固定化脂肪酶催化桐油制备生物柴油的研究

对磁性Fe3O4纳米粒固定化脂肪酶催化桐油制备生物柴油进行了研究，分步探讨了硼酸盐缓冲液用量、固定化酶用量、醇油摩尔比、反应温度、固定化酶清洗与否对转酯反应的影响，以及固定化酶的操作稳定性。结果表明：将正己烷与桐油体积比定为2∶?1，然后加入与桐油等摩尔的甲醇、桐油体积6%的硼酸盐缓冲液及7.5 mg/mL（以桐油体积计）固定化酶，反应5 h和12 h各加入与桐油等摩尔的甲醇（总的醇油摩尔比3∶?1），并每次添加甲醇前用丙酮清洗固定化酶，45?℃、200 r/min 反应26 h后，甲酯转化率可达91.2%。该固定化脂肪酶连续催化10批次反应后，甲酯转化率仍然可达84.1%，具有一定的工业应用价值。     

毕赤酵母展示稻米脂肪酶催化乙酸乙酯研究

论文利用展示在毕赤酵母表面的稻米脂肪酶作为催化剂在正己烷中催化合成乙酸乙酯,考察了稻米脂肪酶的酶活、底物摩尔比、反应温度、反应时间和3A分子筛添加量对合成乙酸乙酯反应的影响.结果表明,在15 m L正己烷体系中,n(乙酸/乙醇,摩尔比)为1.2∶1,稻米脂肪酶的酶活为15 750 U/g,发酵时间为4 h,反应温度为40℃,3A分子筛添加质量为8%是合成乙酸乙酯的最佳条件。在此条件下,稻米脂肪酶催化合成乙酸乙酯的转化率达到90.92%。     

米糠油酶解条件研究

利用脂肪酶催化油脂水解成脂肪酸和甘油,从而得到脂肪酸产品是一种温和的油脂水解方法.为了提高产品质量和水解效率,研究了米糠油酶解时反应温度、水/米糠油摩尔比及脂肪酶用量对米糠油水解效果的影响.试验结果表明,当水/米糠油摩尔比为40:1,加入6%的脂肪酶,在55℃下水解7 h,其水解率可达92.54%,水解较彻底.     

不同反应温度下T1脂肪酶催化油酸与甘油酯化反应动力学研究

T1脂肪酶是一种新型耐热脂肪酶,能催化油酸与甘油的酯化反应。当甘油与油酸物质的量比2∶1、酶的添加量9.7 U/g(占总反应物质量)、缓冲液的添加量5%(占总反应物质量)和转速542r/min时,考察不同反应温度(40、50、60℃)下T1脂肪酶催化油酸与甘油酯化反应的动力学。结果表明,T1脂肪酶催化该酯化反应活化能为20.272 9 k J/mol;当反应温度较低(40℃)时遵循准一级反应;随着反应温度升高到较高范围(50、60℃),则变为准二级反应。     

高活性红曲源脂肪酶抑制剂的初步研究

在红曲霉(Monascusruber)发酵过程中添加氨基酸可提高红曲发酵产物的脂肪酶抑制活力。本研究首先分别考察了20种氨基酸的添加对红曲发酵物的脂肪酶抑制活力的影响。结果表明,苯丙氨酸、甲硫氨酸、色氨酸、缬氨酸、赖氨酸、亮氨酸、组氨酸和谷氨酰胺的添加可显著增强红曲发酵产物的脂肪酶抑制效能,其中以苯丙氨酸的添加效果最为显著。其次,借助高速逆流色谱(HSCCC)对红曲提取物进行初步分离,当分离体系配比为正己烷/甲醇/水(10/8/2,v/v/v)时,提取组分得到较好的分离,脂肪酶活力测定结果表明,组分5、6、7和8的抑制作用较强。最后,采用高效液相色谱(HPLC)方法对比分析添加苯丙氨酸与未添加氨基酸的红曲提取物成分,结果表明,前者存在非已知红曲色素类高活性脂肪酶抑制物质。     

水相体系酶法制备甘油磷脂酰乙醇胺的研究

研究了水相体系中脂肪酶Thermo 4S-3水解大豆粉末磷脂制备甘油磷脂酰乙醇胺(L-α-GPE)的可行性,探讨了反应条件对磷脂酰乙醇胺(PE)转化率和L-α-GPE得率的影响.确定脂肪酶Thermo4S-3催化制备L-α-GPE的最佳反应条件为:pH 7,底物质量浓度5 mg/mL,反应温度40℃,酶添加量30 U/mL,CaCl2添加量3.33 mg/mL,反应时间6h.在最佳反应条件下,PE转化率为95.5％,L-α-GPE得率为93.6％.     

