溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

Immobilization of Pseudomonas cepacia lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

The immobilization of Lipase PS from Pseudomonas cepacia by entrapment within a chemically inert hydrophobic solgel support was studied. The gel-entrapped lipase was prepared by the hydrolysis of tetramethoxysilane (TMOS) with methyltrimethoxysilane (MTMS), isobutyltrimethoxysilane (iso-BTMS), and n-butyltrimethoxysilane. The immobilized lipase was subsequently used in the hydrolysis of soybean oil to determine its activity, recyclability, and thermostability. The biocatalyst so prepared was equal to or better than the free enzyme in its hydrolytic activity. The catalytic activity of the entrapped lipase strongly depended on the type of precursor that was used in its preparation. The lipase entrapped within TMOS/iso-BTMS showed the highest activity. The catalytic activity of the immobilized lipase was more pronounced during the earlier stages of the reaction. Thermostability of the lipase was significantly improved in the immobilized form. The immobilized lipase was stable up to 70°C, whereas for the free enzyme, moderate to severe loss of activity was observed beyond 40°C. The immobilized lipase was consistently more active and stable than the free enzyme. The immobilized lipase also proved to be very stable, as it retained more than 95% of its initial activity after twelve 1-h reactions.     

Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase

Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.     

Utilization of rice hull ash as a support material for immobilization of Candida cylindracea lipase

Rice hull ash was heated in a muffle furnace at 700°C for 2 h and metallic oxides were leached with 10% sulfuric acid. The acid-activated ash was then examined for immobilization of Candida cylindracea lipase. Immobilization was carried out by direct addition of the enzyme solution to the activated ash suspended in hexane. The immobilized lipase retained 30% of its hydrolytic activity, but thermal stability was greatly increased. Half-lives of the immobilized enzyme at 50, 60, and 70°C were 45, 17, and 4 min, respectively. Optimal pH of the immobilized enzyme was 7.2. The apparent K_m and V_max for olive oil were 41 mM and 99.5 μmol/h-mg solid, respectively.     

Immobilization of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

Immobilization of lipase AY from Candida rugosa by entrapment within a chemically inert hydrophobic sol-gel support was studied. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. Certain modifications were incorporated into the conventional immobilization procedure, including the use of glucose as additive and the application of vacuum during the drying and aging stages. The activity and thermostability of immobilized enzyme were subsequently determined in hydrolyzing soybean oil. Hydrolysis results showed more than 95 mol% of the theoretical yield for the formation of FF after 1 h of reaction at 40°C. The level of FFA was 3.3 times greater than that seen when an immobilized enzyme was prepared by the conventional sol-gel process. The immobilized enzyme retained most of its hydrolytic activity compared to the free enzyme and kept more than 95% activity after 120 h of incubation at 40°C, whereas the free enzyme lost 67% of its activity after 24 h of incubation and almost all of its activity after 96 h of incubation at 40°C. The immobilized enzyme also proved to be very stable, as it retained more than 90% of the initial activity after 16 one-hour reactions. Surface characterization studies suggested that the enzyme-containing sol-gel particles have amorphous morphology and are void of micro/meso pores.     

Synthesis of Ascorbyl Palmitate with Immobilized Lipase from Pseudomonas stutzeri

Synthesis of ascorbyl palmitate by enzymatic esterification of palmitic acid and ascorbic acid was conducted in an organic medium with Pseudomonas stutzeri lipase TL immobilized in different supports and its performance was compared with commercial Novozym 435 lipase used as a reference. The enzyme was immobilized in different supports and the best catalyst was selected in terms of immobilization yield and mass specific activity to perform the reactions of synthesis. Synthesis of ascorbyl palmitate was optimized considering temperature, substrate molar ratio and enzyme to limiting substrate mass ratio as variables, and substrate conversion and specific productivity as evaluation parameters. The best reaction conditions for immobilized lipase TL were 55 °C, 1:5 ascorbic to palmitic acid molar ratio, and 1:10 lipase to ascorbic acid mass ratio, obtaining 57 % substrate conversion and a specific productivity of 0.013 [g ascorbic acid/(g enzyme × min)]; the best conditions for Novozym 435 were 70 °C, ascorbic to palmitic acid molar ratio 1:10, and 1:10 lipase to ascorbic acid mass ratio, obtaining 51 % substrate conversion and a specific productivity of 0.016 [g ascorbic acid/(g enzyme × min)].     

