Solvent optimization for efficient enzymatic monoacylglycerol production based on a glycerolysis reaction

This study was aimed at screening solvent systems of varying polarities to identify suitable solvents for efficient and practical enzymatic glycerolysis. Several pure solvents and solvent mixtures were screened in a batch reaction system consisting of glycerol, sunflower oil, and Novozym^® 435 lipase. Out of 13 solvents tested, tert-butanol and tert-pentanol were the only pure solvents suitable for a fast glycerolysis reaction with an acceptably high formation of MAG. In these systems, MAG contents of 68–82% were achieved within a few hours. Mixtures of tert-butanol/hexane, tert-pentanol/hexane, and tert-butanol/tert-pentanol in varying ratios also gave high MAG contents (58–78%). The tertiary alcohols tert-butanol and tert-pentanol, or mixtures of one of them with hexane, seemed to be the best choice among the solvents tested with respect to reaction efficiency, practical industrial applications, and steric hydroxyl group hindrance, which suppresses the ester formation with FA.     

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.     

Biocatalytic Heterogeneous Processes of the Esterification of Saturated Fatty Acids with Aliphatic Alcohols

Heterogeneous biocatalysts prepared by immobilizing a recombinant lipase from Thermomyces lanuginosus on mesoporous inorganic supports—silica (SiO₂), alumina (Al₂O₃), and titania (TiO₂)—are comparatively studied in the esterification of fatty acids with aliphatic alcohols. It is found that the T. lanuginosus lipase adsorbed on silica has the highest esterifying activity, while the lipase adsorbed on titania is completely inactivated. SiO₂-based catalysts have high activity and stability in the esterification of saturated fatty acids containing 4–18 carbon atoms (C4–C18) with aliphatic alcohols (C5–C16) in organic solvents (hexane and diethyl ether). The catalysts operate in this reaction for several tens of reaction cycles (>40) without loss of activity. The recombinant rPichia/lip lipase immobilized on silica exhibits the most pronounced specificity for its first substrate, a fatty acid. For instance, the rate of synthesis for esters of low molecular weight acids (С4–С6) is three to four times slower than for the esters of acids with more than seven carbon atoms. The catalyst has a relatively broad specificity for the second substrate, an aliphatic alcohol. It is found that the ester of enanthic acid (C7:0) and butanol (C4) is synthesized at the maximum rate.     

Lipase-catalyzed synthesis of hydroxy stearates and their properties

Hydroxy fatty acid (HFA) esters of long-chain alcohols, such as hydroxy stearates, have potential applications from lubricants to cosmetics. These esters were synthesized enzymatically to overcome the problems associated with chemical processes. An immobilized lipase, Rhizomucor miehei, was employed as catalyst in the esterification reaction between hydroxy-stearic acid as a source of HFA and monohydric fatty alcohols (C₈–C₁₈). The yields of esters were in the range of 82–90% by conducting the reactions at 65±2°C, 2–5 mm Hg pressure, and 10% lipase concentration. The products were analyzed by infrared spectroscopy, and some of their analytical characteristics were determined.     

Production of a biodiesel additive in a stirred basket reactor using immobilized lipase: Kinetic and mass transfer analysis

?A laboratory-scale stirred basket reactor (SBR) was constructed to study the synthesis of an n-butyl oleate ester using Novozym 435. An ester yield of approximately 98% was obtained after 6 h using an equimolar substrate ratio, 3.5 g of enzyme, a reaction temperature of 40 °C, and an impeller speed of 200 rpm. The kinetic data were modeled as a ping-pong bi-bi mechanism using a non-linear regression technique. Statistical analysis of the results showed that a model that incorporated the inhibitory effect of n-butanol yielded the best fit with the following parameters: V _max =24.8mmol L^?1 min^?1, K _{m, oleic acid} =190.8mM, K _{m, n-butanol} =544.7 mM, and K _{i, n-butanol} =158.3mM. Mass transfer effects on the enzyme kinetics were also studied, and the absence of internal and external diffusion limitations on the reaction in the SBR was confirmed by considering calculated values of the Thiele modulus and the Damkohler number. Novozym 435 exhibited satisfactory performance in repeated-batch experiments using SBR.     

