磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶的非水相激活

研究了kosmotropic型磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶(Pseudomonas cepacia lipase, PCL)非水相催化性能的影响.以往磷酸缓冲盐被用来调控体系的pH值,其掺杂量对酶的催化活性无明显影响,而适量硫酸钠的掺杂则可有效提高酶在非水相的催化活性.本文研究发现,通过精确调控冻干过程,磷酸缓冲盐掺杂能够将PCL在有机相中的转酯化活性提高近10倍,达到其水相本征活性的50%,这一激活效果甚至高于硫酸钠掺杂.利用热重方法分析了盐掺杂PCL的含水量和蛋白结构,并将失重结果同其在有机相中的催化活性相关联,发现PCL在磷酸缓冲盐和硫酸钠掺杂下的催化构型与蛋白含水量及其周围盐环境具有不同的依赖关系.利用2-(4’-氨基-2’-羟基苯基)苯并恶唑作为荧光探针,研究了磷酸缓冲盐和硫酸钠掺杂的PCL悬浮于有机相时对荧光探针发射光谱的影响,发现盐掺杂酶制剂的存在能够大大增加荧光探针稳定于极性溶剂的构型含量,这可能与蛋白周围掺杂盐键和的水分子有关.如果用探针分子稳定于极性溶剂和非极性溶剂的构型比值间接表示悬浮酶制剂的极性结构,在正己烷体系中硫酸钠掺杂的PCL具有比磷酸缓冲盐掺杂的PCL大得多的极性,且酶制剂的极性大小与其非水相转酯化活性之间具有相似的变化趋势.上述研究结果表明,掺杂盐对粗PCL酶制剂的激活可能部分归因于掺杂盐键和的水分子在蛋白周围构筑的极性环境.     

Hydrophobic surface induced activation of Pseudomonas cepacia lipase immobilized into mesoporous silica

Lipase from Pseudomonas cepacia (PCL) was successfully immobilized into siliceous mesocellular foams (MCFs) with various hydrophobic/hydrophilic surfaces. The catalytic performances of immobilized PCL were investigated using the transesterification reaction and hydrolytic reaction as model reactions. The specific activity of immobilized PCL greatly increased with enhanced surface hydrophobicity of MCFs, mainly because of lipase activation via hydrophobic interaction between alkyl groups in MCFs and the surface loop (so-called "lid") of PCL. Conformational changes of immobilized PCL were further investigated using time-resolved fluorescence spectroscopy with Trp as an intrinsic probe. When the immobilized PCL was suspended in phosphate buffer, short-lived τ(1) shortened and the fractional contribution of τ(1) significantly increased with the increasing level of surface hydrophobicity of MCFs. These results revealed that Trp(s) of the immobilized PCL were surrounded by a hydrophilic microenvironment because of the fact that the opened "lid" permitted the diffusion of water to the active site cleft. However, for the immobilized PCL suspended in n-hexane, long-lived τ(3) increased with the increase of surface hydrophobicity of MCFs. The reduced interaction between Trp(s) and the surrounding protein matrix was due to intercalation of n-hexane into the active site cleft when the lipase was in open conformation. The above results demonstrated that PCL immobilized into MCF with hydrophobic surfaces were in an activated open conformation.     

非水相中微生物脂肪酶催化转酯化拆分(R,S)-α-苯乙醇

研究了非水有机溶剂体系中脂肪酶不对称转酯化拆分(R,S)-α-苯乙醇反应,比较了15种不同微生物来源的脂肪酶,从中优选出催化活性及对映选择性较高的脂肪酶Lipase PS,系统考察了影响该酶催化不对称转酯化反应的关键因素,获得了优化的催化拆分工艺条件.结果表明,脂肪酶Lipase PS在非水反应体系中,以正己烷为反应介...     

Improved catalytic performance of lipase accommodated in the mesoporous silicas with polymer-modified microenvironment

The highly ordered mesoporous silicas with elaborately controlled microenvironment were synthesized via covalent incorporation of long-chain polymers (M(w) = 2000 g mol(-1)) bearing specific hydrophilic/hydrophobic balance. The microenvironment (hydrophilicity/hydrophobicity) of the mesoporous silicas was quantitatively determined by gas adsorption experiments and investigated by lysozyme (LYZ) adsorption. The relative activity of lipase from Pseudomonas cepacia (PCL) encapsulated in the mesoporous silica with moderate hydrophobic microenvironment (hereafter denoted as MHM) reaches up to 281% compared with the free PCL, notably higher than that of PCL accommodated in the mesoporous silicas with hydrophilic or strong hydrophobic microenvironment (20.7-26.2% relative to the free PCL). Moreover, PCL entrapped in the nanochannels with MHM affords the highest initial rate in the kinetic resolution of (R,S)-1-phenylethanol relative to other immobilized PCL. The above results suggest that the MHM could render the active center of PCL entirely exposed to the substrates without interrupting its native conformation in the "interfacial activation". In addition, the nanochannels with MHM could markedly improve the thermal stability of PCL (preserving nearly 60% of the initial activity after the incubation at 70 °C for 2 h) and facilitate the recycling of the immobilized PCL in both aqueous and organic media. Our work demonstrates that the subtle modulation of the microenvironment of mesoporous silicas for enzyme immobilization designates a very promising strategy to fabricate the highly active and stable heterogeneous biocatalysts for industrial application.     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

