Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40 degrees C in a mixture of tuna oil and 1 3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40 degrees C for 12 h in a mixture of tuna oil and 1 3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2 3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1 3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40°C in a mixture of tuna oil and 1/3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40°C for 12 h in a mixture of tuna oil and 1/3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2/3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1/3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

生物柴油的双脂酶催化生产工艺研究

以菜籽酸化油为原料,研究两种脂酶顺序催化制备生物柴油的生产工艺。结果表明,固相化细菌A007脂酶催化甘油三酯(TAG)水解的最适条件为:含水量40%、脂酶用量100 U/g、反应温度30℃、反应时间12 h,此时TAG水解率和游离脂肪酸(FFA)含量分别为93.3%和90.1%;在催化FFA甲酯化过程中,固相化Candida antarctica脂酶在FFA与甲醇摩尔比为1∶5时可达到最佳效果;在第二次甲酯化时,加入甘油有利于提高FFA酯化率,经过24 h反应,可将总酯化率从无甘油时的96.9%提高到98.6%。该工艺可操作性强,具有较好的应用价值。     

Interesterification (acidolysis) of butterfat with conjugated linoleic acid in a batch reactor

Six commercial lipases, in free or immobilized form, were tested for their ability to catalyze acyl exchange between conjugated linoleic acid and anhydrous butterfat under solvent-free conditions. Immobilized Candida antarctica lipase exhibited the best activity. Experiments were conducted for this lipase in butterfat to conjugated linoleic acid ratios of 10:1 (vol/vol), temperatures from 30 to 70 degrees C, enzyme concentrations of 50 to 200 mg/g of reaction mixture, and water contents of 0.15 to 2% (wt/wt). At the maximum enzyme concentration used, equilibrium was reached within the first 24 h of reaction. The optimum temperature was 50 degrees C. The triacylglycerol profile of the product butterfat reflected changes in the relative proportions of fatty acid residues as the reaction proceeded, with increases in those triacylglycerols containing 46 to 54 carbon atoms and concomitant decreases in those triacylglycerols containing 34 to 42 carbon atoms.     

酶法全酯化合成富含EPA/DHA甘油酯工艺研究

以富含EPA/DHA的脂肪酸为底物,采用两步酶法合成富含EPA和DHA的甘油酯。首先,以T1脂肪酶为催化剂催化富含EPA/DHA的脂肪酸和甘油反应;在最优条件为:反应温度40℃,水分添加量为底物混合物的3%、甘油与脂肪酸摩尔比3∶1和酶添加量50 U/g底物混合物时,富含EPA/DHA的脂肪酸的转化率达到62%以上,此时产物中甘油三酯、甘油二酯、甘油单酯的质量分数分别为10.52%、38.15%、25.64%。将游离酶催化酯化反应产物中的油相回收,利用自制的固定化CALB(LipozymeCALB L固定于环氧树脂ECR8285上)为催化剂,在真空条件下继续催化未反应的脂肪酸与偏甘油酯(甘油单酯和甘油二酯)继续酯化反应12 h,此时产物中甘油三酯、甘油二酯和甘油单酯的质量分数分别达到38.34%、51.02%、10.63%,没有检测到脂肪酸的存在。     

游离DHA酯化富集的脂酶催化工艺研究

研究了脂酶催化游离DHA和甘油酯化的工艺。结果表明:以Rhizomucor miehei脂酶催化的一步法工艺的最适条件为FFA∶甘油=1∶3、脂酶量1%和水分5%,其酯化率、三酰甘油(TAG)含量和TAG中DHA富集量分别为71.6%、22.98%和47.53%;以Rhizomucor miehei脂酶催化的二步法工艺和以Rhizomucor miehei脂酶、Alcaligenes sp.脂酶共同催化的混合酶法工艺不能改变TAG中DHA的百分含量,但可显著提高反应的酯化率和TAG含量。其中,二步法工艺可将酯化率和TAG含量分别提高到84.66%和91.86%,而混合酶法工艺则可分别将其进一步增加到97.68%和93.39%。     

α-亚麻酸芦丁酯的酶法合成工艺研究

以脂肪酶Novozym435为生物催化剂,建立了新型功能脂质α-亚麻酸芦丁酯的酶促合成工艺,并通过响应面方法系统考察了反应工艺参数对酯化率的影响。得到的最佳合成工艺参数为：反应溶剂为丙酮,脂肪酶Novozym435的添加量为17.9mg/mL, 芦丁的底物浓度为17.3mmol/L, 芦丁与α-亚麻酸的摩尔比为1：4.5,反应时间60h,酯化率高达92.6%,经过提纯后产物纯度可达到95.0%。     