Enzymatic esterification of (−)-menthol with fatty acids in solvent by a commercial lipase from Candida rugosa

Esterification of (−)-menthol with fatty acids in isooctane was successfully catalyzed using a commercial lipase, Lipase AY “Amano” 30 from Candida rugosa in original powder form. The esterification reactions were performed to elucidate the effects of temperature, enzyme load, molar ratio of (−)-menthol/fatty acid, and fatty acid type, keeping the (−)-menthol concentration at 200 mM. At the optimal conditions for (−)-menthol esterification, determined at a (−)-menthol/lauric acid molar ratio of 1∶1 and 35°C [1.5 g enzyme/g (−)-menthol, 0.1 g molecular sieves], the molar conversion of (−)-menthol after 48 h reached 93%. After 24h, the lowest and the highest molar conversions of fatty acids at 2∶1 molar ratio were obtained with myristic acid (71%) and margaric acid (98%), respectively. After 48 h, the molar conversions of lauric acid at molar ratios 2∶1, 1∶1, and 1∶2 were 98, 93, and 49%, respectively.     

Production of structured lipids containing essential fatty acids by immobilizedRhizopus delemar lipase

An attempt was made to produce structured lipids containing essential fatty acid by acidolysis with 1,3-positional specificRhizopus delemar lipase. The lipase was immobilized on a ceramic carrier by coprecipitation with acetone and then was activated by shaking for 2 d at 30°C in a mixture of 5 g safflower or linseed oil, 10 g caprylic acid, 0.3 g water and 0.6 g of the immobilized enzyme. The activated enzyme was transferred into the same amount of oil/caprylic acid mixture without water, and the mixture was shaken under the same conditions as for the activation. By this reaction, 45–50 mol% of the fatty acids in oils were exchanged for caprylic acid, and the immobilized enzyme could be reused 45 and 55 times for safflower and linseed oils, respectively, without any significant loss of activity. The triglycerides were extracted withn-hexane after the acidolysis and then were allowed to react again with caprylic acid under the same conditions as mentioned above. When acidolysis was repeated three times with safflower oil as a starting material, the only products obtained were 1,3-capryloyl-2-linoleoylglycerol and 1,3-capryloyl-2-oleoyl-glycerol, with a ratio of 86∶14 (w/w). Equally, the products from linseed oil were 1,3-capryloyl-2-α-linolenoyl-glycerol, 1,3-caprylol-2-linoleoyl-glycerol, and 1,3-capryloyl-2-oleoly-glycerol (60∶22∶18, w/w/w). All fatty acids at the 1,3-positions in the original oils were exchanged for caprylic acid by the repeated acidolyses, and the positional specificity ofRhizopus lipase was also confirmed to be strict.     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.     

Characterization and application of immobilized lipase enzyme on different radiation grafted polymeric films: Assessment of the immobilization process using spectroscopic analysis

Lipase has been immobilized onto different films, polypropylene and poly(tetrafluoroethylene‐perfluroro‐propyl vinyl ether) using glutalaradehyde as a crosslinker. Differential scanning calorimetery, Fourier transform infrared spectroscopic, x‐ray diffraction, and scanning electron microscopy measurements were carried out to confirm the structure of the polymer films as well as the immobilization process of the enzyme onto the polymeric carrier. The activity and stability of the resulting biopolymers produced by lipase have been compared to those for the native lipase. The experimental results showed that the optimum temperature and pH were 40°C and 8.0, respectively. The activity of the immobilized lipases varied with lipase concentration and with the yield of grafting. Subjecting the immobilized enzymes to a dose of γ‐radiation of (0.5–10 Mrad) showed complete loss in the activity of the free enzyme at a dose of 5 Mrad. A leakage of the enzyme from the irradiated membranes was not observed in the repeated batch enzyme reactions. The operational stability of the free and immobilized lipase in n‐hexane showed that the immobilized enzyme was much more stable than the free one. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 155–167, 2003     

Separation of neutral lipid,free fatty acid and phospholipid classes by normal phase HPLC

Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10 min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100∶5∶0.02) proved effective in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic acid (70∶30∶0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride. A mobile phase consisting of hexane/isopropanol/acetic acid (100∶2∶0.02) effectively separated free fatty acid, cholesterol, 1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid classes were separated using methylteriarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5∶8∶2) as the mobile phase. The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were each greater than 96%.     

