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.     

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.     

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).     

Optimized interesterification of virgin olive oil with a fully hydrogenated fat in a batch reactor: Effect of mass transfer limitations

The lipase‐catalyzed interesterification of virgin olive oil and fully hydrogenated palm oil (FHPO) was studied in a batch reactor operating at 75 °C. The reactions between olive oil {rich in OOO (32.36%), OPO (21.7%) and OLO (11.6%) [L = linoleic; O = oleic; P = palmitic acid]} and the fully hydrogenated fat {(36.5% PSP, 28.8% PPP, 23.2% SPS) [S = stearic acid]} produced semi‐solid fats. For an initial weight ratio of olive oil to FHPO of 60 : 40, the reaction product is a complex mixture of triacylglycerol (TAG) species. The TAG profile of the fat product is time dependent. Because of the high viscosity of the liquid reagent phase, it was important to determine if mass transfer effects were significant. Hence, the reaction was optimized with respect to the type and speed of agitation employed, temperature, use of solvent, and the type of biocatalyst. Three immobilized lipases [from Thermomyces lanuginosus (TL IM), Rhizomucor miehei (RM IM) and Candida antarctica B (Novozym 435)] were compared as catalysts for the interesterification reaction. Equilibrium is reached four times faster (in 1–4 h) with a magnetic stirrer to provide agitation than when agitation is not sufficient, i.e. when orbital agitation is employed. Equilibrium was reached faster with Lipozyme TL IM than with the other two lipases. The effects of all the factors investigated on the composition of the products have also been determined. Semi‐solid fats obtained with the non‐specific Novozym 435 contain levels of unsaturated fatty acid residues on sn‐2 sites that are similar to the products obtained with the 1(3)‐regiospecific enzymes Lipozyme TL IM and RM IM. The chemical properties of the product semi‐solid fat were characterized. The fat prepared using optimal reaction conditions contained 17.20% OPO, 13.61% OOO, 11.09% POP, and 10.35% OSP isomers as the primary products. The induction time obtained in the assay of the oxidative stability of the fat product was 21 h at 98 °C. The lipases Lipozyme TL IM and Novozym 435 were very stable with residual activities of 90 and 100%, respectively, after 15 batch reaction cycles.     

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     

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.     

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.     

Enzymatic Synthesis of an Isopropyl Ester by Alcoholysis of Camellia Oil

A camellia oil-based isopropyl ester (CO-IPE) was successfully synthesized by enzymatic alcoholysis with camellia oil (CO), and its physiochemical properties were analyzed. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best one. The optimal reaction conditions were achieved at 240 U/g of Novozym 435 loading, a substrate molar ratio of 5:1 (isopropanol/CO), and 24 h of reaction time at 55 °C. Under the above conditions, the content of CO-IPE was obtained as 89.83%. Purity of CO-IPE further increased to be 96.95% after separation by rotary evaporation and molecular distillation. The viscosity of the synthesized CO-IPE showed itself to be about six times lower than that of CO, and the refractive index of the CO-IPE (1.449) was nearer to 1 in contrast to that of CO. It suggested that CO-IPE could be more intensively applied in the cosmetic industry.     

Diacylglycerol synthesis by enzymatic glycerolysis: Screening of commercially available lipases

Seven lipases were screened for their ability to synthesize DAG in the glycerolysis of rapeseed oil. In batch reactions with free glycerol, the lipase carrier was of great importance for catalysis. Catalysis did not take place in reactions with lipases having hydrophilic carriers. The best DAG yield (approx. 60 wt%) was achieved with Novozym 435 and Lipase PS-D after 7 h, and an equilibrium was obtained. Stepwise addition of glycerol allowed catalysis with Novozym CALB L (immobilized) to take place in spite of the hydrophilic carrier; however, the DAG yield was only 19 wt%. This result suggests that glycerol forms a layer around the hydrophilic lipase particles, limiting contact between the lipases and the hydrophobic oil phase. With glycerol absorbed on silica gel, all lipases catalyzed the glycerolysis reaction. Faster conversion of TAG was obtained with Lipase PS-D, Lipase AK, and Lipase F-AP15 than in reactions with free glycerol, but the DAG yield remained approximately 60–65 wt%. Nonspecific lipases yielded more 1,3-DAG early in the reaction.     

Enzymatic production of human milk fat substitutes containing γ-linolenic acid: Optimization of reactions by response surface methodology

Structured lipids resembling human milk fat and containing GLA were synthesized by an enzymatic interesterification between tripalmitin, hazelnut oil FA, and GLA in n-hexane. Commercially immobilized 1,3-specific lipases, lipozyme^® RM IM and Lipozyme^® TL IM, were used as the biocatalysts. The effect of these enzymes on the incorporation levels was investigated. A central composite design with five levels and three factors—substrate ratio, reaction temperature, and time—were used to model and optimize the reaction conditions via response surface methodology. Good quadratic models were obtained for the incorporation of GLA (response 1) and oleic acid (response 2) by multiple regression and backward elimination. The determination coefficient (R ²) values for the models were found to be 0.92 and 0.94 for the reactions catalyzed by Lipozyme RM IM, and 0.92 and 0.88 for the reactions catalyzed by Lipozyme TL IM, respecitively. The optimal conditions generated from the models for the targeted GLA (10%) and oleic acid (45%) incorporation were 14.8 mol/mol, 55°C, and 24 h; 14 mol/mol, 55°C, and 24 h for substrate ratio (moles total FA/mol tripalmitin), temperature and time for the reactions catalyzed by Lipozyme RM IM and Lipozyme TL IM, respectively. Human milk fat substitutes containing GLA that can be included in infant formulas were success-fully produced using both Lipozyme RM IM and Lipozyme TL IM enzymes. The effect of the two enzymes on the incorporation of GLA and oleic acid were found to be similar.     

