Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value

One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3 Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials.     

Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil

The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.     

Improving fatty acid methyl ester production yield in a lipase-catalyzed process using waste frying oils as feedstock

The application of waste frying oil (WFO) mixed with rapeseed oil as a feedstock for the effective production of fatty acid methyl esters (FAME) in a lipase-catalyzed process was investigated. The response surface methodology (RSM) was used to optimize the interaction of four variables: the percentage of WFO in the mixed feedstock, the methanol-to-oil ratio, the dosage of Novozym 435 as a catalyst and the temperature. Furthermore, the addition of methanol to the reaction mixture in a second step after 8 h was shown to effectively diminish enzyme inhibition. Using this technique, the model predicted the optimal conditions that would reach 100% FAME, including a methanol-to-oil molar ratio of 3.8:1, 100% (wt) WFO, 15% (wt) Novozym 435 and incubation at 44.5 °C for 12 h with agitation at 200 rpm, and verification experiments confirmed the validity of the model. According to the model, the addition of WFO increased FAME production yield, which is largely due to its higher contents of monoacylglycerols, diacylglycerols and free fatty acids (in comparison to rapeseed oil), which are more available substrates for the enzymatic catalysis. Therefore, the replacement of rapeseed oil with WFO in Novozym 435-catalyzed processes could diminish biodiesel production costs since it is a less expensive feedstock that increases the production yield and could be a potential alternative for FAME production on an industrial scale.     

固定化Enterobacter agglomerans脂肪酶生产菜籽油生物柴油的研究

用硅藻土对实验室筛选得到的成团肠杆菌脂肪酶干燥酶粉进行固定化,固定化酶在有机溶剂体系下催化生产生物柴油。在最佳反应条件,即菜籽油15.47 mL,固定化脂肪酶用量1 000 U,甲醇为酰基受体(7.15 mL,3次等量加入),5 mL正己烷,振荡速度180 r/min,35℃反应48 h时,转化率达91.03%。实验结果表明,油酸含量高有利于生产生物柴油,而芥酸有不利影响。固定化酶稳定性好,重复使用8次,转化率仍大于50%,同时还具有一定的适应性,可催化大豆油和葵花籽油生产生物柴油。研究表明,固定化酶可用于催化生产生物柴油,并有效降低酶催化法的生产成本。     

非水相介质杂醇油酶法转化天然酯类香料的研究

为提高杂醇油的利用率和附加值,研究非水相条件下杂醇油的脂肪酶催化酯化反应,优化其转化为天然酯类香料的工艺条件.结果表明,酶用量、分子筛用量、乙酸加入次数及间隔时间、摇床转速、反应温度、反应时间等因素条件对产物酯的生成有重要影响,采用间歇式加酸、加入适量分子筛吸水剂可明显提高混合酯得率.在正已烷10mL、杂醇油3ml、乙酸1.5mL的反应体系中,正交试验设计优化后的工艺条件(乙酸异戊酯产率最高时):脂肪酶Novozym435FG用量70mg、乙酸分6次加入、加入乙酸间隔时间3h、分子筛用量2g、摇床转速140r/min、反应温度45℃、反应时间36h.该条件下,乙酸异戊酯、乙酸异丁酯和乙酸丙酯的产率分别为90.1％,91.3％,78.1％.     

酶法制备葛根素的丙酯衍生物

本文研究了不同种类的水解酶催化非水相葛根素丙酰化反应的催化效率,并研究以Novozym 435脂肪酶催化葛根素丙酰化反应为模型,几个关键反应因素对该模型的影响规律。研究发现固定化脂肪酶Novozym 435、Lipozyme IMTL、Lipozyme IMRM均能高效催化葛根素丙酰化反应,且转化率高达98%以上,但另外几种游离脂肪酶催化葛根素丙酰化反应活性及效率就特别低。相同条件下,猪胰脂肪酶催化葛根素丙酰化反应的转化率为67%左右;胰脂肪酶和CRL脂肪酶表现很低的催化活性,肽酶和蛋白酶没有表现催化活性。以四氢呋喃为反应溶剂,2 mg/m L Novozym 435脂肪酶,底物:酰基供体之比为1:30,水分含量为0的反应条件下,反应6 h,底物转化率达到99.5%。反应产物经分离纯化后进行了结构鉴定,高效液相色谱、质谱、傅里叶红外光谱结果表明,非水相酶催化葛根素酰化反应主要生成单酯,所得产物酯为葛根素丙单酯,区域选择性达98%。     

