游离脂肪酶NS81006催化含酸油脂制备生物柴油的应用研究

与固定化脂肪酶相比,游离脂肪酶具有反应速率快、成本较低的优势,成为制备生物柴油新的研究方向。前期研究结果表明,游离脂肪酶NS81006可以高效催化大豆油甲醇解制备生物柴油,进一步研究其对含酸油脂的催化,对于促进游离脂肪酶在生物柴油领域中的应用具有重要意义。本文系统研究了甲醇添加策略对游离脂肪酶NS81006催化油酸制备生物柴油的影响,进而考察了NS81006催化模拟酸化油以及实际含酸油脂制备生物柴油的转化情况。研究表明,在优化的甲醇添加策略下,游离脂肪酶NS81006可有效催化油酸、不同含酸量(0~100%,基于总质量)模拟酸化油以及实际含酸油脂进行生物柴油的制备;离心分离可有效实现NS81006的回复使用,连续回用5个批次,游离脂肪酶活性未出现明显下降。     

Influences of operating conditions on biocatalytic activity and reusability of Novozym 435 for esterification of free fatty acids with short-chain alcohols:A case study of palm fatty acid distillate

In the present study, the effects of operating conditions on biocatalytic activity and stability of Novozym 435 for repeated-batch biodiesel production from free fatty acid (FFA) were investigated. Thermal deactivation caused by increased operating temperature from 45 to 50 °C could seriously affect the reusability of Novozym 435. The deactivation of Novozym 435 during the esterification of oleic acid with ethanol tended to be stronger than that in the system with methanol. Under the optimal conditions, considering both biocatalytic activity and stability of the enzyme, Novozym 435 could be reused for 13 cycles for biodiesel productions from oleic acid and absolute alcohols (methanol and ethanol) with FFA conversions of at least 90%. The presence of 4%–5%water in ethanol significantly affected the reusability of Novozym 435. Changes in the surface morphology of Novozym 435 during the esterification with various conditions were observed. It was revealed that the reduc-tion in catalytic activity was related to the swel ing degree of the catalyst surface. Additionally, biodiesel produc-tion from low cost renewable feedstocks, such as palm fatty acid distillate (PFAD) and 95%ethanol was examined. The esterification of PFAD with 95%ethanol catalyzed by Novozym 435 in 10-repeated batch operation showed the similar results in FFA conversion as compared to those using oleic acid. Novozym 435 remained active and could maintain 97.6%of its initial conversion after being used for 10 batches.     

Free Lipase-Catalyzed Esterification of Oleic Acid for Fatty Acid Ethyl Ester Preparation with Response Surface Optimization

Free lipase-mediated alcoholysis for biodiesel production has drawn increasing attention in recent years due to its advantages of lower cost and faster reaction rate compared to immobilized lipase. Ethanol, derived from renewable biomass, has a great potential for biodiesel production. A previous study showed that free lipase NS81006 could effectively catalyze the ethanolysis of triglycerides for biodiesel preparation. Since most crude plant oils always contain an amount of free fatty acids, oleic acid was used as the model substrate for this study on lipase-mediated esterification for biodiesel production. The central composite design of the response surface methodology was adopted for process optimization. A biodiesel yield of over 90 % was achieved under optimal reaction conditions and the repeated use of the free lipase was easily realized through phase separation either by natural gravity force or centrifugation.     

