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.     

Enzymic Hydrolysis of Soybean Phosphatidyl Inositol by Trimeresurus flavoviridis Venom and a Pancreatic Lipase Preparation

We have made an investigation on the specific liberation of fatty acids from the molecules of soybean phosphatidyl inositol by Trimeresurus flavoviridis venom and a pancreatic lipase preparation in order to utilize the reaction for the study of the fatty acid distribution in phosphatidyl inositols. Both the venom and a pancreatic lipase preparation liberated about one half of the total fatty acids in phosphetidyl inositol, leaving probably a lysophosphatidyl inositol which contained mostly saturated fatty acids, whereas the most part of the component unsaturated acids was found in the liberated acids. The formation of the other hydrolysis products by these enzymes is also discussed.     

胰腺脂肪酶相关研究进展

胰腺脂肪酶包括甘油三酯脂酶与其相关蛋白1和2、胆盐刺激性脂酶及磷脂酶A2。这些脂肪酶协调作用,是膳食脂肪消化的主要酶。胰腺脂肪酶的发育不仅与年龄相关,还受到膳食结构、内分泌激素等的调节,其表达的改变与肥胖等代谢性疾病有着密切的关系。胰腺脂肪酶的调节可能是治疗肥胖等代谢性疾病的靶点。     

Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl^−/− macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl^−/− macrophages. Hence, phagocytosis was also decreased in vivo when Atgl^−/− mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.     

Effect of Dimethylsulfoxide on Hydrolysis of Lipase

To establish an industrially feasible reaction process, the effect of dimethylsulfoxide (DMSO) added to an aqueous solution on the hydrolysis of lipase was investigated using fluorescent substrates. Several lipases from microorganisms were improved in their hydrolysis activities against 4-methylumbelliferyl oleate by DMSO. Variation was found in the effect of DMSO depending on the species of lipase. After the high stability of the lipase from Pseudomonas fluorescens in DMSO solution was confirmed, hydrolysis by this lipase of four acyl-4-methylumbelliferones was studied kinetically at different DMSO concentrations. DMSO added to an aqueous solution increased the V_max of this lipase for a substrate with strong hydrophobicity, and decreased that value for a substrate with an opposite property. On the other hand, DMSO had a very small effect on K_m for each substrate. A fluorometric study suggested that DMSO induced a change of the chemical environment that surrounded tryptophan residues of the lipase. Such conformational change would be one of the causes of the DMSO-induced alteration of its reactive property. These results suggest that the addition of DMSO may be a novel method of ‘solvent engineering’ of this enzyme.     

Activities in Crude Porcine Pancreatic Lipase: Enantioselectivity in Hydrolysis of the Diacetate of 2-Phenylpropane-1,3-Diol

The hydrolysis of a prochiral diacetate by porcine pancreatic lipase is catalysed by the purified enzyme, not by an enzyme present in the crude enzyme but absent from the purified enzyme, as previously reported.     

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.     

脂肪酶水解植物油的研究

本文报导了薄层层析法及高压液相色谱法定性定量地测定植物油甘油酯的组成,对不同来源的脂肪酶对植物油的水解进行了研究。实验结果表明,不同来源的脂肪酶对植物油(橄榄油)的水解性不同,同一脂肪酶水解不同种类的植物油,脂肪酶的水解率也不同,脂肪酶水解植物油有最适pH和最适温度。     

米胚芽中胰脂肪酶抑制剂的提取工艺

以米胚芽为原料,提取具有抑制胰脂肪酶活力的胚芽蛋白。经工艺优化得到提取条件为:原料颗粒60目,料液比1∶6,pH 10.18,温度55℃,提取时间4h,水提后离心速度4 456r.min-1,等电点沉降pH 5.1,等电点沉降后离心速度3 692r.min-1。在此条件下提取得到的胰脂肪酶抑制剂的蛋白质纯度为60.7%。15.18g/L的胚芽蛋白对30.1U/mg的猪胰脂肪酶的平均抑制率达71.17%。     

