High yields of ascorbyl palmitate by thermostable lipase-mediated esterification

High yields of ascorbyl palmitate (6-O-palmitoyl-l-ascorbic acid) were obtained by lipase-mediated esterification using Bacillus stearothermophilus SB 1 lipase. The final yield was greatly influenced by the initial water content of the system, quantity of enzyme, and molar ratio of palmitic acid to l-ascorbic acid. Reaction rates increased directly with temperature from 40 to 100°C. Maximum conversion (97%) was achieved after 30 min at 100°C (solvent-free), 1 h at 80°C (solvent-free), and 2 h at 60°C (solvent/hexane). The synthesis was scaled up to 1-l volume with 95% conversion using 50 mmoles of ascorbic acid and 250 mmoles of palmitic acid in hexane. Similar yields of ester were obtained in five repetitive cycles using 5 g enzyme immobilized on Accurel. The present B. stearothermophilus SB 1 lipase was a more efficient catalyst for the synthesis of ascorbyl palmitate than other commercial lipases.     

Optimization of the synthesis of lower glycerides rich in unsaturated fatty acid residues obtained via enzymatic ethanolysis of sesame oil

The lipases Novozym 435, Lipozyme TL IM and Lipozyme RM IM were employed in the production of lower acylglycerols (LG), i.e. mono‐ (MAG) and diacylglycerols (DAG), rich in unsaturated fatty acids from sesame oil in batch reactors. The effect of the molar ratio of ethanol to fatty acids on the reusability of these immobilized lipases was studied in detail. The effects of pretreatment on lipase activity for ethanolysis were investigated. Glycerol had a strong product inhibition effect on the ethanolysis reaction, and a relatively large excess of ethanol was necessary to remove the glycerol adsorbed on these biocatalysts. The enzymatic activity was drastically reduced by addition of water to the reaction medium. The presence of organic solvents (hexane and acetone) did not favor the production of LG. For the Novozym 435‐catalyzed reaction, optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 15 wt‐% lipase and 50 °C. For Lipozyme TL IM, the optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 20 wt‐% biocatalyst, and 30 °C. Novozym 435 and Lipozyme TL IM produced LG with molar ratios of unsaturated to saturated fatty acids of 20.4 in 1 h and 25.3 in 5 h, respectively. In the original oil, this ratio was 5. For trials conducted under optimum conditions, the products from the Novozym 435 trials contained 21.8 wt‐% triacylglycerols (TAG), 24 wt‐% DAG and 54.2 wt‐% MAG. The products of the Lipozyme TL IM trials consisted of 12.9 wt‐% DAG and 87.1 wt‐% MAG. No TAG species were detected.     

Synthesis of 6-fatty acid esters ofl-ascorbic acid

High yields (85%) of 6-fatty acid esters ofl-ascorbic acid were obtained by reactingl-ascorbic acid (1.2 M) in 98~99% sulfuric acid for 36 hr at 20 C with 36% excess fatty acid. In concentrated sulfuric acid,l-ascorbic acid was rapidly sulfated to produce mainlyl-ascorbyl 6-sulfate, which slowly reacted with fatty acid to producel-ascorbyl 6-acylate.     

Lipase-catalyzed alcoholysis of crambe oil and camelina oil for the preparation of long-chain esters

Crambe oil and camelina oil were transesterified with oleyl alcohol, the alcohols derived from crambe and camelina oils, n-octanol or isopropanol using Novozym 435 (immobilized lipase B from Candida antarctica), Lipozyme IM (immobilized lipase from Rhizomucor miehei), and papaya (Carica papaya) latex lipase as biocatalysts. The highest conversions to alkyl esters were obtained with Novozym 435 (up to 95%) in most cases, whereas Lipozyme IM and papaya latex lipase gave lower (40 to 50%) conversions. The conversions with long-chain alcohols (oleyl alcohol, crambe alcohols, and camelina alcohols) were higher (40 to 95%) than with medium-chain n-octanol (30 to 85%). Isopropyl esters of crambe oil and camelina oil were obtained with rather low conversions using Novozym 435 (<40%) and Lipozyme IM (about 10%) as biocatalysts, whereas with papaya latex lipase no isopropyl esters were formed. The conversions of crambe oil and camelina oil to oleyl and n-octyl esters using Novozym 435 as biocatalyst were hardly affected by the ratio of the substrates, but with Lipozyme IM the conversions to alkyl esters distinctly increased with an excess of alcohol substrate Presented as part of the doctoral thesis of Georg Steinke to the University of Münster, Münster, Germany     

