Lipase catalytic synthesis of diacylglycerol from tuna oil and its anti-obesity effect in C57BL/6J mice

Diacylglycerol (DAG) production by glycerolysis of glycerol with tuna oil was performed using lipases from Rhizomucor miehei (Lipozyme RMIM) and Candida antarctica (Novozyme 435). Lipozyme RMIM caused a clear estrific positional specificity in HPLC analysis and then the Lipozyme RMIM was chosen for the production of DAG. Moreover, the reaction parameter for DAG synthesis was determined by measuring mole ratio of glycerol/tuna oil, amount of enzyme, temperature and water contents. The optimal mole ratio for glycerol/tuna oil was established to be 3:1. Optimal conditions of lipase, water and temperature were 10%, 10% and 35 °C, respectively. Therefore, we have synthesized DAG from tuna oil under these optimal conditions and investigated the effect of the synthesized DAG on body weight and plasma biochemical markers of obesity in C57BL/6J mice. The consumption of DAG diet has effectively lessened body weight gain and final plasma total cholesterol, triacylglycerol and glucose levels compared to the high triacylglycerol (TAG) group.     

Different enzyme requirements for the synthesis of biodiesel: Novozym 435 and Lipozyme TL IM

Enzymatic syntheses of biodiesel via alcoholysis of different vegetable oils (sunflower, borage, olive and soybean) have been studied. Loss of lipase activity induced by the nucleophile is greater with methanol than with ethanol, and is greater for Lipozyme TL IM than for Novozym 435. The optimum volume of ethanol depends on the loading of solid biocatalyst and is higher for preparations of Novozym 435 than for Lipozyme TL IM. Maximum rates were obtained with Lipozyme TL IM, for a molar ratio of alcohol to FA residues of 0.33. By contrast, Novozym 435 requires at least a 2:1 ratio. Alcoholysis of the vegetable oils is faster with Lipozyme TL IM than with Novozym 435. Use of a high loading of Novozym 435 (50% w/w) and a large molar excess of ethanol are required to obtain an initial rate similar to that obtained with Lipozyme TL IM at a lower enzyme loading (10% w/w) and an equimolar ratio of ethanol and FA residues. Novozym 435 produces quantitative conversions in only 7h at 25 degrees C, but complete conversions are not obtained with Lipozyme TL IM. Three stage stepwise addition of ethanol yields 84% conversion to ethyl esters for Lipozyme TL IM. Hence use of Novozym 435 is preferred. After nine cycles in a batch reactor Novozym 435 retained 85% of its initial activity.     

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.     

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.     

Optimization of mono and diacylglycerols production from enzymatic glycerolysis in solvent-free systems

This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 °C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.     

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。     

Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

In human milk fat (HMF), palmitic acid (20–30%), the major saturated fatty acid, is mostly esterified at the sn-2 position of triacylglycerols, while unsaturated fatty acids are at the sn-1,3 positions, conversely to that occurring in vegetable oils.This study aims at the production of HMF substitutes by enzyme-catalyzed interesterification of tripalmitin with (i) oleic acid (system I) or (ii) omega-3 polyunsaturated fatty acids (omega-3 PUFA) (system II) in solvent-free media. Interesterification activity and batch operational stability of commercial immobilized lipases from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM) and Candida antarctica (Novozym 435) from Novozymes, DK, and Candida parapsilosis lipase/acyltransferase immobilized on Accurel MP 1000 were evaluated. After 24-h reaction at 60 °C, molar incorporation of oleic acid was about 27% for all the commercial lipases tested and 9% with C. parapsilosis enzyme. Concerning omega-3 PUFA, the highest incorporations were observed with Novozym 435 (21.6%) and Lipozyme RM IM (20%), in contrast with C. parapsilosis enzyme (8.5%) and Lipozyme TL IM (8.2%). In system I, Lipozyme RM IM maintained its activity for 10 repeated 23-h batches while for Lipozyme TL IM, Novozym 435 and C. parapsilosis enzyme, linear (half-life time, t_1/2 = 154 h), series-type (t_1/2 = 253 h) and first-order (t_1/2 = 34.5 h) deactivations were respectively observed. In system II, Lipozyme RM IM showed linear deactivation (t_1/2 = 276 h), while Novozym 435 (t_1/2 = 322 h) and C. parapsilosis enzyme (t_1/2 = 127 h), presented series-type deactivation. Both activity and stability of the biocatalysts depended on the acyl donor used.     

