固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ

对催化合成Ｌ－捩民血酸棕榈酸本反应的脂肪酶（ＮＯＶＯ４３５、ＭＭＬ、ＬＩＰＯＬＡＳＥ、ＰＰＩ）和反应介质进行比较,得出最佳酶为ＮＯＶＯ４３５,最佳介质为椒戊醇,同时对影响合成Ｌ－抗坏血酸棕榈酸酯反应的初速度的因素（转速、温度、水分含量、酶氏物浓度）进行了探讨,确定了最适反应１条件,转速为２００ｔ／ｍｉｎ,温度为５５℃,水分含量为０,酶浓度为１２．５％。     

酶促反应合成棕榈酸Vc酯

讨论了以黑曲霉脂肪酶为催化剂,以抗坏血酸和棕榈酸甲酯为底物的酯交换反应及其影响因素。考察了在摇床速度为200r/min,叔丁醇为溶剂下,底物的摩尔比、温度、脂肪酶浓度、时间、含水量对转化率的影响。结果表明,底物棕榈酸甲酯与Vc的摩尔比为1.3:1.0、反应温度为36℃、反应时间为24h、脂肪酶浓度为15%、含水量为1%时,Vc的转化率为23%。合成的棕榈酸Vc酯,无需和底物分离,可以直接作为油脂食品的添加剂。     

非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ

对催化合成L-抗坏血酸棕榈酸酯反应的脂肪酶(NOVO435、MML、LIPOLASE、PPL)和反应介质进行比较,得出最佳酶种为NOVO435,最佳介质为叔戊醇;同时对影响合成L抗坏血酸棕榈酸酯反应的初速度的因素(转速、温度、水分含量、酶浓度和底物浓度)进行了探讨,确定了最适反应条件：转速为200r/min,温度为55℃,水分含量为0,酶浓度为12.5%。     

改性壳聚糖固定化脂肪酶的研究

以化学改性后的壳聚糖为载体固定假丝酵母99-125脂肪酶,研究了不同的活化剂对壳聚糖表面羟基基团的活化程度,及以活化后壳聚糖为载体采用不同固定化方法对假丝酵母脂肪酶固定效果的影响。结果表明1-乙基-3-(3-甲基氨基)丙基碳二亚胺可有效的活化壳聚糖表面羟基,活化后的壳聚糖表面氨基与戊二醛偶联后形成的壳聚糖为良好的脂肪酶固定化载体,其固定脂肪酶的水解活力可高达86.8U/g。此外,还对影响固定化进程中的各种因素进行了研究,确定最优条件,比较了固定化前后酶的热稳定性、有机溶剂稳定性及最适反应温度。并考察了该固定化脂肪酶催化合成棕榈酸十六酯的操作稳定性,结果表明,连续反应16批之后棕榈酸十六酯的转化率仍能达到85%以上。     

固定化脂肪酶催化聚乙二醇脂肪酸酯的合成

固定化假丝酵母（Ｃａｎｄｉｄａｓｐ．）－１６１９脂肪酶催化脂肪酸和不同聚合度的乙二醇形成酯。其中以聚乙二醇４００（ＰＥＧ４００）的酯化率较高,Ｃ_１０－Ｃ_１８的饱和脂肪酸与ＰＥＧ４００反应的酯化率相仿。反应体系中,月桂酸与ＰＥＧ４００的摩尔比大于２∶１时有利于酯化反应,作用的最适温度为４０℃,最适Ｐｈ为６.０。在比较的３０种添加有机溶剂中,饱和烷烃,芳香烃能促进反应,使酯化率提高２０％左右。反应开始添加５μｌ水有利于酶的作用,外加水量超过适量时,随外加水量的增加酯化率下降。在由２.５ｍｍｏｌ月桂酸,１.２５ｍｍｏｌＰＥＧ４００,１０ｍｇ固定化脂肪酶（１００ｕ）,５ｍｌ己烷,５μｌ水组成的反应体系中,４０℃振荡反应２２ｈ,酯化率达４９.８％。经薄层色谱,气相色谱鉴定,产物为聚乙二醇４００月桂酸酯。     

固定化假丝酵母1619脂肪酶催化油酸油醇酯的合成

比较了14种不同来源的脂肪酶催化油酸油醇酯的合成。其中,假丝酵母(Candidasp．)1619脂肪酶酯化能力最强,以硅藻土为载体,分别按0．1％添加椰子油、吐温80．按l％添加MgSO43种共固定物,醇化反应初速度提高了1．5倍。此固定化酶催化油酸油醇酯合成的最适温度为30℃,0～60℃下反应24h的酯化率均在90％以上,100℃下还有10．25％的酯化率。最适酯化pH6．0。反应中去水,可使终酯化率提高到99％。在添加的23种有机溶剂中,以异辛烷促进酯化的效果最好．正壬烷和正己烷次之。此固定化酶在28℃下批式重复反应的半衰期为990h,柱式固定床反应器中28℃连续运转1000h后酯化率为78％。     