Synthesis of Ascorbyl Palmitate with Immobilized Lipase from Pseudomonas stutzeri

Synthesis of ascorbyl palmitate by enzymatic esterification of palmitic acid and ascorbic acid was conducted in an organic medium with Pseudomonas stutzeri lipase TL immobilized in different supports and its performance was compared with commercial Novozym 435 lipase used as a reference. The enzyme was immobilized in different supports and the best catalyst was selected in terms of immobilization yield and mass specific activity to perform the reactions of synthesis. Synthesis of ascorbyl palmitate was optimized considering temperature, substrate molar ratio and enzyme to limiting substrate mass ratio as variables, and substrate conversion and specific productivity as evaluation parameters. The best reaction conditions for immobilized lipase TL were 55 °C, 1:5 ascorbic to palmitic acid molar ratio, and 1:10 lipase to ascorbic acid mass ratio, obtaining 57 % substrate conversion and a specific productivity of 0.013 [g ascorbic acid/(g enzyme × min)]; the best conditions for Novozym 435 were 70 °C, ascorbic to palmitic acid molar ratio 1:10, and 1:10 lipase to ascorbic acid mass ratio, obtaining 51 % substrate conversion and a specific productivity of 0.016 [g ascorbic acid/(g enzyme × min)].     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Production of Human Milk Fat Substitutes Catalyzed by a Heterologous Rhizopus oryzae Lipase and Commercial Lipases

This study aims to produce human milk fat substitutes by an acidolysis reaction between lard and the free fatty acids (FFA) from a fish oil concentrate rich in docosahexaenoic acid, in solvent-free media. The immobilized commercial lipases from (1) Rhizomucor miehei (Lipozyme RM IM), (2) Thermomyces lanuginosa (Lipozyme TL IM) and (3) Candida antarctica (Novozym 435) were tested as biocatalyst. Also, the heterologous Rhizopus oryzae lipase (rROL), immobilized in Accurel^® MP 1000, was tested as a feasible alternative to the commercial lipases. After 24 h of reaction at 50 °C, similar incorporations of polyunsaturated fatty acids (c.a. 17 mol%) were attained with Novozym 435, Lipozyme RM IM and rROL. The lowest incorporation was achieved with Lipozyme TL IM (7.2 mol%). Modeling acidolysis catalyzed by rROL and optimization of reaction conditions were performed by response surface methodology, as a function of the molar ratio FFA/lard and the temperature. The highest acidolysis activity was achieved at 40 °C at a molar ratio of 3:1, decreasing with both temperature and molar ratio. Operational stability studies for rROL in seven consecutive 24-h batches were carried out. After the fourth batch, the biocatalyst retained about 55 % of the original activity (half-life of 112 h).     

Antioxidative properties and enzymatic synthesis of ascorbyl FA esters

Efficient synthesis of unsaturated FA esters of ascorbic acid is possible with only a small excess of one of the reactants in t-amyl alcohol using Candida antarctica lipase as biocatalyst. Using free acids, we obtained yields that were comparable to yields reached using vinyl-activated acyl donors (71, 80, and 86% yields of esters with FA excesses of 1∶1, 1∶1.5, and 1∶2, respectively). As very low water activity is needed to achieve sufficiently high yields of product, molecular sieves were used to improve the ascorbyl ester yields. Ascorbyl oleate is more amorphous and has a much lower m.p. and lower enthalpy of fusion than ascorbyl palmitate. This leads to a higher solubility of ascorbyl oleate in oil, resulting in an increased antioxidant effect compared to that of the palmitate. In an accelerated storage test using deodorized rapeseed oil, samples incubated with ascorbyl palmitate showed noticeable oxidation after 1 wk of storage, whereas samples incubated with ascorbyl oleate displayed negligible oxidation for 9 and 4 wk at 30 and 40°C, respectively.     

