毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

脂肪酶YCJ01拆分对位取代α-苯乙醇

利用脂肪酶YCJ01催化拆分对位取代α-苯乙醇衍生物。以异丙醚为反应介质,采用乙酸乙烯酯作为酰基供体,对180 mmol/L的1-(4-甲基苯基)乙醇进行选择性酯化,脂肪酶粗酶粉添加量为5 g/L,50℃反应21 h后,底物转化率可达49.96%,对映体过量值e.e.s、e.e.p值分别为97.1%和97.2%,对映体选择性E200;同样,对1-(4-甲氧基苯基)乙醇进行选择性酯化,酰基供体为丁酸乙烯酯,底物浓度150 mmol/L,脂肪酶粗酶粉添加量为2.5g/L,30℃反应12 h后,底物转化率为49.8%,e.e.s、e.e.p值分别为97.7%和98.4%,对映体选择性E200,显示了很好的手性拆分效果。     

硬脂酸和月桂酸在Novozym 435催化下对芦丁酯化及分子筛对酯化率的影响

为了增加芦丁的脂溶性从而使其具有更优秀的抗氧化活性,以硬脂酸和月桂酸为酰基供体,在脂肪酶Novozym 435催化下对芦丁选择性酯化.经色谱柱提纯,得到两种带不同长度烃基的芦丁脂肪酸酯.用红外光谱和核磁共振波谱对芦丁硬脂酸进行了结构鉴定,表明该类酯化物的酯化反应位为鼠李糖的C4′″位羟基.以高效液相色谱监测酯化反应进程,分子筛添加时间对酯化率的研究结果显示,分子筛对酯化率和反应速率有提高的作用.分子筛添加时间对酯化率有影响.对于硬脂酸为酰基供体的情况,反应24h后添加分子筛的酯化反应可以得到最大的酯化转化率46%.以月桂酸为酰基供体的酯化反应,反应11 h后添加分子筛可以得到最大的酯化转化率64.5%.     

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．     

酶法合成头孢环己二烯

以环己二烯甘氨酸甲酯盐酸盐为酰基供体,7-氨基脱乙酰氧基头孢烷酸为酰基受体,γ－氧化铝为载体的固定化巨大芽孢杆菌胞外青霉素G酰化酶为酰化剂,合成了头孢环己二烯。5％酰基供体,2％酰基受体,每毫升反应物加44IU固定化酶,pH7．5,25℃振荡反应5h,头孢环己二烯产率为81％。苯乙酸、苯氧乙酸和头孢霉素G对酶法合成有不同程度的抑制作用。     

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％。     

菜油基长链辣椒素酯小型化合物库的制备与初步生物活性

以脂肪酶在有机介质中催化食用菜油与香草醇直接转酯反应合成了一个含菜油酰基的长链辣椒素酯小型化合物库。反应条件：5mmol香草醇,2．5mmol食用菜油溶于100mL丙酮中,加入0．5g固定化脂肪酶（Nov0435）,置于30℃的摇床上以200r／min反应24h。分离固定化酶后,反应物以硅胶柱色谱分离,得到长链辣椒素酯小型库,收率34．1％。这个化合物库主要由亚麻酸香草醇酯（1）、亚油酸香草醇酯（2）、软脂酸香草醇酯（3）、油酸香草醇酯（4）、硬脂酸香草醇酯（5）、花生-烯酸香草醇酯（6）、芥酸香草醇酯（7）组成;它在50彬mL时对PPARv模型具有弱的激活作用,激活倍数为1．5。用制备性高效液相色谱对这个化合物库进行分离,得到化合物1—4、7,以1HNMR确证了结构,其中化合物7未见文献报道。     

非水体系中脂肪酶催化合成乳酸乙基糖苷酯的工艺研究

在非水体系中 ,通过固定化脂肪酶催化合成一种新型α 羟基酸衍生物 乳酸糖苷酯。考察了常压下有机溶剂、酰基供体、不同种固定化酶、乙基糖苷的浓度、酶量和反应温度对反应的影响。研究表明在无溶剂体系中以乳酸丁酯作为酰基供体可有效地合成乳酸糖苷酯 ,固定化酶Novozym435和来源于Candida sp .菌株的细胞固定化酶 ,化学修饰的干酶粉均是合适的催化剂。最佳反应条件为 :酶浓度 75g L ,乙基葡萄糖苷的浓度为 0.4mol L ,温度为 70℃ ,转速 200r min ,反应 50h ,转化率可达 71%。在真空度为 0.09MPa的压力下 ,反应温度 65℃ ,酶浓度 75g L ,乙基葡萄糖苷 0.35mol L时 ,反应初速率可达到 607(mmol·L^-1·h^-1 ) ,40h后转化率可达到 90%。反应产物经过萃取法和硅胶柱层析方法分离 ,纯度达到 95 % (W/W)。     