Lipase‐mediated methanolysis of soybean oils for biodiesel production

BACKGROUND: Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Biocatalysis of soybean oils using soluble lipase offers an alternative approach to lipase‐catalyzed biodiesel production using immobilized enzyme or whole‐cell catalysis. The central composite design (CCD) of response surface methodology (RSM) was used here to evaluate the effects of enzyme concentration, temperature, molar ratio of methanol to oil and stirring rate on the yield of fatty methyl ester. RESULTS: Lipase NS81006 from a genetically modified Aspergillus oryzae was utilized as the catalyst for the transesterification of soybean oil for biodiesel production. The experimental data showed that enzyme concentration, molar ratio of methanol to oil and stirring rate had the most significant impact on the yield of fatty methyl ester; a quadratic polynomial equation was obtained for methyl ester yield by multiple regression analysis. The predicted biodiesel yield was 0.928 (w/w) under the optimal conditions and the subsequent verification experiments with biodiesel yield of 0.936 ± 0.014 (w/w) confirmed the validity of the predicted model. CONCLUSION: RSM and CCD were suitable techniques to optimize the transesterification of soybean oil for biodiesel production by soluble lipase NS81006. The related lipase NS81006 reuse stability, chemical or genetic modification, and transesterification mechanism should be taken into consideration. Copyright © 2007 Society of Chemical Industry     

Optimized synthesis of lipase‐catalyzed biodiesel by Novozym 435

The ability of immobilized lipase from Candida antarctica (Novozym 435) to catalyze the alcoholysis of canola oil and methanol was investigated. Response surface methodology (RSM) and five–level–five–factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time, temperature, enzyme concentration, substrate molar ratio of methanol to canola oil, and added water content on percentage weight conversion of canola oil methyl ester by alcoholysis. Reaction temperature and enzyme concentration were the most important variables. High temperature and superabundant methanol inhibited the ability of Novozym 435 to catalyze the synthesis of biodiesel. Based on the analysis of ridge max, the optimum synthesis conditions were as follows: reaction time 12.4 h, temperature 38.0 °C, enzyme concentration 42.3%, substrate molar ratio 3.5:1, and added water 7.2%. The predicted value was 99.4% weight conversion, and the actual experimental value was 97.9% weight conversion. Copyright © 2004 Society of Chemical Industry     

Enzymatic Glycerolysis of Palm and Palm Kernel Oils

Glycerolysis of palm and palm kernel oils were carried out using commercial lipases from Candida antarctica (Novozym 435) and Mucor miehei (Novozym 388) as catalyst (500 units lipase/g oil) at 40°C and with an oil:glycerol molar ratio of 1:2 in a solvent-free system. Novozym 435 catalyzed the glycerolysis of palm and palm kernel oils giving reaction products in similar compositions. Partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils were 64% (wt) and 66% (wt), respectively. However, partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils conducted with Novozym 388 as catalyst at the same conditions were 44% (wt) and 56% (wt), respectively. On the other hand, free fatty acid contents of the glycerolysis products of palm and palm kernel oils obtained using Novozym 388 were higher, 25–30% (wt), than those obtained by Novozym 435, 4–5% (wt). The monoacylglycerols fraction with the highest content of oleic acid, 62.7% (wt), was obtained from the palm kernel oil glycerolysis reaction catalyzed by Novozym 435.     

Enzymatic Glycerolysis of Palm and Palm Kernel Oils

Glycerolysis of palm and palm kernel oils were carried out using commercial lipases from Candida antarctica (Novozym 435) and Mucor miehei (Novozym 388) as catalyst (500 units lipase/g oil) at 40°C and with an oil:glycerol molar ratio of 1:2 in a solvent-free system. Novozym 435 catalyzed the glycerolysis of palm and palm kernel oils giving reaction products in similar compositions. Partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils were 64% (wt) and 66% (wt), respectively. However, partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils conducted with Novozym 388 as catalyst at the same conditions were 44% (wt) and 56% (wt), respectively. On the other hand, free fatty acid contents of the glycerolysis products of palm and palm kernel oils obtained using Novozym 388 were higher, 25-30% (wt), than those obtained by Novozym 435, 4-5% (wt). The monoacylglycerols fraction with the highest content of oleic acid, 62.7% (wt), was obtained from the palm kernel oil glycerolysis reaction catalyzed by Novozym 435.     

Effect of methanol content on enzymatic production of biodiesel from waste frying oil

The enzymatic production of biodiesel from waste frying oil with methanol has been studied using immobilized lipase Novozym 435 as catalyst. The effects of methanol to oil molar ratio, dosage of enzyme and reaction time were investigated. The optimum reaction conditions were methanol to oil molar ratio of 25:1, 10% of Novozym 435 based on oil weight and reaction period of 4 h at 50 °C obtaining a biodiesel yield of 89.1%. Moreover, the reusability of the lipase over repeated cycles was also investigated under standard conditions.     