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor. In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.     

Lipase-catalyzed incorporation of conjugated linoleic acid into tricaprylin

Three commercially available immobilized lipases, Novozym 435 from Candida antarctica, Lipozyme IM from Rhizomucor miehei, and Lipase PS-C from Pseudomonas cepacia, were used as biocatalysts for the interesterification of conjugated linoleic acid (CLA) ethyl ester and tricaprylin. The reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The effects of molar ratio, enzyme load, incubation time, and temperature on CLA incorporation were investigated. Novozym 435, as compared to Lipozyme IM and Lipase PC-C, showed the highest degree of CLA incorporation into tricaprylin. By hydrolysis with pancreatic lipase, it was found that Lipozyme IM and Lipase PS-C exhibited high selectivity for the sn-1,3 position of the triacylglycerol early in the interesterification, with small extents of incorporation of CLA into the sn-2 position, probably due to acyl migration, at later reaction times. A small extent of sn-1,3 selectivity during interesterification by Novozym 435 was observed.     

Lipase-catalyzed preparation of palmitic and stearic acid-rich phosphatidylcholine

Saturated FA enhance the oxidative stability of phospholipids. In the present study phosphatidylcholine (PC) rich in palmitic and stearic acids was prepared using lipase-catalyzed transesterification from PC isolated from egg and soybean lecithins. Two different lipases, namely, Novozym 435 and Lipozyme TL IM, were used for the transesterification. The reaction conditions were optimized by varying the lipase dosage, molar ratio of PC to FA, and reaction period. Palmitic acid could be incorporated up to 58.6 and 57.1% using Lipozyme TL IM and 56 and 61% using Novozym 435 in egg and soybean PC from an initial content of 37.4 and 16.8%, respectively. Similarly, stearic acid incorporation was up to 44.7 and 46.3% using Lipozyme TL IM and 37.2 and 55.8% using Novozym 435 in egg and soybean PC from an initial content of 8.6 and 2.1%, respectively.     

Synthesis of n‐butyl acetamide over immobilized lipase

BACKGROUND: The conversion of carboxylic esters to amides can be accomplished efficiently by enzymatic catalysis. Amidation of benzyl acetate with n‐butyl amine was studied in non‐aqueous media using immobilized lipases. RESULTS: The activities of immobilized lipases, Novozym 435, Lipozyme RM IM and Lipozyme TL IM were evaluated in the synthesis of n‐butyl acetamide, among which Novozym 435 was the best. The process was optimized by studying various process parameters. Benzyl acetate conversion of 46% was achieved in 8 h for a mole ratio of 3:1 of n‐butyl amine to benzyl acetate with 3.67 g L^?1 Novozym 435 in toluene at 55 °C. A model based on an ordered bi–bi mechanism fitted the initial rate data very well and the rate constant and inhibition constants were calculated by non‐linear regression analysis. The initial rate studies showed that the Michaelis constant for benzyl acetate was low indicating high affinity between the enzyme and the reactant. CONCLUSION: A novel, efficient and environmentally benign enzymatic process is reported for the synthesis of n‐butyl acetamide. This method is general and can be used to synthesize analogous compounds in optically enriched form, since it is difficult to make such amides directly from carboxylic acids and amines by purely chemical means. The theoretical predictions and experimental data matched very well. Copyright © 2008 Society of Chemical Industry     

Continuous production of acyl <Emphasis Type="SmallCaps">l</Emphasis>-ascorbates using a packed-bed reactor with immobilized lipase

