游离脂肪酶催化蓖麻油制备蓖麻油酸

游离脂肪酶与固定化脂肪酶相比具有成本低、反应速率快等优势,是油脂化工中新的研究方向。前期研究表明,游离脂肪酶NS81006能高效催化多种油脂水解,进一步研究其对含独特羟基的绿色石油材料蓖麻油的水解过程,对于促进游离脂肪酶在新能源领域的应用具有重要意义。本文对影响游离脂肪酶NS81006催化蓖麻油水解过程的主要因素,温度、酶用量、水用量和搅拌速率进行了研究和优化,在优化后的条件下48 h水解率可达94.8%,且发现通过离心分离可有效实现NS81006的重复使用,连续回用5个批次,游离脂肪酶仍能有效催化水解反应。而对比高温高压法水解蓖麻油,发现游离脂肪酶NS81006具有明显优势。     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

Maximization of bioconversion of castor oil into ricinoleic acid by response surface methodology

In this study, response surface methodology was applied to optimize process variables like temperature, pH, enzyme concentration (mg/g oil), and buffer concentration (g/g oil) for hydrolysis of castor oil using Candida rugosa lipase. A 2⁴ full factorial central composite design was used to develop the quadratic model that was subsequently optimized and the optimal conditions were as follows: temperature 40 °C, pH 7.72, enzyme concentration 5.28 mg/g oil, buffer concentration 1 g/g oil and there was 65.5% conversion in 6 h. These predicted optimal conditions agreed well with the experimental results. This is the first report on the application of response surface methodology in castor oil hydrolysis using C. rugosa lipase with higher percentage conversion in 6 h.     

Lipase-induced hydrolysis of castor oil: effect of various metals

The ability of an extracellular lipase from Pseudomonas aeruginosa KKA-5 to commence hydrolysis of castor oil in the presence of various metal chlorides, was investigated. Apart from CaCl₂ (commonly used for castor oil hydrolysis), AlCl₃ (group IIIB), CrCl₃ (group VIA) and MgCl₂ (group IIA) displayed enhanced hydrolysis capability. Specifically, our statistics show that with respect to time, when Cr³⁺ was used, hydrolysis of castor oil was four times faster than that of calcium, and 1.6 times faster with regards to Al³⁺. The chlorides of group VIII and alkali metals had no effect on hydrolysis. Group IV metal chlorides did not enhance lipase activity and inhibited castor oil hydrolysis. The effect of metal ions from other groups on lipase activity is also reported. Received 14 August 1998/ Accepted in revised form 22 October 1998     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Characterization of Betaine Aldehyde Dehydrogenase from Cylindrocarpon didymum M-1

To obtain a lipase which effectively hydrolyzes castor oil, bacteria were isolated from 500 soil samples. The best strain was examined; its microbiological characteristics suggested that it belongs to the genus Pseudomonas. A lipase from this strain was purified by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and DEAE-Toyopearl 650 M. The enzyme was purified about 400-fold with a yield of 13%. The purified enzyme was electrophoretically homogeneous and its molecular weight was 30,000. The optimum pH and temperature for the hydrolysis of olive oil emulsion were 7.0 and 60°C. The enzyme was stable up to 35°C at pH 7.0 for 30min and also stable from pH 9.0 to 10.0 at 4°C for 22 hr. The activity was inhibited by Fe³⁺ , Hg²⁺ , pCMB, and anionic surfactants, and enhanced by nonionic surfactants and bile salts. The enzyme efficiently hydrolyzed castor oil.     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.     

Hydrolysis of soybean oil by lipase with a bioreactor having two different membranes

A membrane bioreactor with 25 sets of two different membranes, one hydrophilic and the other hydrophobic microporous, was used for the hydrolysis of soybean oil by lipase from Candida cylindracea. In the bioreactor, oil, water and enzyme liquid were separately fed into each of 3 clearances between the two membranes. Oil diffused through the hydrophobic membrane and water diffused through the hydrophilic membrane came into contact with the lipase, and hydrolysis subsequently took place. Fatty acid and glycerine produced by the enzymatic reaction diffused through each membrane into the oil and water sides respectively, so that these products were separated from each other. The enzyme was also separated from the products. In this membrane bioreactor, both reaction and separation occurred. Semi-continuous reaction of soybean oil with one cycle of 48 h was carried out for 18 d, at 45°C. Satisfactory performance of the bioreactor, such as a high hydrolysis percentage (92-88%) and enzyme stability over a long-term period was shown. Enzyme stability was found to depend on produced glycerine, as observed by Yamane et al., and the rate of the hydrolysis reaction was controlled by that of the diffusion of fatty acid through the hydrophobic membrane.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

