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)。     

Enzymatic transesterification of purine nucleoside having a low solubility in organic medium

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.     

有机介质中脂肪酶催化转酯化反应拆分苯乙氰醇的研究

研究了有机相中脂肪酶催化苯乙氰醇的转酯化反应,拆分苯乙氰醇。考察了酶、溶剂、溶剂水含量、外加苯甲醛和苯甲酸以及底物浓度等因素对反应的影响,结果表明ZJU008号脂肪酶催化活性最高,经实验确定的最佳反应条件为：乙酸乙烯酯为反应物兼溶剂,利用分子筛去除溶剂中微量水分,40 ℃,200 r/min,酶量为10 mg/mL时的最佳底物浓度为200 mmol/L,在上述条件下反应20 h底物转化率为50%,e.e.值大于99%,能将苯乙氰醇有效拆分。外加苯甲醛和苯甲酸不利于反应的进行。     

Kinetics of lipase-catalysed methyl gallate production in the presence of deep eutectic solvent

Production of methyl gallate (MG), which is an important phenolic acid ester for pharmaceutical industry, was carried out by Novozym 435-catalysed transesterification of propyl gallate (PG) with methanol in a deep eutectic solvent. Reaction parameters governing substrate molar ratio, enzyme concentration, temperature and agitation rate were investigated batch-wise in choline chloride:glycerol-water binary mixture. The results were evaluated in terms of conversion of PG, yield of MG and hydrolysis of PG to gallic acid. 10% (w/w) of water was found to be favourable in the reaction medium for low hydrolysis percent. The highest conversion (17.4%) and yield (60.4%) but the lowest hydrolysis (2%) after 120?h of transesterification were found at PG/methanol molar ratio of 1:6, enzyme concentration of 40?g/L, 50?°C and 200?rpm. A kinetic model based on the Ping-Pong Bi–Bi mechanism for transesterification of PG was proposed with the assumption that there were no internal and external mass transfer resistances.     

Direct preparation of biodiesel from rapeseed oil leached by two-phase solvent extraction

A new method which coupled the two-phase solvent extraction (TSE) with the synthesis of biodiesel was studied. Investigations were carried out on transesterification of methanol with oil-hexane solution coming from TSE process in the presence of sodium hydroxide as the catalyst. Biodiesel (fatty acid methyl esters) were the products of transesterification. The influential factors of transesterification, such as reaction time, catalyst concentration, mole ratio of methanol to oil and reaction temperature were optimized. The results showed that the optimal reaction parameters were sodium hydroxide concentration 1.1% by weight of rapeseed oil, mole ratio of methanol to oil 9:1, reaction time 120 min, and reaction temperature 55-60 degrees C. Under these conditions, the TG conversion would rise up to 98.2%. Based on the new method, biodiesel production process could be simplified and the biodiesel cost could be reduced.     

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from Thermomyces lanuginosus

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.     

Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative

An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.     

Thiol-functionalization of acrylic ester monomers catalyzed by immobilized Humicola insolens cutinase

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0-1.72 M), temperature (22-50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14-0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.     

Markedly improving Novozym 435-mediated regioselective acylation of 1-beta-D-arabinofuranosylcytosine by using co-solvent mixtures as the reaction media

A comparative study was made of Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine with vinyl propionate for the preparation of the 5'-O-monoester in eleven co-solvent mixtures and three pure polar solvents. Novozym 435 displayed low or no acylation activity toward 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly. The polarity of co-solvent mixtures had significant effect on the reaction. Among the solvent mixtures investigated, the higher the polarity of the solvent mixture, the lower the initial reaction rate and the substrate conversion. It was also found that the acylation was dependent on the hydrophobic solvent content, the water activity and the reaction temperature. The most suitable co-solvent, initial water activity, and reaction temperature were hexane-pyridine (28:72, v/v), 0.07, and 50 degrees C, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 91.1 mM h(-1), >97% and >98%, respectively, after a reaction time of 6 h. Among the reaction mediums examined, the lowest apparent activation energy was achieved with hexane-pyridine (28:72, v/v), in which Novozym 435 also exhibited good thermal stability.     