有机溶剂体系固定化脂肪酶催化合成生物柴油

探讨了有机溶剂体系固定化Candida antarctica脂肪酶催化大豆色拉油合成生物柴油的过程。将固定化Candida antarctica脂肪酶置于有机溶剂体系中催化合成生物柴油的效果较好。研究发现,在40℃下反应10 h,固定化Candida antarctica脂肪酶以石油醚作为有机溶剂转化率最高,当总醇油物质的量比为3∶1,固定化酶占5%(相对于油质量),加入5%质量分数的水时固定化酶反应活性最高,酯化率可以达到88.4%。固定化酶重复使用10次仍具有较高活性。     

Production of butyl acetate ester by lipase from novel strain of Rhizopus oryzae

A new lipase preparation from Rhizopus oryzae was used to catalyze the esterification reaction between acetic acid and butanol to produce butyl acetate ester (pineapple flavor). This flavor compound can be used in food, cosmetic and pharmaceutical industries. Only 3% of butyl acetate was obtained when free lipase was used in the synthesis containing only the substrates. In contrast, the conversion yield reached 25% when immobilized lipase was used under the same conditions. The synthesis of butyl acetate catalyzed by immobilized lipase in nonconventional media was optimized. A maximum conversion yield of 60% in a solvent-free system was obtained under the following conditions: amount of immobilized lipase, 500 IU; amount of initially added water, 45%; acetic acid/butanol molar ratio, 1:1; and in incubation temperature, 37 degrees C. Immobilized lipase could be repeatedly used for three cycles without a decrease in synthesis activity. The production of butyl acetate esters by immobilized R. oryzae lipase was also studied in the presence of organic solvents. Compared with a solvent-free system, the synthesis activity was improved in the presence of heptane and hexane with conversion yields of 80% and 76%, respectively. However, solvent-free systems tend to purify more easily the products without any toxicity and inflammability problems.     

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     

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 production of human milk fat substitutes containing palmitic and docosahexaenoic acids at sn-2 position and oleic acid at sn-1,3 positions

This paper studies the synthesis of structured triacylglycerols (STAGs) rich in palmitic and docosahexaenoic acids (PA and DHA) at sn-2 position and oleic acid (OA) at sn-1,3 positions by a four step process. First, triacylglycerols (TAGs) were obtained with 63–66 mol PA/100 mol total fatty acids and 10 mol DHA/100 mol by acidolysis of tuna oil and commercial PA, catalyzed by the non-positionally specific lipase Novozym 435. Then these TAGs were purified neutralizing the free fatty acids (FFAs) by KOH hydroethanolic solutions and extracting TAGs with hexane; these TAGs were completely recovered as pure TAGs (without FFAs). The third step involved the displacement of fatty acids located at sn-1,3 positions by acidolysis of PA and DHA enriched TAGs with OA rich FFAs, catalyzed by the sn-1,3 specific lipase DF from Rhizopus oryzae, immobilized on Accurel MP-1000; TAGs with 67 mol OA/100 mol at sn-1,3 positions and 52.1 and 15.4 mol PA and DHA, respectively, per 100 mol at sn-2 position were obtained. Both acidolysis reactions were carried out in stirred tank reactors (STRs) with lipase both dispersed in the reaction medium and contained in a cartridge filter attached to the stirrer rod. Finally STAGs were purified and obtained with yields of over 80 mol STAGs/100 mol STAGs in the reaction product (no FFAs were detected).     

Resolution of (±)-menthol by immobilized Candida rugosa lipase on superparamagnetic nanoparticles

Lauric acid-stabilized magnetic particles were prepared by coprecipitation in the presence of lauric acid and used for the covalent immobilization of Candida rugosa lipase via carbodiimide activation. Size analysis by transmission electron microscopy (TEM) and measurement of magnetization curves revealed that the immobilized lipase was superparamagnetic. Resolution of (±)-menthol was performed by the immobilized lipase-catalyzed enantioselective esterification with propionic anhydride. Effects of various reaction parameters, such as enzyme load, solvents, water activity, substrate concentration, reaction time and temperature, on the conversion as well as enantioselectivity were investigated. As a result, (−)-menthyl propionate with a yield higher than 96% and over 88% enantiomeric excess of products was obtained. Better conversion and enantioselectivity could be expected for the immobilized lipase-catalyzed reaction performed at 30 °C for 2.5 h with 0.2 mol/l of (±)-menthol. Hexane was found to be the most suitable solvent, and the activity as well as enantioselectivity of the immobilized lipase decreased gradually with increasing water activity. Good durability of the immobilized lipase to catalyze the resolution of (±)-menthol was also observed.     