非水介质中酶催化葡甘聚糖的转酯化反应

葡甘聚糖(KGM)是一种具有优异的生物降解性、生物相容性、独特的生理和药理功能的天然聚多糖。通过环境友好的、高选择性的酶催化转酯化反应引入疏水基团可以制备葡甘聚糖的酯化衍生物,从而拓宽其应用领域。本研究探讨了在非水介质中以脂肪酶Novozym 435、Lipozym RMIM、Lipozym TLIM和Lipase TypeⅦ催化KGM和乙酸乙烯酯的转酯化反应,并且考察了相关因素对转酯化反应的影响。实验结果表明:在非水介质N,N-二甲基乙酰胺、甲苯和异辛烷中,脂肪酶Novozym 435可以较好的催化乙酸乙烯酯和KGM发生转酯化反应。以Novozym 435为催化剂、以异辛烷为反应介质,当底物浓度[S]=30(mg/mL)、酶用量[E]/[S]=0.30(wt/wt)、酰基供体乙酸乙烯酯用量[Acyl]/[OH]=3.0(mol/mol)、50℃、反应72 h的条件下,酯化KGM的取代度(DS)可达到0.49。     

有机介质中脂肪酶催化外消旋苯甘氨酸甲酯的对映体选择性氨解反应

由水解酶催化的酯的对映体选择性水解和醇解反应 ,已在外消旋物质的拆分中得到广泛应用[1 ] 。近年来的一些研究表明 ,某些水解酶还可催化一些非天然酰基受体的转化 ,如过氧化氢、烷基胺、联胺和氨等。这些非天然酰基受体的转化反应在多肽的合成及手性化合物的拆分中显示出巨大的应用前景。其中 ,以氨为酰基受体的酶促氨解反应 ,是继酶促对映体选择性水解、酯化及转酯反应之后的另一制备光学纯化合物的新反应[2 ] 。目前国际上对这一新反应的研究尚属起步 ,国内未见有对该反应研究的报道。(Ｄ ,Ｌ) 苯甘氨酸是半合成 β 内酰胺类抗生素的重…     

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.     

Lipase-catalyzed esterification of conjugated linoleic acid with sorbitol: a kinetic study

The kinetics of esterification of conjugated linoleic acid (CLA) with sorbitol in acetone was investigated. An immobilized lipase from Candida antarctica (Chirazyme L-2) was used as the biocatalyst. A 2(2) x 3 factorial design was employed to find an experimental region in which one obtains a high rate of formation of the diester product. Best results were obtained at 10 degrees C using a CLA to sorbitol molar ratio of 5 and a biocatalyst loading of 150 mg/mL of acetone. Under these conditions, in 72 h one obtains a nearly quantitative yield (ca. 98%) of the diester of sorbitol with CLA. To minimize formation of products with degrees of esterification greater than two, the reaction should be carried out at 10 degrees C. A kinetic model developed using the King-Altman method was employed to fit the data. Use of the steady-state approximation for the monoester and an assumption that the concentration of sorbitol was constant and equal to its solubility limit permit one to minimize the number of parameters necessary to model the reaction network. Nonlinear regression analysis based on either two or three parameters provides very good fits of the multiresponse data in the presence or absence of triesters, respectively.     

Continuous enzymatic esterification of glycerol with (poly)unsaturated fatty acids in a packed-bed reactor

Enzymatic synthesis of mono-, di-, and triacyglycerols from (poly)unsaturated fatty acids (linoleic, oleic, and conjugated linoleic acids) has been studied as a solvent-free reaction in a packed-bed reactor containing an immobilized lipase from Mucor miehei. The extents of the esterification reactions of interest are primarily determined by the molar ratio of glycerol to fatty acid because the presence of excess glycerol as a immiscible phase is responsible for reducing the activity of the water produced by the esterification reactions. For molar ratios of fatty acid to glycerol of less than 1.5, the percentage of the fatty acid esterified decreases quasi-linearly with an increase in this molar ratio. By appropriate manipulation of the fluid-residence time, one can control the relative proportions of the various acylglycerols in the effluent stream. At the outlet of the reactor, one observes excellent spontaneous separation of the glycerol and acylglycerol/fatty acid phases. At 50 degrees C and a fluid residence time of 1 hour, as much as 90% of the fatty acid can be esterified when the molar ratio of fatty acid to glycerol is 0.33 or less.     