Influence of silica gel in production of diacylglycerol via enzymatic glycerolysis of palm olein

Enzymatic glycerolysis was explored in this paper for the production of diacylglycerol (DAG) oils from palm olein. Three commercial enzymes, Lipozyme TL IM, Lipozyme RM IM and Novozym 435 were used for their ability to synthesize DAG in a solvent‐free system. Novozym 435 was found to be the more effective enzyme, resulting in a high DAG production even in the absence of an adsorbent such as silica gel. The yields of DAG were between 43 and 50 wt‐%. Lipozyme TL IM and RM IM, being supported on hydrophilic materials, require an adsorbent to allow slow release of glycerol for reaction with the enzyme and oil. In the absence of silica, no reaction was observed. The success of the reaction is therefore very dependent on the amount of silica used. The yields of DAG using Lipozyme TL IM and RM IM were 52 and 45 wt‐%, respectively. In addition, the degree of reduction in tocopherols and tocotrienols appeared correlated with the efficacy of the glycerolysis reaction. Changes in the slip melting points and solid fat contents of the products are indicative of the reaction occurring.     

Enzyme‐catalyzed synthesis of structured phospholipids with conjugated linoleic acid

Structured phospholipids were synthesized with the functional lipid conjugated linoleic acid (CLA). The lipase‐ and phospholipase A₂‐catalyzed enzymatic acidolysis reaction between phospholipids (PL) and CLA was used for fatty acid modification. Enzymatic processes were an effective way to produce structured PL. Screening of four lipases and immobilized phospholipase A₂ and a combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PL. The maximum incorporation achieved by the latter enzyme was 16% with soy PL in 72 h.     

Sn-1,3-specific Interesterification of Soybean Oil with Medium-chain Triacylglycerol Catalyzed by Lipozyme TL IM

abstract The structured lipids are produced through sn-1,3-specific interesterification of soybean oil with medium-chain triacylglycerol (MCT) in continuous reactions catalyzed by Thermomyces lanuginos...     

Enzymatic Production of Monoacylglycerols with Camellia Oil by the Glycerolysis Reaction

Three commercial immobilized lipases, Lipozyme RM IM, Lipozyme TL IM and Novozym 435, were screened for the production of monoacylglycerols (MAG) by glycerolysis of camellia oil in a solvent medium of tert-butyl alcohol. Novozym 435 showed the best performance and was selected to catalyze the glycerolysis reaction. Different reaction conditions for the batch reaction, substrate mole ratio, substrate concentration and temperature, were investigated. The optimal reaction conditions were determined as 6:1 mole ratio of glycerol to camellia oil at 40% (w/v) of substrate concentration in tert-butyl alcohol at a reaction temperature of 50 °C. Under these optimal conditions, the conversion rate of camellia oil was 98.7% (10 h), and the mixture of acylglycerols contained 82.0% of MAG. A packed-bed reactor (PBR) system with 4.5 g Novozym 435 was employed in continuous production. The resulting product mixture of acylglycerols contained 80.74% of MAG and was obtained at a flow rate of 0.25 mL/min of substrates. The long-term operation of the PBR system gave an average productivity of 0.698 kg MAG/(kg enzyme h) after 38 days of operation.     

Enzymatic Interesterification of Extra Virgin Olive Oil with a Fully Hydrogenated Fat: Characterization of the Reaction and Its Products

The lipase-catalyzed interesterification of extra virgin olive oil (EVOO) and fully hydrogenated palm oil (FHPO) was studied in a batch reactor operating at 75 °C. The compositions of the semi-solid fat products depend on the reaction conditions and the initial ratio of EVOO to FHPO. The dependence of the quasi-equilibrium product TAG profile on the reaction time was determined for initial weight ratios of EVOO to FHPO from 80:20 to 20:80. Lipozyme TL IM, Lipozyme RM IM and Novozym 435 were employed as biocatalysts. The interesterification reaction was optimized with respect to the type and loading of biocatalyst. Equilibrium was approached in the shortest time with Novozym 435 (80% conversion in 4 h). The chemical, physical, and functional properties of the products were characterized. Appropriate choices of the reaction conditions and the initial ratio of EVOO to FHPO lead to TAG with melting profiles and solid fat contents similar to those of commercial products. Differences were observed in the solid fat contents, melting profiles, and oxidative stabilities of the various interesterified products and also between the indicated properties of each category of product and the corresponding physical blend of the precursor reagents.     

