Practical synthesis of 1,3-oleoyl 2-docosahexaenoylglycerol by lipase-catalyzed reactions: An evaluation of different reaction routes

Three new synthetic routes were critically evaluated for the lipase-catalyzed production of 1,3-oleoyl-2 docosahexaenoylglycerol (ODO) in relatively large-scale (approximately 200 g). First, the production of 1,3-diolein by the reaction of glycerol and oleic acid followed by incorporation of docosahexaenoic (DHA) ethyl ester at the sn-2 position was studied. 1,3-Diolein was produced in 68.3% and 84.6% yield when stoichiometric amounts of the substrates were reacted at 25 °C for 8 h in the presence of 10% Novozym 435 and Lipozyme RM IM, respectively. Further increase in reaction temperature and time led to decrease in the 1,3-diolein yield. However, only a 9.4% yield of triacylglycerol was obtained in the subsequent reaction step when the 1,3-diolein was reacted with DHA ethyl ester in the presence of Novozym 435. Secondly, the feasibility of direct acidolysis was studied. Acidolysis of single cell oil (SCO) in excess oleic acid using Novozym 435 as the catalyst occurred twice as fast in solvent (tert-butanol) compared to a solvent-free system, and 63% oleic acid was incorporated into SCO. However, the regio-isomeric purity of the product was poor. Finally, the ethanolysis of SCO to produce DHA-enriched 2-monoacylglycerol followed by esterification with oleic acid or ethyl oleate was investigated. ODO was obtained in 50.9% regio-purity by Lipozyme RM IM-catalyzed esterification. The latter method was the most feasible for preparing ODO in large-scale. This synthetic route could be adapted for related triacylglycerols containing highly polyunsaturated when their productions in large-scale and high regio-purity are required.     

Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435

The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production.     

Improved synthesis of 1,3-diolein by Novozym 435-mediated esterification of monoolein with oleic acid

We successfully developed an efficient and promising bioprocess for 1,3-diolein synthesis by performing Novozym 435-mediated esterification of oleic acid with monoolein in this work. Under the optimized conditions (60 °C, molar ratio of oleic acid to monoolein 1.2:1), a 1,3-diolein yield of 93.7% could be achieved, and the yield of 1,2-diolien was low (2.6%). The high yield of 1,3-diolein and the optimum reaction time were improved remarkably compared with the results of our previous study, which involved the enzymatic esterification of oleic acid with glycerol. An additional advantage of the new process is the fact that 90% original activity of the enzyme was maintained after being used for 100 reactions. The present work could be seen as a useful enzyme-catalyzed process for the industrial production of 1,3-diacylglycerol.     

Enzymatic synthesis of betulinic acid ester as an anticancer agent: Optimization study

Abstract

Immobilized Candida antarctica lipase, Novozym 435, was used to catalyze the esterification reaction between betulinic acid and phthalic anhydride to synthesize 3-O-phthalyl betulinic acid in n-hexane/chloroform. Response surface methodology based on a five-level, four-variable central composite rotatable design was employed to evaluate the effects of synthesis parameters such as reaction time, reaction temperature, enzyme amount and substrate molar ratio on the yield of ester. Based on the response surface model, the optimal enzymatic synthesis conditions were predicted to be: reaction time 20.3 h, reaction temperature 53.9°C, enzyme amount 145.6 mg, betulinic acid to phthalic anhydride molar ratio 1:1.11. The predicted yield was 65.8% and the actual yield was 64.7%.     