Synthesis of triglycerides of phenylalkanoic acids by lipase-catalyzed esterification in a solvent-free system

Immobilized Candida antarctica lipase B catalyzed the synthesis of triglycerides from glycerol and phenylalkanoic acids in a solvent-free system. 4-Phenylbutyric acid was the best acyl donor and displayed the highest synthetic rate of triphenylbutyrin (glyceryl triphenylbutyrate) at 65 degrees C among various phenylalkanoic acids with straight alkyl chains. The external mass transfer between the immobilized lipase and the bulk reaction mixture was limited. Different methods of removing water during the lipase-catalyzed esterification including spontaneous evaporation, the use of saturated salts solutions, and the use of molecular sieves were studied. The highest yield of triphenylbutyrin at 65 degrees C was 98%, by the elimination of water using molecular sieves in a solvent-free system. The glycerol was almost completely esterified to triphenylbutyrin in excess phenylbutyric acid with various substrate molar ratios.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Erratum to: Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40?°C, geraniol to oleic acid molar ratio of 5:1, 150?rpm and 10?wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5?wt%) and temperature of 50?°C afforded nearly complete reaction conversion after 1?h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Synthesis of N-Octyl Oleate with Lipase from Mucor miehei Immobilized onto Polyethylene Based Graft Copolymers

Lpase from Mucor miehei was immobilized onto partially hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE/HEMA) via spacer arms of 1,6-diaminohexane and glutaraldehyde-. The PE/HEMA-lipase system was used for the enzymatic esterification of n-octanol with oleic acid in the absence of organic solvents. The influence of lipase' concentration, in the attachment solution, on the ester production profile and initial reaction rate was studied. It was found that very small amounts of lipase gave preparations which reached good degrees of conversion. The effect of the initial oleic acid concentration on that pseudo-first order reaction, as well as the presence of water in the reactional medium and the influence of temperature were evaluated. It was found that initial oleic acid concentrations lesser than 1.2 M did not inhibit the immobilized lipase activity; the presence of small amount of water (10–30μ) solubilized in the reaction mixture (6.5 cm³) increased the lipase activity and a maximum of activity of the immobilized lipase preparation was found at 55d`C. The operational stability of the preparation was determined at 37d`C in a BSTR type reactor and a half-life time of three days for the immobilized lipase was obtained.     

Lipase mediated simultaneous esterification and epoxidation of oleic acid for the production of alkylepoxystearates

Epoxy alkylstearates were synthesized by lipase catalysed esterification and perhydrolysis followed by epoxidation of oleic acid in a one-pot process. Immobilized Candida antarctica lipase (Novozym^®435) was used as the catalyst. The esterification reaction occurred relatively quickly and was followed by epoxidation of the alkyl ester and the remaining fatty acid. Higher degree of esterification was achieved with n-octanol, n-hexanol and n-butanol as compared to that with ethanol and iso-propanol. The rate and yield of epoxidation was enhanced with iso-propanol but was lowered with the other alcohols. The lipase suffered significant loss in activity during the reaction primarily due to hydrogen peroxide. The presence of alcohols, in particular ethanol, further contributed to the enzyme inactivation. The epoxidation reaction could be improved by step-wise addition of the lipase.     

Esterification of fatty acids using Candida antarctica lipase A in water-abundant systems

The feasibility of using native lipase A from Candida antarctica (CAL-A) to esterify fatty acids with water-insoluble alcohols in the presence of excess water was investigated in stirred-tank reactors. For high reaction rates, a ratio of water:substrates of 0.6-1.4:1 (v/v) was required. CAL-A showed higher substrate selectivity for the esterification of saturated palmitic acid with branched-chain 2-ethyl-1-hexanol than for unsaturated oleic acid with linear alcohol (1-decanol). After 18 h at 70 °C in a 1.5 l bulk stirred-tank reactor, an 2-ethyl-1-hexyl palmitic acid ester was obtained near 100 % yield [molar ratio palmitic acid:2-ethyl-1-hexanol ~1:1.25, with 1.11 % (w/w) Novocor ADL (based on palmitic acid weight)].     

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc-co-DMA-cl-MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification ofmyristic acid and isopropanol in n-heptane at 65 degrees C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n-heptane resulted in formation of isopropyl myristate (66.0 +/- 0.3 mM) in 15 h. The reaction temperature below or higher than 65 degrees C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 A x 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 +/- 0.2 mM isopropyl myristate after the 3rd cycle of esterification.