Enzyme-Catalyzed Synthesis of Monoacylglycerols Citrate: Kinetics and Thermodynamics

The kinetics and thermodynamics of the esterification of citric acid (CA) and monoglycerides (MGs) for citrate esters of monoacylglycerols catalyzed by Novozym 435 in tert-butyl alcohol system was studied in this work. The relationship between initial reaction rate and temperature was established, based on the Arrhenius law. A linear relationship was established between the initial reaction rate and enzyme load up to 3 g/L, which demonstrated that the influence of external mass transfer limitations on the reaction could be eliminated. The reaction kinetics agreed with the Ping-Pong Bi–Bi mechanism with CA inhibition characterized by V _max, K _B, K _A, and K _iA , values of 0.7092 mmol/(min g), 0.0553, 0.0136 and 0.1948 mol/L, respectively. The model was used to simulate the reaction process. The values calculated from the kinetic mode agreed well with the experimental rate data under the different MG and CA concentrations.     

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     

Utilization of acid oils in making valuable fatty products by microbial lipase technology

Microbial lipase-catalyzed hydrolysis, esterification, and alcoholysis reactions were carried out on acid oils of commerce such as coconut, soybean, mustard, sunflower, and rice bran for the purpose of making fatty acids and various monohydric alcohol esters of fatty acids of the acid oils. Neutral glycerides of the acid oils were hydrolyzed byCanadida cylindracea lipase almost completely within 48 h. Acid oils were converted into fatty acid esters of short- and long-chain alcohols like C₄, C₈, C₁₀, C₁₂, C₁₆, and C₁₈ in high yields by simultaneous esterification and alcoholysis reactions withMucor miehei lipase as catalyst. Acid oils of commerce can be utilized as raw materials in making fatty acids and fatty acid esters using lipase-catalyzed methodologies.     

Gas Chromatographic Separation and Identification of Jacaric and Punicic 2-Ethyl-1-Hexyl Esters

The present study demonstrates the separation of a critical pair of conjugated linolenic acid (CLN) isomers—jacaric acid (JA; c8, t10, c12-18:3) and punicic acid (PA; c9, t11, c13-18:3)—on a 60-m conventional Supelcowax 10 column. The alkyl esters of different alcohols (C₁–C₈) of JA and PA were prepared and analyzed isothermally at 220, 230 and 240 °C. The adequacy of their separation was determined from the separation factors (α) and peak resolutions (R _s). Acceptable resolution (R _s = 1.01) of JA and PA was obtained with their 2-ethyl-1-hexyl ester derivatives at a column temperature of 230 °C. In addition, the Gibbs energy of transfer from solution to gas of the three double bonds \((\Delta_{\text{sln}}^{\text{g}} G_{\text{u}}\)) could be used to describe the interactions of the double bond with the stationary phase. Characterization of 2-ethyl-1-hexyl esters of Jacaranda mimosifolia seed oil at 230 °C demonstrates that the oil contains JA and α- and β-calendic acid as a CLN without the presence of PA. The results suggested that JA could be resolved from PA on a 60-m Supelcowax 10 column as the ethyl hexyl ester.     

Candida sp. 99-125脂肪酶及其在化学品合成中的应用

传统的酯化或转酯化产品的合成通常需要高温、强酸、强碱等相对苛刻的条件,脂肪酶由于其生物催化过程具有高效、高选择性、条件温和和环境友好等特点,在化学品的合成中越来越受到人们的关注。本课题组开发了一种可以用于酯类合成的新脂肪酶,并且实现了该酶的工业化生产。来源于Candida sp. 99-125的脂肪酶在非水相中对酯化和转酯化反应具有高效的催化活性和稳定性。本文介绍了该脂肪酶的发酵生产及其在中长链脂肪酸酯、二元酸酯、维生素A棕榈酸酯、手性化合物以及生物柴油等多种化学品的合成中的应用。     