解脂耶氏酵母lip2脂肪酶水解谷维素产生阿魏酸的研究

对解脂耶氏酵母(Yarrowia lipolytica)脂肪酶水解谷维素产生阿魏酸的酶反应体系进行了研究。实验发现解脂耶氏酵母全脂肪酶粉(105U/mg)在50mmol/L p H7.0 Tris-HCl(含7.5mmol/L黄胆酸钠),100mmol/L p H6.0磷酸钠缓冲液(含1000U脂肪酶)的体系中,水解产生阿魏酸的得率为2.94%。为了进一步提高脂肪酶水解效率,对解脂耶氏酵母脂肪酶中主要组分lip2脂肪酶基因进行了克隆,整合至毕赤酵母GS115基因组后发酵制取lip2脂肪酶粉(70.1U/mg),于上述酶解体系中进行水解谷维素实验。实验结果表明阿魏酸产率为2.87%。获得的lip2脂肪酶催化效率略低于全脂肪酶粉催化效率,但是获得了单一的脂肪酶基因,为进一步采取分子进化技术提高其催化能力奠定了基础。     

正己烷体系中酶催化菜籽油制备乙酯生物柴油

研究了脂肪酶催化菜籽油乙醇解反应的几个主要影响因素,即醇油摩尔比、催化剂用量、反应温度和反应时间,并研究了正己烷对反应的影响。得出脂肪酶催化菜籽油乙醇解制备生物柴油的最佳反应条件为:醇油摩尔比5∶1,催化剂用量10%,反应温度40℃,反应时间36 h、正己烷用量15%。在最佳反应条件下,1次加入乙醇时转化率为86%;采用3次加醇能很好改善催化环境,转化率达到93%。利用气相色谱分析了产物中脂肪酸乙酯的组成,结果表明,产物中主要是十八碳酸乙酯。     

橄榄油乳化法测定脂肪酶活性的优化研究

以猪胰脂肪酶为例,针对传统橄榄油乳化法存在准确度较低、重现性不佳等问题,对底物浓度及用量、均质时间、缓冲液添加量、反应体系温度、反应环境等反应条件进行了优化研究:以20%橄榄油乳化液5mL代替25%橄榄油乳化液4mL,以3mL磷酸盐缓冲液代替5mL磷酸盐缓冲液,将均质处理的速度与时间由高速(>10000r/min)、6min降低为5000r/min、2min,将反应体系温度由40℃降低为35℃,改变静止的反应环境为在100r/min下振荡反应,选择在空白组反应体系中加入1mL0.025mol/LpH7.5磷酸缓冲液代替1mL酶液。优化后的条件下测得的脂肪酶活力较优化前提高了35%以上,平行测定的RSD值为5.5%,方法的精密度较高。     

白地霉脂肪酶固定化及其催化光皮树油合成生物柴油的研究

本文采用响应面分析法对树脂NKA-Ⅱ固定脂肪酶的条件进行优化,并对其酶学性质和在催化光皮树油合成生物柴油中的操作稳定性进行了简单的探讨。首先通过单因素试验研究酶液/载体比、温度、缓冲液pH值等条件对固定化脂肪酶酶活力的影响,在此基础上,利用响应面试验设计对树脂NKA-Ⅱ固定化脂肪酶的条件进行优化,用制备的固定脂肪酶催化光皮树油和甲醇反应合成生物柴油。结果表明：酶液/载体比、温度和缓冲液pH值对固定化酶的活力都有影响,树脂NKA-Ⅱ固定化脂肪酶的最佳固定条件是：酶液/载体比为4∶1、温度为39℃、缓冲液pH值为7.5,固定化脂肪酶湿重活力高达216.2±4.5U/g,生物柴油转化率达90%以上。将响应面法应用于树脂NKA-Ⅱ固定化脂肪酶条件的优化是可行的,制备的固定脂肪酶具有工业化生产的潜力。     

Lyophilization of lipase with cyclodextrins for efficient catalysis in ionic liquids

Lipase was lyophilized with cyclodextrins to prepare lipase formulation suitable for the efficient resolution of allethrolone in ionic liquids. The effects of the type and amount of cyclodextrin used on lipase preparation were evaluated, and the properties of lyophilized lipase such as thermostability and pH sensitivity were investigated and compared with those of native lipase. The results showed that lipase lyophilized with cyclodextrins can achieve a higher conversion rate than the native one, and that lipase lyophilized with inorganic salts cannot improve the conversion rate of the resolution reaction. The catalytic behavior of the lyophilized lipase was strongly dependent on cyclodextrin type and reaction media. The activity of the lyophilized lipase increased as the amount of added cyclodextrins increased. The activity of the lipase lyophilized with cyclodextrins was optimum at pH 7 and 40 degrees C, which was similar to that of the native one, but the half-life of the lyophilized lipase was low compared with that of the native one.     