Enzymatic Synthesis of Lipophilic Caffeoyl Lipids Using Soybean Oil as the Novel Acceptor

Soybean oil-based caffeoyl lipids are the novel lipophilic derivatives of caffeic acid, which can be used as UV absorbers and antioxidants in the food and cosmetic industries. In the work, the novel lipophilic structured lipids were prepared using soybean oil as the novel caffeoyl acceptor by enzymatic transesterification. The effects of the reaction variables on the transesterification were investigated, and response surface methodology was used to optimize the reaction variables. Reactions were monitored by HPLC-UV. Different enzymes (Novozym 435, Lipozyme RMIM, and Lipozyme TLIM) were used as biocatalysts, and Novozym 435 showed the best performance for the reaction. The results showed that a high lipophilic soybean oil-based caffeoyl lipids yield (73.5 ± 1.2%) was achieved under the optimal conditions (reaction temperature 85°C, substrate molar ratio 1:6 (ethyl caffeate (EC)/soybean oil), enzyme load 25% (w/w), and 60 h at atmosphere pressure). The activation energies of EC conversion, hydrophilic glyceryl caffeates (GC) and lipophilic caffeoylated acylglycerol (CAG) formations were 32.92 kJ/mol, 17.21 kJ/mol and 57.36 kJ/mol, respectively. Km and Vm were 0.022 mol/L and 0.033 × 10^-3 mol/(Lmin), respectively.     

Contribution of response surface design to the development of glycerolysis systems catalyzed by commercial immobilized lipases

Two commercial immobilized lipases (“Lipozyme® IM” and “Novozym® 435”) were tested as biocatalysts for the glycerolysis of olive residue oil in n-hexane aimed at the production of monoglycerides (MG) and diglycerides (DG). A central composite rotatable design (CCRD) was followed to model and optimize glycerolysis as a function of both the amount of biocatalyst (L) and of the molar ratio glycerol/triglycerides (Gly/TG). For both biocatalysts, the production of free fatty acids (FFA) was described by second order models. In terms of MG and DG production, as well as of TG conversion, the best fits were obtained with first-order models. The highest MG productions were in the range 43–45% (w/w, on the basis of total fat) for both biocatalysts tested at a (Gly/TG) ratio of one. In the case of “Novozym 435”, the lowest load used (12%, w/w) gave the best results, in contrast with “Lipozyme IM” with which a concentration of about 26% (w/w) was necessary to obtain the highest production. Under these conditions, the amount of FFA produced was about 2% and 10% (w/w), respectively, for “Novozym 435” and “Lipozyme IM” catalyzed systems. Considering both FFA production and lipase loading, “Novozym 435” was shown to be a better biocatalyst for the glycerolysis of olive residue oil in n-hexane, aimed at the production of MG, than “Lipozyme IM”.     

Improved high-pressure enzymatic biodiesel batch synthesis in near-critical carbon dioxide

The enzymatic synthesis of biodiesel by a high-pressure semi-continuous process in near-critical carbon dioxide (NcCO(2)) was studied. Biodiesel synthesis was evaluated in both batch and semi-continuous systems to develop an effective process. Batch processing demonstrated the advantageous properties of NcCO(2) as an alternative reaction medium. Three immobilized lipases (Novozym 435, Lipozyme RM IM, and Lipozyme TL IM from Novozymes) were tested, with Lipozyme TL IM the most effective, showing the highest conversion. Biodiesel conversion from several edible and non-edible oil feedstocks reached >92%. Higher conversion (99.0%) was obtained in a shorter time by employing repeated batch processes with optimized conditions: 44.3 g (500 mM) canola oil, a substrate molar ratio (methanol:oil) of 3:1, an enzyme loading of 20 wt% (of the oil used), at 30 °C, 100 bar, and 300 rpm agitation. The enzyme maintained 80.2% of its initial stability after being reused eight times. These results suggest that this method produces biodiesel energy-efficiently and environment-friendly.     