Optimisation of enzymatic esterification of soybean oil deodoriser distillate

The enzymatic esterification of free fatty acids from soybean oil deodoriser distillate with ethanol, catalysed by immobilised fungal lipase, was studied. The extent of conversion of free fatty acids to ethyl esters was optimised using a response surface methodology obtained through a second‐order factorial experimental design. The variables studied were reaction temperature (30–70 °C), enzyme concentration (7–23%) and ethanol/free fatty acid molar ratio (0.3–3.7:1). The optimal reaction conditions achieved were temperature from 46.4 to 53.6 °C, enzyme concentration from 13.6 to 16.5% and ethanol/free fatty acid molar ratio from 1.7 to 2.3:1, with conversions above 88%. No significant tocopherol losses were observed during the process. In conclusion, enzymatic fatty acid esterification was shown to be a technically viable process. © 2001 Society of Chemical Industry     

酵母展示脂肪酶催化合成葡萄酒香料己酸丁酯

以毕赤酵母展示的南极假丝酵母脂肪酶B为催化剂,已酸和丁醇为反应底物,合成葡萄酒用香料己酸丁酯。结果表明,酵母展示脂肪酶是合成己酸丁酯的良好催化剂,其最适催化工艺条件为：酸醇物质的量比为1：1．2,催化剂展示脂肪酶用量为2％,反应时间为4h,酯化率可达97．2％。     

Effect of reaction rate on converted products from wheat germ oil by immobilized lipase ethanolysis

Monoglycerides and diglycerides containing function of wheat germ oil were to produce and to assess the influence of various conditions on the ethanolysis activities of immobilized lipases. Immobilized lipases like lipozyme (Thermonuces lanuginose immobilized on silica gel and Rhizomucor miehei immobilized on an ion exchange resin were used for enzymatic ethanolysis. Ethanolysis was carried out in different processes (non-pressured and pressured system) to compare the reaction rate and yield. For immobilized lipase, the optimal condition was found at 1.0 of ethanol mol ratio, temperature of 60°C, and lipases amount of 4% in non-pressured system. However, in pressured system, the optimal temperature was found at 50°C. The enzyme activity was changed depending on the enzyme source, reaction time, pressure, and temperature. Changing experimental parameters (temperature, ethanol mol ratio, enzyme amount, and reaction time) affecting wheat germ oil ethanolysis reaction, the optimal reaction conditions were established.     

Regeneration of immobilized Candida antarctica lipase for transesterification

Immobilized lipase from Candida antarctica was employed to convert triglycerides to biodiesel using alcohol. Immobilized lipase is frequently deactivated by lower alcohols with deactivation being caused by the immiscibility between triglycerides and methanol or ethanol. When the lower alcohol is adsorbed to the immobilized enzyme, the entry of triglycerides is blocked, which causes the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-butanol, can regenerate the deactivated immobilized enzyme. The present work established that the activity of immobilized lipase could be significantly increased when such alcohols were used for an immersion pretreatment of the enzyme. The activity of the commercially available immobilized enzyme, Novozyme 435, increased about tenfold in comparison to the enzyme not subjected to any pretreatment. Following complete deactivation of the enzyme by methanol, washing with 2-butanol and tert-butanol successfully regenerated the enzyme and restored it to about 56% and 75% of its original activity level, respectively.     

脂肪酶催化鱼油醇解富集EPA和DHA的研究

研究了固定化脂肪酶Lipozyme RM IM催化鱼油部分醇解反应,探讨了油醇比、酶加量等因素对反应的影响,在优化条件下,可以使鱼油中EPA、DHA的含量由26.1%升至43.O%,得率大于75%,对鱼油醇解产物进行了分子蒸馏提纯,得到了富含EPA、DHA的甘油酯型鱼油产品.     

交联剂固定化假丝酵母脂肪酶及其催化反应研究

以价格低廉的无纺布为固定化载体,添加聚丙烯酸酯作为交联剂,生产固定化脂肪酶。这种固定化方法对脂肪酶的纯度等性质要求不高,制备工艺操作简单。交联剂对酶液的活力没有影响,且具有一定的成膜特性,能够为脂肪酶分子催化提供良好的微环境。固定化脂肪酶酶活损失较少,在25%以内。制备的固定化脂肪酶在棕榈酸异辛酯酯化反应体系可以重复催化达到47批次,酯化率在80%以上;在酯交换生产脂肪酸甲酯反应体系可以连续反应25批次,转酯化率在90%以上。     

非水介质中脂肪酶催化合成正戊酸异戊酯的研究

德氏根霉菌(Rhizopusdelemar) 经固态发酵生产脂肪酶,以此酶为催化剂,在非水介质中合成了正戊酸异戊酯。研究了反应温度、溶剂、底物浓度、底物摩尔比、吸水剂用量等因素对酯化反应的影响,确定了正戊酸异戊酯的最佳合成条件为:反应温度为5 0℃,异辛烷为反应介质,底物浓度为0 15mol/L ,酸醇摩尔比为1∶1 4。在反应体系中需加入0 2 5 g/mL的5 分子筛,以吸收酯化反应生成的水。在优化的条件下,反应6h后,酯合成转化率达98%。