Long Chaina-phosphono fatty acids,salts and esters

A series of α-phosphono fatty acids and their salts and esters was prepared from pelargonic, capric, lauric, myristic, palmitic, and stearic acids. In comparison to correspondingα-sulfo fatty acids theα-phosphono fatty acids are white solids of higher melting point, weaker acids, less hygroscopic, have a lower critical micelle concentration and are less resistant to hard water. Methyl, isopropyl, and amyl esters RCH. [PO (OH)₂] CO₂R′, were prepared from the α-phosphono fatty acids ; a sulfuric acid catalyst was required in the case of lower boiling alcohols. Hydrolysis studies with sodium methylα-phosphonomyristate showed the a-phosphono ester to be 50 times as stable towards alkali as the correspondingα-sulfo ester, but only one-tenth as stable toward acid hydrolysis. Wetting, foaming, detergent, and other surface active properties of theα-phosphono fatty acids, salts, and esters were measured and compared with those of analogousα-sulfo compounds.     

Synthesis of (Z)‐3‐hexen‐1‐yl acetate by lipase immobilized in polyvinyl alcohol nanofibers

Polyvinyl alcohol (PVA)‐nanofibers‐immobilized lipase were formed by electrospinning. The specific surface area of the nanofiber (5.96 m²/g) was about 250 times larger than that of PVA‐film‐immobilized lipase (0.024 m²/g). The PVA‐nanofibers‐immobilized lipase were used as the catalyst for the esterification of (Z)‐3‐hexen‐1‐ol (leaf alcohol) with acetic acid in hexane. The activity of the nanofiber is equivalent to that of commercially available immobilized lipase (Novozym‐435). The ester conversions of the nanofibers, Novozym‐435, the film and lipase powder reached 99.5% at 5 h, 100% at 5 h, 11.5% at 6 h, and 81.1% at 5.75 h, respectively. The nanofibers‐immobilized lipase showed higher activity for the esterification than the film‐immobilized lipase and lipase powder, probably because it has high specific surface area and high dispersion state of lipase molecules in PVA matrix. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Lipase‐catalyzed synthesis of butyl esters by direct esterification in solvent‐free system

BACKGROUND: Reactions performed under solvent‐free conditions give processes that are environmentally friendly, since most solvents are polluting agents. In this work, the performance of Candida rugosa lipae (CRL) immobilized on styrene‐divinylbenzene (STY‐DVB) or controlled pore silica (CPS), and the commercial lipase Novozym 435, was evaluated for the synthesis of butyl esters in solvent–free systems (SFS). A 2² full factorial design was used to study the influence of the organic acid chain length and the biocatalyst concentration on the esterification performance. RESULTS: When CRL on STY‐DVB was used, the ester formation was influenced by both variables and their interaction. The reaction conversion was higher (63%) using 10% of immobilized system and lauric acid, corresponding to a productivity of 3.62 g L^?1 h^?1 For CRL on CPS, only the effect of biocatalyst concentration was significant, and the highest yield was attained using 20% of immobilized system and caprilic acid. In the case of Novozym 435, the highest yield (49%) was obtained using butyric acid as acyl donor at 15% of immobilized lipase. CONCLUSION: The results allowed better understanding of the influence of important parameters in this environmentally friendly process, which also has the process advantage of a higher volumetric productivity when compared with a solvent system. Copyright © 2007 Society of Chemical Industry     

Synthesis of (Z)-3-hexen-1-yl butyrate in hexane and solvent-free medium using Mucor miehei and Candida antarctica lipases

(Z)-3-Hexen-1-yl butyrate is an important flavor and fragrance compound as it represents the model of a natural herbaceous (green) note. Two immobilized lipases from Mucor miehei (Lipozym IM) and from Candida antarctica (Novozym 435) were investigated for their use in the synthesis of (Z)-3-hexen-1-yl butyrate by direct esterification in n-hexane. To determine optimal conditions for esterification, we examined the following parameters: temperature, amount of lipase, acid/alcohol ratio, and absence of solvent. In n-hexane, bioconversion yields reached 95 (after 4 h) and 92% (after 6 h) for, respectively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2% (w/w reactants)]. In the absence of solvent, at 60°C, Novozym 435-catalyzed esterification afforded the title compound in 80% yield. Up to 250 g (in hexane) and 160 g (without solvent) of ester were easily prepared, in a single operation, at a laboratory scale, in few hours, using 2% (w/w reactants) lipase.     