有机相脂肪酶催化合成柠檬酸甘油酯

以脂肪酶为催化剂,在有机介质中合成柠檬酸甘油酯。对催化合成反应的脂肪酶和反应介质进行了筛选,最佳溶剂为叔丁醇,固定化于大孔丙烯酸树脂的Candida antarctica脂肪酶B(Novozym 435)的催化活性最好。同时对底物浓度、底物摩尔比、脂肪酶用量、吸水剂用量、反应温度和反应时间等条件进行了优化,确定最佳工艺参数为:酸浓度0.12mol/L,单甘酯和柠檬酸摩尔比2∶1,脂肪酶用量为质量分数8%,吸水剂用量0.12g/mL,50℃反应48h,柠檬酸转化率可达70.97%。经电喷雾质谱和红外光谱分析,反应产物主要是α-柠檬酸单硬脂酸甘油酯。     

Enzymatic Synthesis of Ascorbyl Palmitate in Organic Solvents: Process Optimization and Kinetic Evaluation

This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents. The sequential strategy of experimental designs proved to be useful in maximizing the conditions for product conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production were achieved at ascorbic acid to palmitic acid mole ratio of 1:9, stirring rate of 150 rpm, 70 °C, enzyme concentration of 5 wt.% at 17 h of reaction, resulting in an ascorbyl palmitate conversion of about 67%. Reaction kinetics for ascorbyl palmitate production showed that very satisfactory reaction conversions (∼56%) could be achieved in short reaction times (6 h). The kinetic empirical model proposed showed ability to satisfactory represents and predict the experimental data.     

有机相脂肪酶催化合成阿魏酸乙酯

本实验研究了有机溶剂中脂肪酶催化阿魏酸乙酯合成反应。对催化合成阿魏酸乙酯反应的脂肪酶和反应介质进行了比较,最佳溶剂为叔丁醇,在所选的6种脂肪酶中,固定化于大孔丙烯酸树脂的南极假丝酵母脂肪酶B(Novozym 435)的催化活性最好。同时对影响合成阿魏酸乙酯反应的因素(底物浓度、底物摩尔比、温度、初始水含量、反应时间等)进行了探讨,优化了反应条件:在10ml无水叔丁醇中,当酸醇摩尔比为1:1,酸浓度为0.1mol/L,反应温度为60℃,反应时间为120h时产率达到最高,脂肪酶Novozym 435具有较高的稳定性,重复使用六次后产率仍然可达到23%。     

Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4–30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80–90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.     

Physico‐enzymatic production of monoacylglycerols enriched with very‐long‐chain polyunsaturated fatty acids

BACKGROUND: Monoacylglycerols (MAG) containing polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have interesting applications. The enzymatic processing of such MAG directly from fish oils is highly interesting, integrating the processing of MAG and concentration of EPA and DHA. The aim of this study was to develop an efficient enzymatic glycerolysis system together with physical fractionation for the production of PUFA‐MAG from tuna oil. RESULTS: Novozym 435 was eventually selected after evaluation together with immobilized lipase AK in a tertiary alcohol‐based system. A further evaluation of solvent mixtures involving tertiary alcohols was made, taking ease of operation into consideration. It turned out that a number of mixtures gave a similar performance to that of tert‐butanol (TB). Basic reaction parameters were thoroughly evaluated. In the batch reaction system with TB as solvent, the recommended conditions were: glycerol/tuna oil 4:1 (mol/mol), TB/tuna oil 2:1 (wt/wt), 15 wt% Novozym 435, and temperature 40 °C. Under these conditions, the yield of MAG was up to 90% after 3 h incubation. Crude MAG from the production was fractionated to produce MAG with higher EPA and DHA content. Using acetone as solvent at 0 °C led to ca 50% yield of MAG but contained EPA and DHA up to 71% in comparison with ca 30% in tuna oil. CONCLUSION: Potentially practical process steps have been developed for the production of MAG containing a high content of EPA and DHA from natural fish oils with high efficiency and simplicity. Copyright © 2007 Society of Chemical Industry     

Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil

BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn‐2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn‐3 position. RESULTS: Two types of lipase‐catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn‐2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn‐2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase‐catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase‐catalysed re‐esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn‐2 position was synthesised from pine nut oil via lipase‐catalysed redistribution and re‐esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts. Copyright © 2011 Society of Chemical Industry     

Enzymatic synthesis of lard-based ascorbyl esters in a packed-bed reactor: Optimization by response surface methodology and evaluation of antioxidant properties

An optimal continuous production of lard-based ascorbyl esters (LBAEs) by transesterification of lard with l-ascorbic acid in a packed bed reactor (PBR) was developed using immobilized lipase (Novozym 435) as a catalyst in a tert-amyl alcohol solvent system. Response surface methodology (RSM) and central composite design (CCD) were employed to evaluate the effects of substrate flow rate, reaction temperature and substrate molar concentration ratio on the molar conversion of LBAEs. The optimum conditions were as follows: substrate flow rate 1.07 ml/min, reaction temperature 56.44 °C, and substrate molar concentration ratio 2.24:1. The optimum predicted LBAEs yield was 50.83% and the actual value was 50.50%. The above results shows that the RSM study based on CCD is adaptable for LBAEs yield studied for the current transesterification system. The antioxidant activities of LBAEs has also been studied. LBAEs represented positive antioxidant potential on superoxide anion radical and hydroxyl radicals and satisfactory antioxidant activity in lard and soybean oil. The results suggest that LBAEs has the potential to serve as natural antioxidant in food system.     

预处理固定化脂肪酶催化合成生物柴油

探讨了预处理固定化Candida antarctica脂肪酶催化餐饮废油合成生物柴油的过程。将固定化Candida antarctica脂肪酶用叔丁醇处理3h后再用废油浸泡4h，用于催化酯交换反应，酯交换反应速率明显加快。研究发现，固定化Candida antarctica脂肪酶预处理后，过量甲醇对酶的抑制作用仍然存在。采用分步添加甲醇工艺，按总醇油摩尔比3：1，分别在0、0．5、1．0、1．5、2．0、2．5h等量加入甲醇，反应4h后，体系中甲酯含量达到97．86％，反应：效率是未处理固定化酶催化合成生物柴油体系的6倍。固定化酶重复使用仍具有较高活性。     

乌桕梓油理化特性及其酶法制备生物柴油的研究

测定了乌桕梓油常规理化特性.采用柱层析、薄层层析和气相色谱法,研究了乌桕梓油中甘油酯种类及其脂肪酸组成.采用高温气相色谱/EI质谱联用仪直接分析了乌桕梓油中甘油酯结构.结果表明,乌桕梓油中性脂占98.79%,磷脂占0.22%,糖脂占0.99%.乌桕梓油中性脂的脂肪酸组成主要有C12:0、C14:0、C16:0、C17:0、C18:0、C18:1、C18:2和C18:3,磷脂中没有检出C12:0和C17:0,糖脂中则没有检出C17:0.乌桕梓油中甘油酯组成主要有月桂酸棕榈酸亚油酸甘油三酯、硬脂酸亚油酸亚麻甘油三酯、双棕榈酸亚油酸甘油三酯、棕榈酸亚油酸亚麻酸甘油三酯和双亚油酸亚麻酸甘油三酯等5种.最后,比较了无溶剂体系和叔丁醇体系中,以杂醇油为酰基受体,初步研究了脂肪酶法制备乌桕梓油生物柴油效果.结果表明,对于无溶剂体系和叔丁醇体系而言,当Novozym435与Lipozyme TLIM脂肪酶复合比例为2:4时,生物柴油转化率达到最高,分别为98.28%和76.33%.     