Characteristics of lipase-catalyzed hydrolysis of olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

Mechanism study on NS81006-mediated methanolysis of triglyceride in oil/water biphasic system for biodiesel production

Compared to immobilized lipase, soluble lipase has the merits of lower cost and faster reaction rate, thus much attention has been paid to soluble lipase-mediated methanolysis for biodiesel (fatty acid methyl ester, FAME) production in recent years. Our previous study showed that soluble lipase NS81006 could effectively catalyze the methanolysis of soybean oil (triglyceride, TG) for FAME preparation in oil/water biphasic system. Study on the related mechanism of soluble lipase NS81006-mediated methanolysis of TG was carried out in this paper. Based on the analysis of substances change in the reaction process, mechanism model was hypothesized and the model parameters were simulated by Matlab. The simulated model was validated further. The results showed that in the reaction process of soluble lipase NS81006-mediated methanolysis of TG in oil/water biphasic system, TG proceeded three-step hydrolysis to generate FFA (free fatty acid), and then FFA transformed into FAME by esterification with methanol. During the whole process, FFA is mainly generated through the hydrolysis of TG and intermediate DG (diglyceride), while the hydrolysis of FAME could be ignored.     

Interesterification of fats and oils by immobilized fungus at constant water concentration

The kinetics of enzymatic interesterification of oils and fats, using acetone-dried cells of Rhizopus chinensis immobilized on biomass support particles as a lipase catalyst, were investigated in batch operations at several constant water concentrations.Even under microaqueous (i.e., low-water-content) conditions, not only interesterification but also hydrolysis occured, and the water content in the reaction system decreased. The reaction rates of interesterification and hydrolysis at constant water concentrations were determined.For the reactions between olive oil and methyl stearate at several water concentrations, the parameters involved in the reaction model were determined by a trial-and-error method so as to make the calculated results correlate with the experimental data. The relationship between the parameters obtained and water concentration were examined.The rate constants involved in the reaction model of both interesterification and hydrolysis increased or decreased monotonically with the increasing water content, while the apparent activity of the lipase catalyst for interesterification had a maximum value at a water concentration of about 50 ppm. This suggests that when the water content is excessive the hydrolysis activity of lipase is accelerated more than its interesterification activity, and that when the water content is too little lipase activity can not be activated for either hydrolysis or interesterification.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Exploring a new,soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock

Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides (TAGs, DAGs, MAGs) variation during 24 h reaction. An excellent 96% FAMEs release was achieved when low (3–5%) water concentrations were used in the conversion of crude soybean oil. Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs, DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.     

Use of a low-cost methodology for biodetoxification of castor bean waste and lipase production

The castor bean (Ricinus communis) represents a potential candidate for biodiesel production. The Petrobras Research Center is developing a biodiesel production process from castor bean seeds, in which an unwanted byproduct named castor bean waste is produced. This extremely alkaline waste is toxic and allergenic and, as such, poses a significant environmental problem. Solid-state fermentation (SSF) of castor bean waste was carried out to achieve ricin detoxification, reduce allergenic potential and stimulate lipase production. The fungus, Penicillium simplicissimum, an excellent lipase producer, was able to grow and produce lipase enzyme. After an optimization process, the maximum lipase activity achieved was 44.8 U/g. Moreover, the fungus P. simplicissimum was able to reduce the ricin content to non-detectable levels in addition to diminishing castor bean waste allergenic potential by approximately 16%. In this way, SSF of castor bean waste by P. simplicissimum may increase the utility of the waste by promoting enzyme production and eliminating the principal toxic element, ricin.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

Optimization of process variables in castor oil hydrolysis by Candida rugosa lipase with buffer as dispersion medium

In this study, castor oil is hydrolyzed in presence of Candida rugosa lipase, while in the buffer (aqueous) phase as a dispersion medium. The following conditions were used to optimize the process: speed of agitation, initial pH of buffer phase, temperature, and ratio of buffer phase volume to oil weight. The optimal conditions are 1,100 rpm, pH 6.5, temperature 35°C, and 3:1 buffer phase volume to oil weight ratio. Under these described conditions, the reusability of lipase was tested and it was found that nearly 80% of original hydrolysis percentage was achieved after the first recycle.