Enzymatic Production of Alkyl Esters Through Lipase-Catalyzed Transesterification Reactions in Organic Solvents: Solvent Effects and Prediction Capabilities of Equilibrium Conversions

The solvent effect on the equilibrium position of the transesterification reaction of hexanol with ethyl acetate catalyzed by a lipase has been investigated in a variety of non-polar and polar solvents - and binary mixtures. The results obtained indicate that the solvent effect on the equilibrium conversion is very small as compared to that for the direct esterification reactions.

Equilibrium conversions were then predicted using the equilibrium constant for the reaction obtained from Gibbs free energy of formation information for reactants and products in combination with the UNIFAC activity coefficient model. A solvent independent equilibrium conversion was obtained, which was in good agreement with the observed average value for all solvents. This indicates that UNIFAC provides satisfactory estimates of the activity coefficients but its group contribution structure does not allow the prediction of the small differences in conversion among the solvents examined.

Finally plots of these conversions versus the solvent octanol/water partition coefficient or the solubility of water in the solvent, that provide the correct trend in direct esterification reactions, did not achieve the same for transesterification.     

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris , was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g^?1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi‐Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L^?1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.     

Solvent Effects on Equilibrium Position and Initial Rate of Lipase-catalyzed Esterification Reactions in Organic Solvents: Experimental Results and Prediction Capabilities

The solvent effect on the equilibrium position and the initial rate of esterification of 1-hexanol with acetic acid catalyzed by a lipase has been experimentally investigated. A variety of non-polar and polar solvents have been considered and the results obtained indicate that the solvent effect on the equilibrium conversion is very important compared to that for transesterification reactions. A theoretically sound methodology using the group-contribution UNIFAC model for the prediction of solvent effects on the equilibrium position of enzymatic reactions is presented and it is applied to the reaction of 1-hexanol with acetic acid as well as to a similar reaction from the literature. The results obtained are better than those from empirical methods proposed in the literature such as correlations with the octanol-water partition coefficient of the solvent, as well as the solubility of water in the solvent. Moreover, the proposed methodology can be used for the determination of the equilibrium constant of the reaction. For the prediction of the solvent effect on the initial rate of enzymatic reactions it is found that it is more accurately determined using the product of the activities of the reactants, which can be predicted by the UNIFAC model, than the octanol-water partition coefficient of the solvent or the solubility of water in the solvent.     

Solvent Effects on Equilibrium Position and Initial Rate of Lipase-catalyzed Esterification Reactions in Organic Solvents: Experimental Results and Prediction Capabilities

The solvent effect on the equilibrium position and the initial rate of esterification of 1-hexanol with acetic acid catalyzed by a lipase has been experimentally investigated. A variety of non-polar and polar solvents have been considered and the results obtained indicate that the solvent effect on the equilibrium conversion is very important compared to that for transesterification reactions. A theoretically sound methodology using the group-contribution UNIFAC model for the prediction of solvent effects on the equilibrium position of enzymatic reactions is presented and it is applied to the reaction of 1-hexanol with acetic acid as well as to a similar reaction from the literature. The results obtained are better than those from empirical methods proposed in the literature such as correlations with the octanol-water partition coefficient of the solvent, as well as the solubility of water in the solvent. Moreover, the proposed methodology can be used for the determination of the equilibrium constant of the reaction. For the prediction of the solvent effect on the initial rate of enzymatic reactions it is found that it is more accurately determined using the product of the activities of the reactants, which can be predicted by the UNIFAC model, than the octanol-water partition coefficient of the solvent or the solubility of water in the solvent.     