脂肪酶协同催化猪油合成生物柴油工艺研究

探讨了以乙酸甲酯为酰基受体两种脂肪酶协同催化猪油转酯合成生物柴油的工艺条件。首先利用单因子试验确定2种固定化脂肪酶Novozym435、Lipozyme TLIM单独作为催化剂时的最佳酶用量为40％,反应温度为50℃,乙酸甲酯用量为14（相对于油的摩尔比）。在此基础上,采用3因素5水平和3个中心点的中心组分旋转设计法研究了上述2种脂肪酶协同使用时脂肪酶用量（g/g）、混合酶的配比（%/%）以及乙酸甲酯用量诸因素共同作用对转酯反应转化率的影响。优化后的反应条件为：总酶用量为40％,混合酶配比为50/50,乙酸甲酯用量为14,在该条件下甲酯得率可达97.6％,比同质量的Novozym435、Lipozyme TLIM的催化活性分别高出7.6％、22.3％。表明脂肪酶协同催化猪油合成生物柴油工艺可以较好地提高甲酯得率,并且节约生产成本。     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Modeling and optimization of lipase-catalyzed production of succinic acid ester using central composite design analysis

Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different combinations of the four parameters including temperature (35–65°C), time (30–450 min), enzyme amount (20-400 mg), and alcohol:acid molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R² of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min, 20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid means for estimating the conversion yield of succinate ester within the selected ranges.     

A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium

A new enzymatic route for biodiesel production from soybean oil was developed using methyl acetate as a novel acyl acceptor. Novozym 435 (immobilized Candida antarctica lipase) gave the highest methyl ester (ME) yield of 92%. The optimum conditions of the transesterification were 30% enzyme based on oil weight; a molar ratio of methyl acetate/oil of 12:1; temperature 40 °C and reaction time 10 h. Since no glycerol was produced in the process, this method is very convenient for recycling the catalyst and by-product triacetylglycerol showed no negative effect on the fuel property.     

Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology

An operation mode with N(2) bubbling under vacuum was employed for the solvent-free synthesis of 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) from conjugated linoleic acid (CLA) catalyzed by Novozym 435. The response surface methodology (RSM) was adopted for the optimization of the reaction conditions with five major factors (incubation time, temperature, enzyme load, substrate mole ratio, and system vacuum) and three responses (CLA conversion, 1,3-dCLG yield, and acyl migration). Two sets of optimal conditions were recommended. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of 1,3-dCLG yield. Under the optimal conditions, the yield of 1,3-dCLG up to 93% was obtained. The reaction was scaled up to a production level of 100 g of 1,3-dCLG at a yield of 90.7%, indicating a promising feature of the technology in industrial applications.     

Enrichment of pinolenic acid from pine nut oil via lipase-catalyzed ethanolysis with an immobilized Candida antarctica lipase

Abstract

Pinolenic acid (PLA) enrichment as an ethyl ester from pine nut oil was successfully accomplished in a batch reactor by lipase-catalyzed ethanolysis using Novozym 435 lipase from Candida antarctica as a biocatalyst. PLA is predominantly an sn-3 substituent of the pine nut oil triacylglycerol (TAG), where it accounts for about 39 mol% of the fatty acids esterified at that position. In the presence of ethanol, Novozym 435 exhibited sn-3 regiospecificity with respect to the TAG of pine nut oil. The effect of the molar ratio of reactants on PLA enrichment by ethanolysis was investigated. The molar ratios of pine nut oil to ethanol were varied from 1:20 to 1:100. A fatty acid ethyl ester (FAEE) fraction with higher PLA content was obtained in the early stage of the reaction, although the yield of PLA was small. However, the PLA content of the FAEEs decreased with increasing reaction time, while the yield of PLA increased. The molar ratio of pine nut oil to ethanol that produced the optimum content and yield of PLA in FAEEs was 1:80.     

Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium

tert-Butanol, as a novel reaction medium, has been adopted for lipase-catalyzed transesterification of rapeseed oil for biodiesel production, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to catalyze the methanolysis and the highest biodiesel yield of 95% could be achieved under the optimum conditions (tert-butanol/oil volume ratio 1:1; methanol/oil molar ratio 4:1; 3% Lipozyme TL IM and 1% Novozym 435 based on the oil weight; temperature 35 °C; 130 rpm, 12 h). There was no obvious loss in lipase activity even after being repeatedly used for 200 cycles with tert-butanol as the reaction medium. Furthermore, waste oil was also explored for biodiesel production and it has been found that lipase also showed good stability in this novel system.     

Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane (R134a)

The stability and activity of commercial immobilized lipase from Candida antarctica (Novozym 435) in subcritical 1,1,1,2-tetrafluoroethane (R134a) was investigated. The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions. The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435, and a maximum residual activity of 300% was measured at 4 MPa, 30 °C after 7 h incubation. No deactivation of Novozym 435 treated with subcritical R134a under different operation factors (pressure 2–8 MPa, temperature 30–60 °C, incubation time 1–12 h, water content 1:1, 1:2, 1:5 enzyme/water, depressurization rate 4 MPa/1 min, 4 MPa/30 min, 4 MPa/90 min) was observed. While the initial reaction rate was high in subcritical R134a, higher conversion was obtained in solvent-free conditions. Though the apparent conversion of the reaction is lower in subcritical R134a, it is more practicable, especially at low enzyme concentrations desired at commercial scales.     

Enzymatic production of glycerol acetate from glycerol

In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Modeling and optimization of lipase-catalyzed esterification of policosanols with conjugated linoleic acid by response surface methodology

Abstract

The aim of this study was to model the lipase-catalyzed esterification of policosanols with conjugated linoleic acid (CLA) in a solvent-free system to produce wax esters which had a lower melting point than that of their corresponding policosanol forms and to optimize the reaction conditions by response surface methodology (RSM). Novozym 435 was selected as a suitable biocatalyst for the reaction. The molar ratio of substrates (policosanols to CLA) was 1:2. A well-fitting quadratic polynomial regression model for the degree of esterification (DE) of policosanols with CLA was established with regard to temperature (35–65°C), enzyme loading (1–5% of weight of total substrates), and reaction time (10–50 min). Optimal reaction conditions were 61.3°C for temperature, 3.7% for enzyme loading, and 34.1 min for reaction time, and the DE was ? 95 mol% under these conditions. The policosanols and wax esters synthesized under optimal conditions had melting points of 79°C and 57°C, respectively.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Enzyme catalyzed transesterification of waste cooking oil with dimethyl carbonate

The detrimental effects of waste cooking oil on sewer system attracted attention toward its proper management and reusing this waste oil for making biodiesel provides commercial and environmental advantage. In the present study, biodiesel has been successfully produced from waste cooking oil and dimethyl carbonate by transesterification, instead of the conventional alcohol. In this optimization study, the effect of various reaction conditions such as solvent, time and temperature, molar ratio of DMC to oil, enzyme loading and reusability, on the yield of fatty acid methyl ester (FAME) has been studied. The Maximum conversion of FAMEs achieved was 77.87% under optimum conditions (solvent free system, reaction time of 24 h, 60 °C, molar ratio of DMC to oil 6:1, catalyst amount 10% Novozym 435 (based on the oil weight)). Moreover, there was no obvious loss in the conversion after lipases were reused for 6 batches under optimized conditions.     

Novozym 435-catalyzed 1,3-diacylglycerol preparation via esterification in t-butanol system

1,3-Diacylglycerol (1,3-DAG) oil has beneficial effects on suppressing the accumulation of body fat and preventing the increase of body weight. So, more and more attention has been paid to enzyme-mediated 1,3-DAG production in recent years due to its mild reaction condition and safe products. In this work, t-butanol was adopted as the reaction medium for lipase-catalyzed esterification for 1,3-DAG preparation. In t-butanol system, the harmful effects on lipase caused by glycerol could be eliminated completely, so the high enzymatic activity was maintained and the stability of the lipase could be improved significantly. Under the optimum conditions (60 °C, 1.00 g Novozym 435, 2.5:1 molar ratio of oleic acid to glycerol (10.0 g oleic acid and 1.3 g glycerol) and 6.0 g t-butanol), 1,3-DAG concentration of 40% was achieved and Novozym 435 can be used 100 times. A simplified model based on Ping-Pong Bi-Bi with substrate competitive inhibition by glycerol was found to fit the initial rate data and the kinetics parameters were evaluated by nonlinear regression analysis.