Saturated acyl (6-O-caproyl, lauroyl, and myristoyl) and unsaturated acyl (6-O-oleoyl, linoleoyl, and arachidonoyl) l-ascorbates were continuously synthesized at 50°C using a system where a column packed with ascorbic acid powder and a packed-bed reactor with an immobilized lipase from Candida antarctica were connected in series. A productivity of 1.6–1.9 kg/L reactor·d was achieved for at least 11 d. The surface tension of the caproyl or lauroyl l-ascorbate in aqueous solution was measured at various temperatures and pH to estimate the critical micelle concentration (CMC) of the acyl l-ascorbate. The CMC values were independent of temperature but dependent on the pH. The value of the caproyl ascorbate increased with an increase in pH.     

Synthesis of Fatty Acid Ethyl Ester from Acid Oil in a Continuous Reactor via an Enzymatic Transesterification

Synthesis of a fatty acid ethyl ester via the lipase‐catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by‐product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half‐life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.     

C18 Unsaturated Fatty Acid Selectivity of Lipases During the Acidolysis Reaction Between Tripalmitin and Oleic,Linoleic, and Linolenic Acids

The C18 unsaturated fatty acid (UFA) selectivity of three immobilized lipases, namely, Lipozyme TL IM from Thermomyces lanuginosa, Lipozyme RM IM from Rhizomucor miehei, and Novozym 435 from Candida antarctica, was determined in acidolysis conducted in hexane. Tripalmitin with a mixture of equimolar quantities of C18 UFAs was used as the substrate. Significantly different incorporation rates were observed for C18 UFAs used (p < 0.05). The highest incorporation was obtained for all three C18 UFAs with Novozym 435 followed by Lipozyme RM IM and Lipozyme TL IM catalyzed acidolysis under default conditions (substrate mole ratio 1:1; temperature 50 °C; reaction time 6 h; enzyme dosage 10%). Incorporation of the equimolar quantities of C18 UFAs was in the order C18:3 > C18:2 > C18:1 which also reflects C18 UFAs preferences of the lipases. The effects of operating variables on incorporation or UFA selectivity of lipases were also investigated. Among the experimental parameters including the mole ratio of fatty acid to triolein, temperature, enzyme dosage, and time on incorporation, the effect of the substrate mole ratio on UFA selectivity was greater than those of the others.     

Synergism of microwave irradiation and enzyme catalysis in synthesis of isoniazid

Isoniazid is a useful antitubercular drug widely employed in combination therapy with rifampicin. The synthesis of isoniazid from ethyl isonicotinate and hydrazine hydrate was studied in non‐aqueous media via lipase‐catalyzed hydrazinolysis under both conventional heating and microwave irradiation by using different supported lipases. Among three different commercial lipases used, namely Novozym 435 (Candida antarctica lipase), Lipozyme RM IM (Rhizomucor miehei lipase) and Lipozyme TL IM (Thermomyces lanuginosus lipase), Novozym 435 was found to be the most effective, with conversion of 54% for equimolar concentrations at 50 °C in 4 h. The rate of reaction as well as final conversion increased synergistically under microwave irradiation in comparison with conventional heating, which showed 36.4% conversion, even after 24 h, for the control experiment. Effects of various process parameters such as speed of agitation, catalyst loading, substrate concentration, product concentration and temperature were studied. A kinetic model is also described. Copyright © 2007 Society of Chemical Industry     

Chemical and enzymatic interesterification of a beef tallow and rapeseed oil equal‐weight blend