Attachment of Lipase on Amino Functionalized Titania Submicrospheres via Covalent Binding

A facile and effective method for immobilized lipase was presented. The titania submicrospheres were synthesized via a modified sol-gel method followed by amino functionalization through the chelation between dopamine and titania. Lipase was covalently attached on the functionalized titania surface using glutaraldehyde as the cross linking agent. The loading ratio and relative activity of the immobilized lipase were 230 mg/g titania submicrospheres and 65%, respectively. The kinetic parameters including the Michaelis constant (K_m) and the maximum reaction rate (V_max) changed slightly after immobilization. Compared to free lipase, the immobilized lipase showed favorable pH stability, thermostability, recycling stability and storage stability. The immobilized lipase retained 90% activity after incubation at 50 ℃ for 2 h, while the free lipase retained only 60% activity. The immobilized lipase retained more than 80% activity after 8 batches.     

Quantitative determination of Tri-, Di-,monooleins and free oleic acid by the thin layer chromatography-flame lonization detector system using internal standards and boric acid impregnated chromarod

The separation conditions for hydrolysates of triglycerides by lipase and their quantitative determination are discussed for a thin layer chromatography-flame ionization detector system utilizing internal standards. The complete separation of glyceride hydrolysis mixtures (triolein 1,3-diolein, 1,2-diolein, 1-monoolein and oleic acid) was achieved on a 3% boric acid-impregnated Chromarod S-II by development with benzene/chloroform/acetic acid (70∶30∶2, v/v/v) (mobile phase A) or hexane/ ether/acetic acid (70∶30∶1, v/v/v) (mobile phase B). Mobile phase B had an advantage over mobile phase A in terms of free space to add internal standards for simultaneous quantitation and was employed.p-Hydroxybenzoic acid andp-carboethoxy benzyl alcohol, which appeared between 1,2-diolein and 1-moloolein, were adopted as the internal standards. The calibration curves relating internal standards to each glyceride were all approximated by the equations Y=aX^b giving high correlations. The method was applied to hydrolysis of triolein by pancreatic lipase. Part of this investigation was reported at the annual meeting of JOCS in Tokyo, November 1982.     

Immobilization of Candida rugosa lipase on modified natural wool fibers

A method has been developed to immobilize lipase from Candida rugosa on modified natural wool fibers by means of graft copolymerization of poly ethylacrylate in presence of potassium persulphate and Mohr’s salt redox initiator. The activities of free and immobilized lipase have been studied. FTIR spectroscopy, scanning electron microscopy, and the Bradford method were used to characterize lipase immobilization. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of free enzyme before and after the immobilization of lipase. The results showed that the optimum temperature of immobilized lipase was 40 °C, which was identical to that of the free enzyme, and the immobilized lipase exhibited a higher relative activity than that of free lipase over 40 °C. The optimal pH for immobilized lipase was 8.0, which was higher than that of the free lipase (pH 7.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value (km) of immobilized lipase was higher than that of the free lipase. However, the thermal and operational stabilities of immobilized lipase have been improved greatly.     

Acidolysis of babassu fat catalyzed by immobilized lipase

The lipase-catalyzed interesterification of oils and fats gives products that are unobtainable by chemical interesterification methods. Acidolysis of babassu fat and palmitic acid, catalyzed by immobilized lipase (Lipozyme; Novo Industri, Bagsveard, Denmark), was studied. The reactions were performed at 65°C with 5% w/w enzyme for 4 h. The molar proportions of babassu fat/palmitic acid were 1∶0.1, 1∶0.3 and 1∶0.5. At the end of the reaction period, the catalyst particles were removed by filtration, and the residual oil was extracted with organic solvent (diethyl ether). The recovered particles were then reused. The palmitic acid content of babassu fat before and after acidolysis changed from 10 to 22% at a molar proportion of 1∶0.5. The equilibrium was attained in about 4 h. The original water and enzymatic activities of Lipozyme were maintained after acidolysis. Water sorption isotherms of the immobilized enzyme were determined at 25, 35 and 45°C. From the temperature dependence of the isotherms, isosteric heats of sorption were evaluated by means of the Clausius Clapeyron equation. Monolayer moisture content was calculated by means of the B.E.T. and Guggenhein-Anderson-De Boer analyses. Paper presented at the International Meeting on Fats and Oils Technology, Universidade Estadual de Campinas, Brazil, 1991.     