Study of Some Experimental Parameters in the Synthesis of Triacylglycerols with CLA Isomers and Structural Analysis

The present research deals with the synthesis of structured triacylglycerols (TAG) by enzymatic treatment of sn-1,3-diacylglycerol (sn-1,3-DAG) with conjugated linoleic acid (CLA) isomers using the immobilized lipase from Rhizomucor miehei (Lipozyme^® IM) under different experimental conditions. In particular, the influence of reaction parameters, such as temperature, enzymatic load, reaction time and DAG/CLA ratio has been evaluated using an experimental design software with a screening objective. Two responses have been selected, they are the percentage of CLA isomers in total TAG and in the sn-2- position and a three-level-4-factor fractional factorial experimental design was used to screen the variables. The results showed that the selected experimental variables have an influence on the enzymatic reaction, in particular, the DAG/CLA substrate ratio and the temperature, both of which inversely correlated with CLA incorporation, but also the enzymatic load and the reaction time, both directly correlated with CLA incorporation. The best results for CLA isomer % content both in total TAG (46.3%) and in the sn-2- position (52.2%) were obtained at 40 °C for 96 h, with 20% enzymatic load and a 0.5 reactive ratio.     

Study of TAG ethanolysis to 2-MAG by immobilized Candida antarctica lipase and synthesis of symmetrically structured TAG

Regiospecific ethanolysis of homogenous TAG with immobilized Candida antarctica lipase (Novozym 435) was studied using trioleoylglycerol (TO) as a model substrate. Optimization of the reactant weight ratio revealed that the 2-MAG reaction yield increased when a larger amount of ethanol was used. These results suggested that Novozym 435 showed strict regiospecificity in an excess amount of ethanol. The process optimization (reaction temperature and reactant molar ratio) and a study of lipase specificity for various substrates were performed. Under the optimized conditions (ethanol/TO molar ratio=77∶1 and 25°C), 2-monooleoylglycerol (2-MO) was obtained in more than 98% content among glycerides of the reaction mixture and approximately 88% reaction yield in 4 h. The above reaction conditions were applied for ethanolysis of tridocosahexaenoylglycerol, trieicosapentaenoylglycerol, triarachidonoylglycerol, tri-α-linolenoylglycerol, and trilinoleoylglycerol. Reaction yields ranging from 71.9 to 93.7% were obtained in short reaction times (2.5 to 8 h). Purified (>98%) 2-MO and 2-monodocosahexaenoylglycerol (2-MD) were reesterified with caprylic acid by immobilized Rhizomucor miehei lipase (Lipozyme IM) to afford symmetrical structured TAG. At a stoichiometric ratio of 2-MAG/caprylic acid, 25°C and 2–5 mm Hg vacuum, the glyceride composition of the esterification mixture was approximately 95% 1,3-dicapryloyl-2-oleoylglycerol (COC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 8 h. The regioisomeric purity of both COC and CDC was 100%.     

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.     

Enzymatic synthesis of butyrates of fusel oil

Many esters derived from lower aliphatic acids and alcohols have pleasant fruity aromas. These compounds are common in the plant kingdom. Some are chemically synthesized and used as fragrances. In this work, fruity flavored butyric esters are produced using lipase agents. The substrate is an alcohol-containing fraction obtained by distilling fusel oil, a waste product of the alcohol industry (T _b = 120–140°С). The yield of the target products in the presence of pancreatic lipase and the lipase from the Candida rugosa yeast is ~94.0% under the determined optimum conditions.     

Sulfonic acid cation-exchange resins in the synthesis of straight-chain alkylphenols

Kinetic characteristics of reactions occurring in the alkylation of phenol with straight-chain alkenes (C₉, C₁₆) over the Amberlyst 15 Dry, Amberlyst 35 Dry, Amberlyst 36 Dry, Amberlyst 70, Amberlyst DT, Tulsion 66 MP, Lewatit K2640, Lewatit K 2431, and Relite EXC8D sulfonic acid cation-exchange resins have been determined experimentally. The activities of these catalysts in different types of reactions have been compared. The rate constants of all reactions decrease on passing from 1-nonene to straight-chain hexadecene. In the alkylations of phenol with straight-chain alkenes, the ortho-alkylphenol/para-alkylphenol ratio is practically equal to the statistical ratio for all of the resins examined in their operation range.     

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.