Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

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％。     

The Influence of Fatty Acid and Glycerol on the Kinetics of Fat Hydrolysis by Candida Rugosa Lipase in a Membrane Reactor

The kinetics of lipid-hydrolysis by Candida rugosa lipase was investigated in a membrane reactor and in an emulsion system. Two models were chosen to describe the kinetics of the enzyme:

(1) The hydrolysis of triglycerides to fatty acids was considered to be a chain reaction with the intermediary products di- and mono-glyceride; each step was assumed to be a reversible second-order reaction. The reaction rate constants were determined from batch experiments. The experimental results could be described with this model.

(2) For process optimization and control, a model based on the power law was developed. For this model, the rate of hydrolysis was measured as a function of fatty acid and glycerol concentrations. Relations for the initial rate and equilibrium ester fraction as a function of the glycerol concentration were determined. Further, the reaction rate could be described with the power-law model with a power of 1.75 in the hydrolyzable ester fraction for a wide range of glycerol concentrations. The model with power 1.75 gave much better results when compared to a similar first order model. Although simpler, the first order model can not be used. The power law model was applied in the simulation of a reactor composed of three modules. The fatty acid production rate was calculated for this reactor system as a function of the outgoing glycerol concentration at different conditions.     

Production of Ethyl Butyrate by Candida rugosa Lipase Immobilized in Polyurethane

The enzymatic production of ethyl butyrate was studied: the lipase of Candida rugosa (E.C. 3.1.1.3.) was immobilized in a polyurethane matrix and subsequently introduced in an organic medium containing the substrates in appropriate concentrations. The large majority of experiments was carried out in n-hexane. Two further solvents were tested, namely n-heptane and n-dodecane. The partition coefficients matrix/solvent were estimated for the various solvent systems. The initial esterification rate, the molar yield ester/acid and the degree of conversion were found to be solvent independent when the reaction media were designed so that similar concentrations were created in the microenvironment. Initial rate experiments indicated that in n-hexane the threshold of inhibitory substrate concentrations lies (i) between 0.40 M and 0.50 M for butyric acid, according to the purity of the enzyme preparation and (ii) at 0.30 M for ethanol. Batch operational stability tests indicate that no enzyme deactivation occurs after 20 consecutive batches.     

The Use of Fatty Acid Esters to Enhance Free Acid Sophorolipid Synthesis

Fatty acid esters were prepared by transesterification of soy oil with methanol (methyl-soyate, Me-Soy), ethanol (ethyl-soyate, Et-Soy) and propanol (propyl-soyate, Pro-Soy) and used with glycerol as fermentation substrates to enhance production of free-acid sophorolipids (SLs). Fed-batch fermentations of Candida bombicola resulted in SL yields of 46 ± 4 g/l, 42 ± 7 g/l and 18 ± 6 g/l from Me-Soy, Et-Soy, and Pro-Soy, respectively. Liquid chromatography with atmospheric pressure ionization mass spectrometry (LC/API-MS) showed that Me-Soy resulted in 71% open-chain SLs with 59% of those molecules remaining esterified at the carboxyl end of the fatty acids. Et-Soy and Pro-Soy resulted in 43% and 80% open-chain free-acid SLs, respectively (containing linoleic acid and oleic acid as the principal fatty acid species linked to the sophorose sugar at the omega-1 position), with no evidence of residual esterification. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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．     

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 Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

Lipase-Catalyzed Synthesis of Fatty Acid Esters Useful in the Food Industry

Enzyme reactions are very attractive in food technology because they can be carried out under mild conditions and without toxic solvents and other catalysts. Lipases can esterify various alcohols with fatty acids. There are opportunities to synthesize useful compounds with special functions as food materials by using the catalytic function of lipase. Reverse micellar systems are discussed as reaction systems for lipases in organic solvents, especially in triacylglycerol synthesis using phosphatidylcholine as the surfactant. Syntheses of some amphiphilic substances including O-acyl-L-homoserine are also discussed.     

Syntheses and Biological Activities of 1-O-Glucopyranosyl Fatty Acid Esters

1-O-Palmitoyl-d-glucopyranose was prepared by the selective 1-O-acylation of 4,6-O-benzylideneglucose followed by hydrogenolysis of the protecting group. 1-O-Oleoyl-d-glucopyranose was synthesized from the corresponding benzylidene derivative by selective hydrolysis in acetic acid. This procedure constitutes a useful method for the synthesis of 1-O-acyl-d-glucopyranoses containing unsaturated carboxylic acids. However, 4,6-O-benzylidene-l-O-linolenoyl-d-glucopyranose was converted to 3-O-linolenoyl-d-glucopyranose by the acidic hydrolysis due to acyl migration.

Synthesized glucosyl esters were inactive in the bean second-internode bioassay. However, it was found that 3-O-linolenoyl-d-glucopyranose had a promoting activity on germination of pollen and growth of pollen tube.     

非水相脂肪酶催化有机硅醇的酯化反应

非水相酶催化是酶工程研究热点之一。本文介绍了来自Ｃ．ｃｙｌｉｎｄｒａｃｅａ的脂肪酶催化有机硅醇和脂肪酸的酯化反应。该酶可催化有机硅醇与脂肪酸的酯化反应,并对不同链长的脂肪酸底物、有机溶剂极性及水含量等进行了初步研究。     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

氨基载体共价结合固定化海洋假丝酵母脂肪酶

共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。     

Studies on the Substrate and Stereo/Regioselectivity of Adipose Triglyceride Lipase,Hormone-sensitive Lipase,and Diacylglycerol-O-acyltransferases

Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization.     

Kinetics of Enantioselective Esterification of Naproxen by Lipase in Organic Solvents

The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

酶法水解油脂生产脂肪酸的研究

豆油和猪油用解脂假丝酵母脂肪酶水解,酶解产物的酸值分别达到196和194.酶解条件是:豆油酶量100单位/克油,猪油酶量250单位/克油,豆油:水=1:1,猪油:水=0.6:0.4,豆油温度40℃,猪油温度42℃,摇床转速180r/min,水解时间36h,酶解过程中加20%NaOH溶液1%.这样的条件适合脂肪酸生产.