A mixture of beef tallow and rapeseed oil (1:1, wt/wt) was interesterified using sodium methoxide or immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as catalysts. Chemical interesterifications were carried out at 60 and 90 °C for 0.5 and 1.5 h using 0.4, 0.6 and 1.0 wt‐% CH₃ONa. Enzymatic interesterifications were carried out at 60 °C for 8 h with Lipozyme IM or at 80 °C for 4 h with Novozym 435. The biocatalyst doses were kept constant (8 wt‐%), but the water content was varied from 2 to 10 wt‐%. The starting mixture and the interesterified products were separated by column chromatography into a pure triacylglycerol fraction and a nontriacylglycerol fraction, which contained free fatty acids, mono‐, and diacylglycerols. It was found that the concentration of free fatty acids and partial acylglycerols increased after interesterification. The slip melting points and solid fat contents of the triacylglycerol fractions isolated from interesterified fats were lower compared with the nonesterified blends. The sn‐2 and sn‐1,3 distribution of fatty acids in the TAG fractions before and after interesterification were determined. These distributions were random after chemical interesterification and near random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at the sn‐2 position remained practically unchanged, compared with the starting blend. The interesterified fats and isolated triacylglycerols had reduced oxidative stabilities, as assessed by Rancimat induction times. Addition of 0.02% BHA and BHT to the interesterified fats improved their stabilities.     

Chemoenzymatic epoxidation of unsaturated fatty acid esters and plant oils

In the presence of an immobilized lipase fromCandida antacrtica (Novozym 435^R) fatty acids are converted to peroxy acids by the reaction with hydrogen peroxide. In a similar reaction, fatty acid esters are perhydrolyzed to peroxy acids. Unsaturated fatty acid esters subsequently epoxidize themselves, and in this way epoxidized plant oils can be prepared with good yields (rapeseed oil 91%, sunflower oil 88%, linseed oil 80%). The hydrolysis of the plant oil to mono- and diglycerides can be suppressed by the addition of a small amount of free fatty acids. Rapeseed oil methyl ester can also be epoxidized; the conversion of C=C-bonds is 95%, and the composition of the epoxy fatty acid methyl esters corresponds to the composition of the unsaturated methyl esters in the substrate. Based partly on a lecture at the 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 7–11, 1995.     

固定化脂肪酶催化制备香叶树籽生物柴油研究

研究了Novozym 435和Lipozyme TLIM混合脂肪酶催化香叶树籽油制备生物柴油,2种酶按1:3质量比混合使用时,既可提高反应转化率,又可降低酶的使用成本.应用响应面优化法确定了固定化酶催化香叶树籽生物柴油的最优工艺参数,采用叔丁醇作为反应体系的溶剂,最优反应条件为反应温度38.5℃、甲醇与油摩尔比4:1、叔丁醇与油体积比1:1.5、酶用量为油质量的4%,此时反应转化率达90.09%.分析表明香叶树籽油的甘油三酯主要由短链脂肪酸甘油酯组成,生物柴油中原油的甘油三酯已完全转变成脂肪酸甲酯.     

Chemo-enzymatic synthesis of amino acid-based surfactants

The application of lipases to the synthesis of amino acid-based surfactants was investigated. Low yields (2–9%) were obtained in the acylation of free amino acids, such as l-serine and l-lysine, as well as their ethyl esters and amides with fatty acids, owing in part to low miscibility of the reactants. When the N-carbobenzyloxy (Cbz)-l-amino acids were used in an effort to improve miscibility of the amino acid derivatives with the acyl donor, a dramatic improvement was observed for N-Cbz-l-serine (92% yield) but not for N _α-Cbz- or N _ζ-Cbz-l-lysine (7 and 2% yield, respectively). As an alternative, and efficient synthesis of N _ζ-acyl-l-lysines was developed, based on the regiospecific chemical acylation of copper(II) lysinate. In pursuit of a general route to amino acid-fatty acid surfactants, the utility of a polyol linker was investigated. Thus, the glycerol ester of N _α′ N _ζ-di-Cbz-l-lysine was prepared and evaluated as a substrate for acylation. As expected, this and other glycer-1-yl esters of N-protected amino acids were excellent substrates for lipase-catalyzed acylation. Their reaction with myristic acid in the presence of Novozyme resulted in the regioselective acylation of the primary hydroxyl group of the glycerol moiety to afford the corresponding 1-O-(N-Cbz-l-aminoacyl)-3-O-myris-toylglycerols with conversions of 50–90%. These were readily deprotected to give a range of 1-O-(aminoacyl)-3-O-myristoyl-glycerols with overall yields of 27–71%.