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。     

固定化脂肪酶催化酯交换合成生物柴油研究

以苯乙烯为单体,二乙烯苯为交联剂,过氧化苯甲酞为引发剂,明胶为分散剂,采用悬浮聚合法制备St-DVB-CBA三元共聚高分子微球,将其作为固定化脂肪酶载体,通过共价结合法进行脂肪酶固定化,探讨固定化脂肪酶催化大豆油酯交换反应活性。实验结果表明:固定化脂肪酶在醇油摩尔比为3∶1,分三次加入;固定化酶加入量15wt.%(油重);反应时间24 h;反应温度40℃;正己烷加入量15wt.%(油重);水分含量4wt.%(油重);转化率最高,可达90.08%;固定化脂肪酶重复使用时显示出较好稳定性和催化活性。     

活性嫩黄K-6G的水解及醇解动力学研究

 针对活性染料在纤维上染色的动力学性能，研究不同条件下一氯均三嗪型活性染料活性嫩黄K-6G(C.I.Reactive Yellow 2)在甲醇碱性水溶液中的醇解和水解反应动力学，并确定了pH值为10时，不同温度下该染料的醇解与水解反应速率常数。研究表明，在80℃，pH值为9～12之间，醇解效率随时间延长逐渐变少，且pH值越高，醇解效率减少的越快， pH值的升高不利于醇解反应进行，即染料的醇解反应优先性下降；在50～70℃，甲醇化反应占相对优势，其速率为水解速率的2～3倍；80～95℃，甲醇化反应速率常数和水解速率常数基本相等。同时实验中还发现染料的醇解和水解反应都是准一级反应。     

Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system

Methyl esters synthesized from plant oil and methanol by the methanolysis reaction are potentially important as a biodiesel fuel. The methanolysis of soybean oil by lipases from various microorganisms was investigated. Several of the lipases were found to catalyze methanolysis in a water-containing system without an organic solvent. The lipases from Candida rugosa, Pseudomonas cepacia, and Pseudomonas fluorescens displayed particularly high catalytic ability. The reaction rates of methanolysis catalyzed by the C. rugosa and P. fluorescens lipases decreased significantly when the water content was low, showing that water prevents the inactivation of these lipases by methanol. On the other hand, the methanolysis reaction rate catalyzed by the P. cepacia lipase remained high even under a low water content. In addition, the P. cepacia lipase gave high methyl ester contents in the reaction mixture up to 2 or 3 molar equivalents of methanol to oil, which is attributed to the P. cepacia lipase having substantial methanol resistance. For the same methanol content, the reaction rates of methanolysis catalyzed by the P. cepacia lipase increased with decreasing water content, and hence lipases strongly resistant to high methanol, such as that from P. cepacia, are desirable for use in methanolysis reaction processes.     

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.     

Lipase-catalysed acidolysis of lard with caprylic acid to produce structured lipid

Enzymatic acidolysis of lard with caprylic acid was investigated. Of the five lipases that were tested in the initial screening, immobilised lipase TL IM from Thermomyces lanuginosus resulted in the highest incorporation of caprylic acid into lard. This enzyme was further studied for the effect of enzyme load, organic solvent, substrate ratio, reaction time and temperature. HPLC was used to analyse the products from the acidolysis reaction. The highest incorporation was attained at 15% enzyme load. Among the solvents tested, n‐hexane was the best reaction medium for the acidolysis of lard with caprylic acid. Time course studied suggests that the incorporation of caprylic acid into lard was increased up to 37.7 mol% after 24 h. Desirable mole ratio of lard to caprylic acid was 1:2, caprylic acid incorporation up to 34.2 mol%. Temperature had no significant effect on enzyme activity in the range of 40–80 °C.     

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.     

Study of lipase‐catalysed synthesis of ascorbyl benzoate in cyclohexanone using response surface methodology

BACKGROUND: Compared with ascorbic acid, ascorbyl esters exhibit many advantages. Their biosynthesis in organic media has been widely studied owing to its many advantages over chemosynthesis. However, since the natural substrates of lipase are fatty acid triacylglycerols, few data are available on the lipase‐catalysed esterification of ascorbic acid with aromatic acids. Furthermore, although many reports can be found on the biosynthesis of ascorbic acid with fatty acids, few deal with the interactions between important reaction factors. The aim of the present study was to optimise the biosynthesis process of ascorbic acid with an aromatic acid, benzoic acid, in cyclohexanone using a statistical approach and to investigate the interactions among variables. RESULTS: Among the five factors studied, only substrate concentration, water activity and temperature had a significant effect on the reaction. Enzyme concentration affected the process slightly, while the effect of reaction time became minor after equilibrium. For the first time, using a partial derivative method, it was quantitatively demonstrated how the optimum of the variable studied depended on the constant values of fixed variables used in a single‐factor experiment. Statistical analysis predicted a maximal conversion rate (47.66%) at enzyme concentration 10 g L^?1, substrate concentration 0.1031 mol L^?1, water activity 0.4896, reaction time 48 h and temperature 66.63 °C. CONCLUSION: This study illustrated possible factor interactions during the lipase‐catalysed synthesis of ascorbyl benzoate in cyclohexanone, and a maximal conversion rate was achieved under optimal reaction conditions. Copyright © 2007 Society of Chemical Industry     

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%。