Lipase-mediated synthesis of neopentyl glycol diester using a combination of reduced and standard pressure

Neopentyl glycol (NPG) diester is a biodegradable lubricant used in the machinery industry as it is suitable for the low-temperature environment. NPG diester was successfully synthesized with fatty acid and NPG using immobilized lipase. Immobilization was carried out with a liquid enzyme, Eversa® Transform 2.0 (from Thermomyces lanuginosus) and Lewatit VP OC 1600 as a carrier. The Eversa immobilized lipase prepared in this study was compared with commercial lipases, such as Novozym 435, Lipozyme RM IM, and Lipozyme TL IM. The Eversa immobilized lipase was the most effective biocatalyst for the synthesis of NPG diesters. The effects of enzyme loading and temperature on conversion were explored at a standard pressure of 101.3 kPa. And then, the effect of reduced pressure was investigated in the range of 1.3 to 40.0 kPa under optimum conditions obtained at standard pressure. The maximum conversion of ca. 63% was achieved at the optimal temperature of 50°C and the enzyme loading of 5% (based on the total weight of the substrate) at standard pressure after 10 h. However, the conversion to NPG diester increased markedly up to 97% after 10 h, even though reduced pressure as low as 26.7 kPa was applied. No significant differences in the conversion to NPG diester between the reactions at constant reduced pressure and the combined reaction of reduced pressure and standard pressure were observed. This pressure combination method employed in this study demonstrated a novel, energy-saving strategy for the synthesis of NPG diester.     

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     

Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids

Structured lipids were synthesized by acidolysis of perilla oil and caprylic acid using two lipases, Lipozyme RM IM from Rhizomucor miehei and Lipozyme TL IM from Thermomyces lanuginosa. Effects of molar ratio, reaction time, reaction temperature, enzyme load, and solvent content on acidolysis reactions were studied. The solvent content ranged from 0.0 (solvent-free) to 85.3%. The results showed that the incorporation increased in parallel with solvent content to 49.0% with Lipozyme RM IM and to 63.8% with Lipozyme TL IM. After 24 h incubation in n-hexane, caprylic acids were incorporated to 48.5 mol% with Lipozyme RM IM and to 51.4 mol% with Lipozyme TL IM, respectively, whereas linolenic acid content was reduced from 61.4 to 31.5 mol% with Lipozyme RM IM and to 28.4 mol% with Lipozyme TL IM, respectively. Lipozyme TL IM showed a higher acyl migration rate than Lipozyme RM IM when acidolysis was performed in the reaction system containing n-hexane as a solvent, whereas the difference in acyl migration between the two lipases in the solvent-free system was negligible.     

Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols

This paper focuses on a detailed evaluation of commercially available immobilized lipases and simple monohydric alcohols for the production of alkyl esters from sunflower oil by enzymatic alcoholysis. Six lipases were tested with seven alcohols, including straight and branched-chain primary and secondary alcohols. The reactions were conducted in a batch stirred reaction vessel using stoichiometric amounts of substrates under solvent-free conditions. Dramatic differences in alcoholysis performance were observed among the different lipases. For most of the alcohols, Novozym 435 produced the highest yield of FA alkyl esters, with yields well over 90% for methanol, absolute ethanol, and 1-propanol. Overall, 96% ethanol was the preferred alcohol for all lipases except Novozym 435, and ethanolysis reactions reached the maximal conversion efficiency. Increasing the water content in the system resulted in an increased degree of conversion for all lipases except Novozym 435. The secondary alcohol 2-propanol significantly reduced the alcoholysis reaction with all lipases; however, the branch-chain isobutanol was more advantageous than linear 1-butanol for Novozym 435, Lipozyme RMIM, and Lipase PS-C. Many commercial immobilized lipases are highly efficient and promising for the production of alkyl esters, offering high reaction yields and a simple operation process.     

Applications of immobilized <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis> lipase in interesterification

The aim of this work was to investigate the catalytic functions of a new immobilized Thermomyces lanuginosa lipase in interesterification and to optimize the conditions of interesterification for the production of human milk fat substitutes (HMFS) containing n−3 PUFA by response surface methodology (RSM). Thermomyces lanuginosa lipase had an activity similar to that of immobilized Rhizomucor miehei lipase (Lipozyme RM IM) in the glycerolysis of sunflower oil, but the former had higher activity at a low reaction temperature (5°C). Thermomyces lanuginosa lipase was found to have much lower catalytic activity than Lipozyme RM IM in the acidolysis of sunflower oil with caprylic acid. However, the activity of T. lanuginosa lipase was only slightly lower than that of Lipozyme RM IM in the ester-ester exchange between tripalmitin (PPP) and the ethyl esters of EPA and DHA (EE). For this reason, the new immobilized T. lanuginosa lipase was used to produce HMFS from PPP by interesterification with EE. The optimization of major parameters was conducted with the assistante molar ratio of 5 (EE/PPP), a lipase load of 20 wt% (on substrates), and a reaction time of 20 h, with acyl incorporation up to 42%. The model generated significantly represented real relationships between the response (incorporation) and reaction parameters.