Study on Free Lipase-Catalyzed Ethanolysis for Biodiesel Preparation in an Oil/Water Biphasic System

Using free lipase as the catalyst for biodiesel production has drawn increasing attention in recent years due to its advantages of lower cost and faster reaction rate compared to immobilized lipase. Our previous study showed that free lipase NS81006 could effectively catalyze the methanolysis of renewable oil. Ethanol, derived from renewable biomass, has a greater potential for biodiesel production. In this paper, ethanol was explored for the first time as the acyl acceptor for free lipase-mediated biodiesel preparation. The effect of stirring rate, water content, molar ratio of ethanol to oil and ethanol adding strategy was investigated systematically during the process of free lipase NS81006-catalyzed ethanolysis. An ethyl ester yield of 90% was obtained under the optimized conditions. Further study showed that the free lipase could be repeatedly used by simple separation of the water phase from the oil phase and there was no obvious loss in lipase activity after five repeated uses.     

Lipase‐catalyzed production of biodiesel from rice bran oil

Biodiesel has attracted considerable attention as an alternative fuel during the past decades. The main hurdle to the commercialization of biodiesel is the cost of the raw material. Use of an inexpensive raw material such as rice bran oil is an attractive option to lower the cost of biodiesel. Two commercially available immobilized lipases, Novozym 435 and IM 60, were employed as catalyst for the reaction of rice bran oil and methanol. Novozym 435 was found to be more effective in catalyzing the methanolysis of rice bran oil. Methanolysis of refined rice bran oil and fatty acids (derived from rice bran oil) catalyzed by Novozym 435 (5% based on oil weight) can reach a conversion of over 98% in 6 h and 1 h, respectively. Methanolysis of rice bran oil with a free fatty acid content higher than 18% resulted in lower conversions (<68%). A two‐step lipase‐catalyzed methanolysis of rice bran oil was developed for the efficient conversion of both free fatty acid and acylglycerides into fatty acid methyl ester. More than 98% conversion can be obtained in 4–6 h depending on the relative proportion of free fatty acid and acylglycerides in the rice bran oil. Inactivation of lipase by phospholipids and other minor components was observed during the methanolysis of crude rice bran oil. Simultaneous dewaxing/degumming proved to be efficient in removing phospholipids and other minor components that inhibit lipase activity from crude rice bran oil. Copyright © 2005 Society of Chemical Industry     

固定化脂肪酶催化制备香叶树籽生物柴油研究

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

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     

叔戊醇体系酶促大豆油制备生物柴油

叔戊醇作为反应介质,固定化脂肪酶Novozym 435催化大豆油与甲醇的转酯反应制备生物柴油。叔戊醇消除了反应底物甲醇及反应副产物甘油对酶活的负面影响。定量分析表明,叔戊醇与油脂的体积比为1,甲醇与油脂的摩尔比为3,2%脂肪酶,反应体系含水量2%,40 ℃、180 r/min条件下反应15 h,生物柴油得率可达97%。在最适条件下反应进行160批次,酶仍保持了较高的活性和良好的稳定性。     

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.     

新型反应介质中脂肪酶催化多种油脂制备生物柴油

用叔丁醇作为反应介质,利用固定化脂肪酶催化油脂原料甲醇醇解反应制备生物柴油,消除了甲醇和甘油对酶的负面影响,酶的使用寿命显著延长. 用菜籽油作原料,叔丁醇和油脂的体积比为1:1,甲醇与油脂的摩尔比为4:1,3%的Lipozyme TLIM和1%的Novozym 435结合使用,35℃下130 r/min反应12 h,生物柴油得率可达95%. 该工艺在200 kg/d的规模下制得的生物柴油产品完全满足美国和德国生物柴油标准,脂肪酶重复使用200批次,酶活性基本没有下降. 且在叔丁醇介质体系中大豆油、桐籽油、棉籽油、乌桕油、泔水油、地沟油和酸化油都能被有效转化成生物柴油且脂肪酶保持很好的稳定性.     