Biodiesel fuel production by transesterification of oils

Biodiesel (fatty acid methyl esters), which is derived from triglycerides by transesterification with methanol, has attracted considerable attention during the past decade as a renewable, biodegradable, and nontoxic fuel. Several processes for biodiesel fuel production have been developed, among which transesterification using alkali-catalysis gives high levels of conversion of triglycerides to their corresponding methyl esters in short reaction times. This process has therefore been widely utilized for biodiesel fuel production in a number of countries. Recently, enzymatic transesterification using lipase has become more attractive for biodiesel fuel production, since the glycerol produced as a by-product can easily be recovered and the purification of fatty methyl esters is simple to accomplish. The main hurdle to the commercialization of this system is the cost of lipase production. As a means of reducing the cost, the use of whole cell biocatalysts immobilized within biomass support particles is significantly advantageous since immobilization can be achieved spontaneously during batch cultivation, and in addition, no purification is necessary. The lipase production cost can be further lowered using genetic engineering technology, such as by developing lipases with high levels of expression and/or stability towards methanol. Hence, whole cell biocatalysts appear to have great potential for industrial application.     

Lipase-catalyzed synthesis and characterization of flaxseed oil-based structured lipids

The biosynthesis of structured lipids (SLs) was carried out by the interesterification of flaxseed oil (FO) and tricaprylin (TC) in an organic solvent medium (OSM), using selected commercial lipases, including Amano DF, Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM. The fatty acyl chains of the synthesized triacylglycerols (TAGs) were identified by atmospheric pressure chemical ionization/mass spectrometric (APCI/MS) analysis, while the fatty acid positional distribution of the MLM- and MML-SLs (M-medium and L-long chain fatty acids) was determined by silver-ion high-performance liquid chromatographic (Ag⁺/HPLC) analysis. The effects of reaction temperature (T_r, 30–50 °C), enzyme concentration (E_c, 0.5–4%, w/v), initial water activity (a_w, 0.05–0.43) and reaction time (R_t, 0–72 h) on the efficiency of the enzymes, were studied. The bioconversion yield (%) of the synthesized MLM- and MML-SLs was monitored under the established reaction parameters for each lipase. The maximum yield of MLM-SLs was obtained in the order, of Novozym 435 > Lipozyme TL-IM > Lipozyme RM-IM > Amano DF. Moreover, considering the ratio of the MLM- to MML-SLs produced by each enzyme, Novozym 435 and Lipozyme TL-IM were selected as the most effective enzymes for interesterification of FO and TC.     

正己烷反应体系中酶法转化乙酸3-甲硫基丙醇酯的工艺条件

研究了脂肪酶催化合成天然香料乙酸3-甲硫基丙醇酯（EMTP）的关键因素及条件。以发酵3-甲硫基丙醇、乙酸为主要原料酶法合成EMTP的反应中,固定化Novozym435脂肪酶为最佳催化剂,正己烷作为溶剂有利于提高酶活力;在正己烷反应体系中,Novozym435用量、分子筛吸水剂用量、乙酸加入次数、反应温度、反应时间等因素对EMTP合成有重要的影响,添加Novozyme43550mg、分子筛2g、反应温度40℃、乙酸分3次加入、摇瓶速率100～150r/min,反应30h为较佳的反应条件,其EMTP产量为6.5mg/mL。      

响应面法优化生物酶法合成植物甾醇酯

采用Novozym 435脂肪酶催化大豆油与植物甾醇的酯交换反应,对其技术参数和条件进行探索。通过单因素与响应面分析,确定最佳工艺参数：反应温度101℃、酶用量1%、甾醇添加量5.2%、反应时间3h。在最佳条件下酯交换反应的转换率高达85.67%。     

Discrimination against diacylglycerol ethers in lipase-catalysed ethanolysis of shark liver oil

Lipase-catalysed ethanolysis of squalene-free shark liver oil was investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to 10 different lipases were compared. The ratio of oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed, although poor discrimination against diacylglycerol ether was observed. The best results in terms of selectivity and stability were obtained with immobilised lipase from Candida antarctica (Novozym 435). Ethanolysis reaction after 24 h in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacylglycerol ethers (10.6%), monoacylglycerol ethers (32.9%) and fatty acid ethyl esters (46.0%). In addition, when an excess of ethanol was used, diacylglycerol ethers completely disappeared after 15 h, giving a final product mainly composed of monoacylglycerol ethers (36.6%) and fatty acid ethyl esters (46.4%).