固定化脂肪酶催化毛棉籽油制备生物柴油

研究了固定化脂肪酶Lipozyme TL IM和Novozym435催化毛棉籽油和乙酸甲酯制备生物柴油的过程。通过向反应体系中添加甲醇,可减少乙酸的抑制,明显提高生物柴油得率,确定最佳反应条件为:正己烷作溶剂,乙酸甲酯与油摩尔比9:1,添加油重3%的甲醇、油重10%的LipozymeTLIM和5%的Novozym435复合使用,温度55°C,反应8h,生物柴油得率达到91.83%。最后探索了酶催化毛棉籽油合成生物柴油的动力学,得到动力学方程。     

Combining regio- and enantioselectivity of lipases for the preparation of (R)-4-chloro-2-butanol

Preparation of 98% ee (R)-4-chloro-2-butanol was carried out by the enzymatic hydrolysis of chlorohydrin esters, using fungal resting cells and commercial enzymes. Hydrolyzes were carried out using lipases from Candida antarctica (Novozym 435), C. rugosa, Rhizomucor miehei (Lipozyme IM), Burkolia cepacia, and resting cells of Rhizopus oryzae and Aspergillus flavus. The influence of the enzyme, the solvent, the temperature, and the alkyl chain length on the selectivity of hydrolyzes of isomeric mixtures of chlorohydrin esters is described. Regioselectivity was higher than 95% for some of the tested lipases. Novozym 435 allowed preparation of the (R)-4-chloro-2-butanol after 15 min of reaction at 30-40 degrees C.     

Immobilized lipase-catalysed synthesis of cinnamyl laurate in non-aqueous media

Esters of cinnamyl alcohol find many applications in food, cosmetic and pharmaceutical industries as flavor and fragrance compounds. The current work focuses on the synthesis of cinnamyl laurate from cinnamyl alcohol and lauric acid, including screening of various immobilized lipases and optimization of reaction conditions such as catalyst loading, speed of agitation, mole ratio and temperature. Among different lipases screened such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM, Novozym 435 was found to be the best catalyst with 60% conversion in 2 h at 30 °C for equimolar quantities of the reactants using 0.33% (w/v) of catalyst and toluene as solvent. An ordered bi–bi mechanism with dead-end complex of lauric acid was found to represent the kinetic data.     

Process intensification of immobilized lipase catalysis by microwave irradiation in the synthesis of 4-chloro-2-methylphenoxyacetic acid (MCPA) esters

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.     

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.     

Continuous lipase-catalyzed production of esters from crude high-oleic sunflower oil

The objective of this work was to develop an economically relevant enzymatic process of butyl ester production using crude high-oleic sunflower oil. Novozym 435, a non-regiospecific biocatalyst, provided the best compromise between activity and butyl-ester yield. The inhibition caused by the presence of phopholipids in crude oil was eliminated by using tert-butanol. It demonstrates the key role of the medium polarity in order to insure the stability of a process. Initial substrate concentrations and their molar ratio were optimized in a continuous packed-bed reactor to maximize product yield and productivity. The best compromise was obtained for an initial oil concentration of 500 mM and a molar ratio of 5. It enabled a high productivity of 13.8 tons year⁻¹ kg Novozym 435⁻¹ with a butyl-ester purity of 96.5% to be obtained. Experiments with the continuous reactor were performed over 50 days without any loss of enzyme activity.     

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.     

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.     

Resolution of racemic ketoprofen in organic solvents by lipase from Burkholderia cepacia G63

A lipase from the Burkholderia cepacia strain G63 immobilized on resin was used for the resolution of ketoprofen. To study its catalytic properties in enantioselective esterication, different alcohols and solvents were tested to select the most suitable acyl acceptor and reaction medium. Compared with the low activity of the free lipase, the enzyme activity and E value of the immobilized lipase were significantly enhanced. The enantioselectivity of the immobilized lipase could also be markedly improved by adding a small amount of 18-crown-6. RSM was employed to optimize the reaction parameters. The optimal reaction conditions were: reaction time 22.50 h, additives dosage 0.4322 g (0.33 mmol/mL), and substrate molar ratio 54.11:1. Under optimal conditions, the maximal E value was up to 10.01, which exhibited a better enantioselectivity than some commercial lipases, such as Novozym 435, Lipozyme RM IM and LipozymeTL IM.     

Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation

Both stability and catalytic activity of two commercial immobilized lipases were investigated in the presence of different organic solvents in ultrasound-assisted system. In a general way, for Novozym 435, the use of ethanol as solvent led to a loss of activity of 35% after 10 h of contact. The use of iso-octane conducted to a gradual increase in lipase activity in relation to the contact time, reaching a maximum value of relative activity of 126%. For Lipozyme RM IM, after 5 h of exposure, the enzyme presented no residual activity when ethanol was used as solvent. The solvents tert-butanol and iso-octane showed an enhancement of about 20 and 17% in the enzyme activity in 6 h of exposure, respectively. Novozym 435 and Lipozyme IM presented high stability to storage after treatment under ultrasound-assisted system using n-hexane and tert-butanol as solvents.