Extraction of Pancreatic Lipase Inhibitor from Rice Germ

以米胚芽为原料,提取具有抑制胰脂肪酶活力的胚芽蛋白.经工艺优化得到提取条件为:原料颗粒60目,料液比1:6,pH 10.18,温度55℃,提取时间4h,水提后离心速度4 456 r·min-1,等电点沉降pH5.1,等电点沉降后离心速度3 692r·min-1.在此条件下提取得到的胰脂肪酶抑制剂的蛋白质纯度为60.7％.15.18 g/L的胚芽蛋白对30.1 U/mg的猪胰脂肪酶的平均抑制率达71.17％.     

Kinetics of Lipase Catalyzed Resolution of Racemic Alcohols by Reversible Interesterification

In this paper a predictive model for the lipase-catalyzed resolution of racemic alcohols by reversible interesterification is presented. The approach takes into account the acyl transference from the acyl donor to the enzyme and from the acyl-enzyme complex to the acyl acceptor. Resolution of (R,S)-2-phenyl-l-propanol by interesterification using n-butyl-butyrate as acyl donor has been experimentally studied. The reaction mechanism was determined as ping-pong with inhibition by n-butanol. The model is based on reaction constants which can be calculated from a few long term experiments. The reaction constants calculated in this way were able to reproduce the results made in other experimental conditions. The extension of this technique to other reaction systems is straight forward.     

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.     

Continuous Hydrolysis of Olive Oil by Lipase in Microporous Hydrophobic Hollow Fiber Bioreactor

Olive oil was hydrolyzed continuously at 40ºC by Candida cylindracea lipase in a small hollow fiber bioreactor (total area of hollow fibers was about 0.11 m²) in which the hollow fibers were made of microporous polypropylene. The lipase could be adsorbed easily onto oil-impregnated hollow fibers from its aqueous solution. The continuous feedings of olive oil inside the hollow fibers and of the buffer solution containing 18% glycerol as a stabilizer outside the hollow fibers were started after the enzyme-glycerol solution was removed from the bioreactor and the buffer-glycerol solution was added. An unvaried half life of 14 days of the adsorbed enzyme was observed when increasing amounts of the enzyme (1.0~5.0mg/ml) were put in, but its half-life was lowered to 6 days when the amount of the added enzyme was less (0.05mg/ml). Free enzymes in the enzyme solutions with and without 18% glycerol retained their initial activities equally for at least 3 months at temperatures below 4ºC. This suggests the feasibility of reuse of the enzyme-glycerol solution that was used in the preceding adsorption procedure after the solution was stored and supplemented with fresh enzyme. It was demonstrated by three successive cleanings and continuous hydrolyses that the used hollow fibers were regenerable. The productivity number was 0.81 mg-(unit)^-1 -h^-1, which was 26 times as great as that of the gel-entrapped lipase.     

Pervaporation Aided Esterification of Carboxylic Acids with Ethanol Catalyzed by Porcine Pancreatic Lipase

The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation.     

人参皂苷Rb3对胰脂肪酶的抑制作用

采用硅胶柱层析、JTY树脂分离制备人参皂苷Rb3,薄层板(TLC)跟踪,高效液相色谱法(HPLC)检测。目的是研究西洋参叶中不同浓度的人参皂苷Rb3对胰脂肪酶的抑制作用影响,从而表明人参皂苷Rb3在抗肥胖中的作用。用制得的人参皂苷Rb3,在卵磷脂所乳化的甘油三酯的检测系统中进行体外胰脂肪酶抑制实验。结果表明:制得的人参皂苷Rb3经过三种不同展开剂,薄层板展开,均为一个点,进一步经HPLC检测其纯度为93.2%。胰脂肪酶抑制试验结果表明人参皂苷Rb3在浓度为0～1 mg/mL时,其抑制率达到85.11±0.409%,而且远远高于西洋参叶提取物。本文通过胰脂肪酶抑制试验表明Rb3具有抗肥胖作用,其抑制机制有待进一步研究。