Enzyme immobilization: Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase

Immobilization of Candida rugosa lipase onto modified and unmodified bentonites is described. The effect of hydrophilic or hydrophobic nature of the support, the reuse efficiency, and kinetic behavior of immobilized lipase were studied. The modified bentonite with monolayer surfactant (BMS), was the best support, for immobilization. The activity of the immobilized enzyme was examined under varying experimental conditions. The effect of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were also evaluated. The activity of lipase on Na-bentonite, on BMS and on bentonite with bilayer surfactant (BBS) at the optimum pH was 7.2%, 56.6% and 3.6%, respectively. The adsorption isotherm was modelled by the Langmuir equation. The amounts of immobilized lipase on Na-bentonite, BMS and BBS at the highest activity were 42.6%, 61.2% and 28.3%, respectively. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited an activity comparable to the free enzyme after storage at 30 °C. The thermal stability of free and immobilized lipase were also studied.     

Immobilized lipase-catalyzed ethanolysis of sunflower oil

The ethanolysis of sunflower oil catalyzed by an immobilized 1,3-specific porcine pancreatic lipase in a medium composed solely of substrates was investigated. The effects of the oil/ethanol molar ratio, temperature, amount of added water, and amount of enzyme used [respectively, 1∶3, 45°C, 0% (vol/vol), and 0.5 g of lipase, i.e., 10% w/w of total substrate]. To investigate the reusability of the lipase, the four-step ethanolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentage of conversion after the first usage decreased markedly.     

磁性纳米粒子的制备及脂肪酶的固定化

建立了以纳米级磁性粒子为载体固定化脂肪酶的方法,优化了脂肪酶的固定化条件,考察了固定化酶的性质. 制备的磁性载体平均粒径20 nm,具有超顺磁性,分散和再分散效果好. 固定化酶的最适吸附时间为60 min,酶用量:载体量为1:1,固定化酶的酶活达到718 U/g. 结果表明,经纳米磁性粒子固定化后,脂肪酶得到活化,固定化酶比活为游离酶的1.8倍. 同时,固定化脂肪酶的pH稳定性显著提高.     

Stability of Immobilized Candida sp. 99–125 Lipase for Biodiesel Production

The stability of the immobilized lipase from Candida sp. 99–125 during biodiesel production was investigated. The lipase was separately incubated in the presence of various reaction components such as soybean oil, oleic acid methyl ester, n‐hexane, water, methanol, and glycerol, or the lipase was stored at 60, 80, 100 and 120 °C. Thereafter the residual lipase activity was determined by methanolysis reaction. The results showed that the lipase was rather stable in the reaction media, except for methanol and glycerol. The stability study performed in a reciprocal shaker indicated that enzyme desorption from the immobilized lipase mainly contributed to the lipase inactivation in the water system. So the methanol and glycerol contents should be controlled more precisely to avoid lipase inactivation, and the immobilization method should be improved with regard to lipase desorption.     

Immobilization of porcine pancreatic lipase on celite for application in the synthesis of butyl butyrate in a nonaqueous system

For immobilization of lipase, the use of a porous support material is recommended so that suitable amounts of lipase can be spread on a surface area without conformational changes. In this work, porcine pancreatic lipase was deposited on Celite, either by direct binding from aqueous solution or by deposition from aqueous solution by the addition of organic solvent. The influence of the immobilization procedure on the activities of the derivatives has been studied regarding their ability to synthesize butyl butyrate. The reaction rates were compared with the rate of esterification with free lipase. Better properties were displayed when the immobilized lipase form was prepared in an apolar solvent such as hexane. Under suitable reaction conditions, esterification yields as high as 90% were attained. Batch operational stability tests indicated that no enzyme deactivation occurs after 15 consecutive batches of 24 h each.