月桂酸生物印迹对脂肪酶酯化活力的影响

生物印迹是改良酶学特性,扩大脂肪酶工业应用领域的新兴技术。本研究结合溶胶-凝胶脂肪酶固定化工艺,以甲基三甲氧基硅烷（MTMS）和四甲氧基硅烷（TMOS）为前驱体,月桂酸为印迹分子,考察了月桂酸生物印迹对脂肪酶PS酯化活力的影响。脂肪酶酯化活力测定及扫描电镜观察表明生物印迹能显著提高脂肪酶的活性及稳定性。印迹体系经正交试验优化获得的最优条件为：水和硅烷摩尔比（R）为12,聚乙二醇（PEG）加入量为120μl,月桂酸加入量为0.15mmol。在最优反应条件下,印迹酶相对于游离酶比活力提高了44.3倍,相对于未印迹固定化酶提高了2.4倍;印迹酶具有较好的热稳定性,在80℃下处理0.5h后,残余酶活分别为58%,而游离酶未检测到活性。     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Comparison of differently modified Pseudomonascepacia lipases in enantioselective preparation of a chiral alcohol for agrochemical use

Three methods for enzyme modification/immobilization were compared to enhance the catalytic performance of a commercially available lipase, Lipase PS from Pseudomonascepacia, in highly enantioselective transesterification of an agrochemically useful sec-alcohol, (R,?S)-HMPC [=(R,?S)-4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopenten-1-one], with vinyl acetate as both acyl donor and reaction medium. The stearic acid-coated lipase showed the highest catalytic activity, with a specific activity improved by 54 times over the native lipase. The microcrystal salt-supported lipase and celite-adsorbed lipase also displayed much better performance as compared with the native lipase. All the three modified lipase preparations showed a similar thermal stability to that of the native enzyme. The enantioselectivity (E-value) was also quite satisfactory in all the cases (E>100 at 30°C), though a trend of slight decline was also observed with the temperature increase in the range of 25–60°C. The optimum aqueous pH, from which the modified lipases were prepared, was 6.0–7.0. A low water activity (a_w) of ca. 0.1 was favorable for all the three modified lipases. The stearic acid-coated lipase displayed prominent advantages in catalyzing the transesterification reaction at a very high (R,?S)-HMPC concentration up to 1.0?M.     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Production and partial characterisation of extracellular lipase from Aspergillus niger

Summary The production and certain kinetic characteristics of extracellular lipase from Aspergillus niger were investigated. It was possible to substantially enhance the activity of excreted lipase by optimising the interaction between carbon and nitrogen sources applying a two-parameter complete experimental design and response surface analysis. The enzyme was partially purified and a number of kinetic characteristics such as optimum pH and temperature, thermal and pH stability and K_m were determined and discussed. The elevated levels of lipase activity (40.5 U/ml) found in this work competed favourably with most of those reported for lipase hyperproducing fungi.     

Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

In-situ crosslinking of Aspergillus flavus lipase: Improvement of activity, stability and properties

Summary The mycelium-bound lipase of Aspergillus flavus was stabilised by cross-linkage using glutaraldehyde or methylglyoxal. Lipase activity was enhanced by up to 48% by treatment with methylglyoxal but not by glutaraldehyde. Cross-linking with methylglyoxal increased the thermal stability of the lipase by 58% at 50 °C     

Serratia marcescens H30脂肪酶的重组可溶表达、固定化及其酶学性质

为了提高Serratia marcescens H30脂肪酶的可溶表达水平,分别将目的基因与p GEX-4T-1、p ET28a和p ET32a构建重组表达载体,转入大肠杆菌BL21(DE3),通过优化诱导过程,发现可溶性酶的最高活性可达25 000 U/L。再经Ni2+亲和柱纯化、LH-EP固定化后,固定化酶的比酶活为214 U/g(以1 g湿质量计),酶活回收率为51%。固定化后重组脂肪酶的最适温度由30℃提高到35℃,最适p H从7.0偏移至8.0左右,并且稳定性也有所增加。该固定化重组脂肪酶同样能够拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,光学选择性没有改变。反应14 h,转化率为48.5%,底物的e.e.值为99.2%,表明该固定化脂肪酶能有效拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,为工业生物催化制备地尔硫卓提供了可能。     