神经网络优化非水相脂肪酶催化合成抗坏血酸油酸酯的条件研究

抗坏血酸油酸酯具有强抗氧化作用．为了获得脂肪酶催化合成抗坏血酸油酸酯的最适条件,主要研究了反应温度、脂肪酶量、油酸量对抗坏血酸油酸酯合成效果的影响．采用中心组合设计和动量梯度下降神经网络对反应条件网络进行训练仿真,并利用训练好的网络对催化酯化工艺条件进行预测．研究结果表明：经过训练的网络可以很好的模拟反应条件,得到了脂肪酶催化反应的最佳工艺参数．当抗坏血酸0．8g时,反应温度56℃,油酸量0．95g,固定化脂肪酶量0．74g,添加分子筛条件下,抗坏血酸油酸酯的转化率为46.5％．该方法为抗坏血酸酯化催化效果的预测提供了一条可行的途径．     

脂肪酶固定化及其稳定性研究

目的:研究脂肪酶的固定化工艺及其稳定性。方法:以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定化黑曲霉属脂肪酶。结果:最优固定化条件是:TMOS 0.5mmol、MTMS 2.5mmol,水与硅烷前驱体摩尔比(R)12,PEG400 120μL,给酶量120mg。酶的固定化效率为93.7%,比活力为游离酶的2.2倍。固定化酶和游离酶在60℃处理2h,其残余酶活分别为91.8%和0;在pH 11的缓冲液中处理2h,其残余酶活分别为95.2%和82%。结论:酶经固定化后其活力、热稳定性和pH稳定性均有提高。     

Immobilization and Characterization of a Thermostable Lipase

Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe₃O₄@SiO₂ supermagnetic nanoparticles. Based on analyses with tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (±2.4)?nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K _m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application.     

Immobilization by Adsorption of Hydrophobic Lipase Derivatives to Porous Polymer Beads for Use in Ester Synthesis

A novel procedure for attaching lipase to certain kinds of hydrophobic surfaces is described. The procedure involves covalent derivatization of the protein molecule by reaction in solution with a hydrophobic imidoester, aldehyde or activated polyethylene glycol. The resulting protein derivative is then allowed to adsorb onto an insoluble hydrophobic surface. Quantitative adsorption is observed and the enzyme is bound very strongly on the support The number and nature of the hydrophobic substituents introduced in the chemical derivatization step can be easily controlled. The adsorption step occurs spontaneously upon exposure of the modified protein to a variety of hydrophobic materials. The hydrophobic lipase derivative obtained by reaction with PEG activated with p-nirrophenyl chloroformate, for example, adsorbs readily onto polyacrylate and polystyrene beads, with most of its esterification activity in organic solvent intact. Its thermostability is also greatly enhanced. Derivatization of lipase with hydrophobic groups greatly enhances its esterification activity in organic solvent, and its immobilization in this manner enables the preparation of a highly reactive biocatalyst for biotechnological application.     

Immobilization of Candida antarctica Lipase B on fumed silica

Enzymes are usually immobilized on solid supports or solubilized when they are to be used in organic solvents with poor enzyme solubility. We have reported previously on a novel immobilization method for subtilisin Carlsberg on fumed silica with results that reached some of the best previously reported catalytic activities in hexane for this enzyme. Here we extend our method to Candida antarctica Lipase B (CALB) as an attractive target due to many potential applications of this enzyme in solvents. Our CALB/fumed silica preparations approached the catalytic activity of commercial Novozym 435 for a model esterification in hexane at 90 wt.% fumed silica (relative to the mass of the preparation). An intriguing observation was that the catalytic activity at first increases as more fumed silica was made available to the enzyme but then decreased precipitously when fumed silica exceeded 90 wt.%. This was not the case for s. Carlsberg where the catalytic activity leveled off at high relative amounts of fumed silica. We determined adsorption kinetics, performed variations of the pre-immobilization aqueous pH, determined the stability, and applied fluorescence microscopy to the preparations. A comparison with recent concepts by Gross et al. may point towards a rationale for an optimum intermediate surface coverage for some enzymes on solid supports.     

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

Nylon membrane was used to immobilize Mucor miehei lipase. Variables that affect this immobilization procedure were studied by experimental design. A 2³ full factorial design was employed for this purpose. The protein retention and hydrolytic activity of the immobilized lipase were used as response variables. The rapid loss of enzyme activity was the main problem during repetitive use. Two strategies were used to improve the low operational stability: nylon treated with HCl and nylon coated with polyvinyl alcohol (PVA). Lipase-nylon-PVA was the best enzyme derivative, allowing performance of five consecutive assays, with a retained activity of 0.5 U mg of protein⁻¹ g of support⁻¹.     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

Enzymic Interesterification of fats: Immobilization and Immunogold Localization of Lipase on Ion-Exchange Resins

A 1,3 specific lipase from Mucor miehei has been immobilized to two phenolformaldehyde resins with active tertiary amine groups and different porous structures (Duolite ES562 and ES568N). Duolite ES568N has a more uniform pore structure, allowing more rapid uptake of lipase. Immobilized lipase particles were treated by washing, dehydration with glycolmethacrylate and embedding in polymer. Following immunogold staining of thin sections, examined by electron or light microscopy showed that the location of the lipase was dependent on the pore structure of the support material and the immobilization time.     

Effect of the Immobilization Method of Lipase from Thermomyces lanuginosus on Sucrose Acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 &#117 mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 &#117 h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 &#117 h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.