Transesterification for biodiesel production catalyzed by combined lipases: Optimization and kinetics

Preparation of biodiesel from waste cooking oil catalyzed by combined lipases in tert‐butanol medium was investigated. Several crucial parameters affecting biodiesel yield were optimized by response surface methodology, such as dosage of combined lipases of Novozym 435 and Lipozyme TLIM, weight ratio of Novozym 435 to Lipozyme TLIM, amount of tert‐butanol, reaction temperature, and molar ratio of oil to methanol. Under the optimized conditions, the highest biodiesel yield was up to 83.5% The proposed model on biodiesel yield had a satisfactory coefficient of R² (= 94.02%), and was experimentally verified. The combined lipases exhibited high‐operational stability. After 30 cycles (300 h) successively, the activity of combined lipases maintained 85% of its original activity. A reaction kinetic model was proposed to describe the system and deduced to be a pseudo‐first‐order reaction, and the calculated activation energy was 51.71 kJ/mol. © 2009 American Institute of Chemical Engineers AIChE J, 2010     

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.     

Enzymatic synthesis of monoacylglycerol citrate optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of citric acid (CA) and monoglyceride (MG) for citrate esters of monoacylglycerols (CITREM) preparation was investigated. The effects of substrate concentration, molecular‐sieve amount, substrate molar ratio, reaction temperature, time, and enzyme load on the conversion of CA in the reaction were investigated. Enzyme screening and the effect of solvent on the esterification were also investigated. RSM was used to optimize the effects of the reaction temperature (45–55°C), the enzyme load (6–10%; relative to the weight of total substrates), and the reaction time (24–48 h) on the conversion of CA. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of CA conversion. The optimum preparation conditions were as follows: CA concentration 0.12 mol/L, molecular‐sieve 120 g/L, molar ratio of MG/CA 2:1, temperature 54.18°C, enzyme load 9.0% (relative to the weight of total substrates), and reaction time 47.5 h. Under the suggested conditions, the conversion of CA was 77.4%. Repeated reaction tests indicated that Novozym 435 could be used eight times under the optimum conditions with 92% of its original catalytic activity still retained.     

Continuous enzymatic production of biodiesel from virgin and waste sunflower oil in supercritical carbon dioxide

A continuous process for biodiesel production in supercritical carbon dioxide was implemented. In the transesterification of virgin sunflower oil with methanol, Lipozyme TL IM led to fatty acid methyl esters yields (FAME) that exceeded 98% at 20 MPa and 40 °C, for a residence time of 20 s and an oil to methanol molar ratio of 1:24. Even for moderate reaction conversions, a fractionation stage based on two separators afforded FAME with >96% purity. Lipozyme TL IM was less efficient with waste cooking sunflower oil. In this case, a combination of Lipozyme TL IM and Novozym 435 afforded FAME yields nearing 99%.     

Synthesis and component confirmation of biodiesel from palm oil and dimethyl carbonate catalyzed by immobilized-lipase in solvent-free system

The transesterification of palm oil and dimethyl carbonate (DMC) for preparing biodiesel has been carried out at the catalysis of immobilized-lipase in solvent-free system. The components were all confirmed by GC and GC–MS analysis. The fatty acid methyl esters (FAMEs) were analyzed with internal standard method. The fatty acids glycerol carbonate esters (FAGCs) were characterized as the intermediates. And, glycerol dicarbonate (GDC) was confirmed as the byproduct by comparing with the model compound. Moreover, the effects of the reaction conditions (type of lipases, molar ratio of DMC and palm oil, amount of catalyst, reaction temperature and time) on the yield of FAMEs were investigated. The yield of FAMEs could reach 90.5% at 55 °C for 24 h with the molar ratio of DMC to oil 10:1 and the catalyst amount of 20% Novozym 435 (based on the oil weight). There was no obvious loss in the FAMEs yield after Novozym 435 having been used for eight cycles.