Effect of moisture on enzymatic reaction in supercritical carbon dioxide

The continuous acidolysis of triolein and stearic acid was carried out by an immobilized lipase to elucidate the characteristics of supercritical carbon dioxide (SC-CO₂) as a reaction medium. At first, an effect of temperature and pressure on the water adsorption to the immobilized lipase in the SC-CO₂ was examined. Then, the continuous interesterification of triolein and stearic acid by the moist immobilized lipase was examined. The amount of water adsorption to the immobilized lipase in the supercritical carbon dioxide measured under the condition of a different temperature and pressure has been expressed by a correlation equation of Freundlich type by using relative water standardized with the solubility of water in each condition. Optimum operating conditions of the interesterification by immobilized lipase in the SC-CO₂ was 323 K, 16.9 MPa and adsorbed-water concentration of 2 wt%. The production rate obtained by enzymatic acidolysis in the SC-CO₂ was found to be about 0.03 mmol/h2g-immobilized enzyme, leaving 74% residual triglyceride at the optimum operating conditions.     

Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. (c) 1993 John Wiley & Sons, Inc.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Kinetics of tributyrin hydrolysis by lipase

The kinetics for the tributyrin hydrolysis using lipase (Pseudomonas fluorscenes CCRC-17015) were investigated in the liquid–liquid and liquid–solid–liquid reaction systems in a batch reactor. The lipase was covalently immobilized onto the surface of porous polymethylacrylamide (PMAA) crosslinking with N,N-methylene biacrylamide with a spacer of ethylenediamine actived by glutaraldehyde. The conditions such as tributyrin concentration, temperature, agitation, and pH value, were evaluated to achieve the optimum reaction conditions for both free lipase and immobilized lipase. The kinetic parameters in the reaction system were also obtained for two reaction systems. The turnover numbers calculated for free lipase and immobilized lipase were 29 and 5.7 s⁻¹, respectively. The parameters of k and k_m obtained using Lineweaver-Burk plot method were 26.2 mol/(mg min) and 1.35 mol/dm³ for free lipase, 5.2 mol/(mg min) and 0.2 mol/dm³ for immobilized lipase, respectively. The experimental results revealed good thermal stability, with greater stability at higher pH value for immobilized lipase in the liquid–solid–liquid reaction.     

疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础.方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的...     

A thermostable variant of P. aeruginosa cold-adapted LipC obtained by rational design and saturation mutagenesis

Cold-adapted Pseudomonas aeruginosa LipC is a secreted lipase showing differential properties compared to its well-known counterpart LipA. LipC is fundamentally a cold-acting lipase, capable of tolerating high concentrations of ions and heavy metals, and showing a shift in substrate specificity when incubated at higher temperatures. These properties make LipC an interesting enzyme, well suited for biotechnological or environmental applications, where activity at low temperatures would be required. However, a relatively low thermal resistance constitutes the main drawback for using this enzyme in long-term operational processes. To overcome the lability of LipC, we developed a rational design system to modify specific sites on the enzyme structure to obtain an improved variant of the lipase bearing higher thermal stability, but without loss of its cold-adapted properties. Eight mutant libraries plus two point mutations were constructed affecting those amino acids showing the highest flexibility on the 3D model structure. After screening more than 3000 mutant clones, a LipC variant bearing two amino acid changes and the required thermostability and cold-adapted properties was obtained. The new variant D2_H8, with a 7-fold increased thermal stability in comparison to wild type LipC, will guarantee the use and maintenance of such a lipase in a number of processes being performed at low (4–20 °C) temperatures.     

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.     

Characterization of three distinct forms of lipolytic enzyme in a commercialCandida lipase preparation

Summary Three distinct forms of lipolytic enzyme were identified in a commercialCandida lipase preparation. Two of these lipases (lipases A & C) were isolated and characterized. Lipase A had a higher optimal reaction pH and a better thermal stability than those of lipase C. Lipase A and C displayed different acyl chain length specificity on the lipolysis of p-nitrophenol esters.     

介孔分子筛MCM-41固定中性脂肪酶条件的研究

以介孔分子筛MCM-41材料为载体,采用物理吸附法对中性脂肪酶进行了固定化处理,并研究不同条件对固定化脂肪酶催化活性的影响,从而得到该种材料对脂肪酶的最佳固定化条件。给酶量为45960 U/g,固定化温度为45℃,pH值为7.5,时间为3 h,此时固定化酶的活力约为4666 U/g。固定化酶和游离酶的最适反应温度都为40℃,最适pH值为7.5,比游离酶低。固定化酶温度稳定性和pH稳定性较游离酶有所提高。