In situ alkaline transesterification of cottonseed oil for production of biodiesel and nontoxic cottonseed meal

The production of fatty acid methyl ester (FAME) by direct in situ alkaline-catalyzed transesterification of the triglycerides (TG) in cottonseeds was examined. The experimental results showed that the amount of cottonseed oil dissolved in methanol was approximately 99% of the total oil and the conversion of this oil could achieve 98% under the following conditions: less than 2% moisture content in cottonseed flours, 0.3-0.335mm particle size, 0.1mol/L NaOH concentration in methanol, 135:1 methanol/oil mole ratio, 40 degrees C reaction temperature and 3h reaction time. Further, the effects of co-solvent petroleum ether and methanol recycling on the cottonseed oil extraction and conversion were also investigated. The use of alkaline methanol as extraction and reaction solvent, which would be useful for extraction oil and gossypol, would reduce the gossypol content in the cottonseed meal. The free and total gossypol contents in the cottonseed meal obtained from in situ alkaline transesterification were far below the FAO standard. And the nontoxic cottonseed meal could be used as animal protein feed resources.     

Transesterification of phosphatidylcholine with eicosapentaenoic acid ethyl ester using phospholipase A2 in organic solvent

Phospholipase A₂-catalysed transesterification of phosphatidylcholine (PC, 99%) with eicosapentaenoic acid ethyl ester (EPAEE, 95%) was carried out in organic solvent. The maximum yield was 14.3% (w/w). The optimum reaction condition was 50°C, 48 h, initial water activity 0.25 and molar ratio of PC to EPAEE 1:10 in 5 ml toluene.     

Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media

Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L-lactate by transesterification between retinol and L-methyl lactate with yield reaching 90% and the synthesis of ascorbyl L-lactate by transesterification between ascorbic acid and L-methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L-methyl lactate in organic media has been carried out and agrees with ping-pong-ordered Bi-Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping-pong-ordered Bi Bi or hybrid ping-pong-random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.     

Enzymatic production of glycerol carbonate from by-product after biodiesel manufacturing process

Glycerol carbonate is one of the higher value-added products derived from glycerol. In this study, glycerol carbonate (GC) was synthesized by transesterification of glycerol and dimethyl carbonate (DMC) using Novozym 435 (Candida antarctica Lipase B) at various conditions. For the enzymatic production of GC, the optimum conditions were the amount of enzyme (75 g/L), DMC/glycerol molar ratio (2.00), reaction temperature (60°C) and organic solvent (acetonitrile). Experimental investigation of the effect of water content revealed that the conversion of GC was maximized with no added water. The addition of surfactant such as Tween 80 increased the GC conversion, which finally reached 96.25% under the optimum condition and with surfactant addition.     

Optimization Studies and Chemical Kinetics of Silica Sulfuric Acid-Catalyzed Biodiesel Synthesis from Waste Cooking Oil

In the present study conversion of waste cooking oil to biodiesel has been carried out via simultaneous esterification and transesterification reaction over silica sulfuric acid as a solid acid catalyst. The process variables that influence the fatty acid methyl ester (FAME) conversion, such as reaction temperature, reaction time, catalyst concentration and methanol to oil molar ratio were investigated and optimized using Taguchi method. Highest FAME production obtained under the optimized condition was 98.66 %. Analysis of variance revealed that temperature was the most significant factor effecting the FAME production among four factors studied. From the kinetic study, the reaction was found to follow pseudo first-order kinetics and rate constant of the reaction under optimum condition was 0.00852 min^?1.     

双极膜电渗析分离发酵液中L-乳酸

采用三室型双极膜电渗析装置将发酵液中的L-乳酸钠转化为L-乳酸。探讨操作电压、流速、进料L-乳酸钠质量浓度等工艺参数对转化过程的影响,考察电渗析过程参数对转化率、物料损失率、电流效率和能耗等技术指标的影响。在最优操作条件下（流速40L／h,电压15V）对2L的100．25g／L乳酸钠发酵液进行分批重复电渗析处理。结果表明：整个过程的转化率为81．22％,损失率为1．5％,能耗为0．81kW·h／kg,电流效率为91．8％,得到的L-乳酸质量浓度可达144．31g／L．电渗析残液补糖后可回到发酵罐中用于发酵生产L-乳酸.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.