirring rates led to faster conversions. The minimal amount of lipase needed was 20 mg/mL buffer. Reaction of (R)-ibuprofen methyl ester under the optimized conditions excluding the lipase led to no racemization, indicating that the conversion of (R)-ibuprofen methyl ester to (S)-ibuprofen is catalyzed by the enzyme, thus, indicating Candida rugosa lipase possess Isomerase activity.     

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.     

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 of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.