A comparison of lipase and trypsin encapsulated in mesoporous materials with varying pore sizes and pH conditions

Immobilized enzymes have an advantage over enzymes free in solution in that they are easily recovered after completed reaction. In addition, immobilization often gives enhanced stability. Entrapment of an enzyme in the pores of a mesoporous material is an attractive procedure since the enzyme is immobilized without any covalent bonding to a support which may be detrimental to the catalytic performance. The objective of this work is to compare the encapsulation and catalytic performance of lipase from Mucor miehei and trypsin from bovine pancreas, two hydrolases with rather dissimilar properties and structures. We also demonstrate the importance of the pore dimensions and the pH for proper function of the encapsulated enzyme. Mesoporous silica particles (SBA-15) with three different pore sizes (50 Å, 60 Å and 89 Å) were synthesized and hexagonal structures with narrow pore size distributions were confirmed with TEM, SAXS and N₂-adsorption. Lipase and trypsin were encapsulated separately in the silica particles and the results indicate distinct differences between the two enzymes, both in loading capacity and catalytic activity. For trypsin the encapsulation rate and the loading capacity were large with a maximum reached at pH 7.6. The largest product yield was obtained with the particles with 60 Å pores, however, the yield was significantly lower than with free trypsin. For lipase optimal encapsulation rate and loading capacity were reached with the particles with 89 Å pores at pH 6.0 but were low compared to trypsin. However, the catalytic activity of the encapsulated lipase was more than twice as large as for free lipase, which can be explained by an interfacial activation of lipase at the silica surface.     

华根霉全细胞脂肪酶催化合成生物柴油

比较了5种不同商品化脂肪酶和自制的华根霉CCTCCM201021全细胞脂肪酶(RCL)催化油脂合成生物柴油的转化效果,结果表明,RCL能有效应用于无溶剂体系催化合成生物柴油.在无溶剂体系中对该酶催化生物柴油的转酯化反应工艺进行优化,考察了甲醇用量、体系含水量、酶的添加量和反应温度对生物柴油收率的影响,使生物柴油最终收率大于86.0%.在有机溶剂体系中选择不同有机溶剂作为助溶剂进行转酯化反应,发现logP值在4.0～4.5的有机溶剂具有较好的转化效果.其中以正庚烷为助溶剂的转酯化反应具有最高的生物柴油收率86.7%.在无溶剂体系中RCL催化转化油酸和模拟高酸价油脂合成脂肪酸甲酯的研究表明,该酶具有很好的催化合成生物柴油的潜力.     

Synthetic reactions catalyzed by immobilized lipase from oilseed rape (Brassica napus L.)

Lipase from rape (Brassica napus L., immobilized onto celite, catalyzes esterification and transesterification reactions in hexane. The activity of the lipase is stimulated up to 35 fold by the addition of water (1.3% w/v). The activity of the lipase in hydrolysis is about 8 times higher than in the esterification reactions in hexane. Interesteri-fication reactions between mono- and diacylglycerols and transesterification reactions of mono- and diacylglycerols with alcohols were also catalyzed at relatively high rates but transesterification/esterification of these acylglycerols with fatty acids was comparatively slow. In transesterification reactions, triacylglycerols reacted rather slowly.     

Synthesis and characterization of poly(β‐hydroxybutyrate) and poly(ϵ‐caprolactone) copolyester by transesterification

To synthesize the copolyester of poly(β‐hydroxybutyrate) (PHB) and poly(?‐caprolactone) (PCL), the transesterification of PHB and PCL was carried out in the liquid phase with stannous octoate as the catalyzer. The effects of reaction conditions on the transesterification, including catalyzer concentration, reaction temperature, and reaction time, were investigated. The results showed that both rising reaction temperature and increasing reaction time were advantageous to the transesterification. The sequence distribution, thermal behavior, and thermal stability of the copolyesters were investigated by ¹³C NMR, Fourier transform infrared spectroscopy, differential scanning calorimetry, wide‐angle X‐ray diffraction, optical microscopy, and thermogravimetric analysis. The transesterification of PHB and PCL was confirmed to produce the block copolymers. With an increasing PCL content in the copolyesters, the thermal behavior of the copolyesters changed evidently. However, the introduction of PCL segments into PHB chains did not affect its crystalline structure. Moreover, thermal stability of the copolyesters was little improved in air as compared with that of pure PHB. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1893–1903, 2002     

Surfactant tail length-dependent lipase activity profile in cationic water-in-oil microemulsions

The catalytic activity of Chromobacterium viscosum lipase (CV-lipase) was estimated across varying surfactant tail lengths (C-10-C-18) in water-in-oil (w/o) microemulsions of cationic surfactants containing four different hydroxyethyl-substituted head groups. An attempt to find a correlation, if any, between the activity of interfacially solubilized lipase and the varying surfactant tails was made for the first time in micellar enzymology. The second-order rate constant, k2, in lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate at pH 6.0 and 25 degrees C shows an improvement in enzyme activity (approximately 30-140%) across different head groups of amphiphiles with increasing tail lengths in varying solution compositions. Improvement of enzyme activity is prominent in ascending from C-10 to C-14/C-16, depending on the nature of polar head group. The hydrolytic activity of lipase in different surfactant (50 mM)/water/isooctane/n-hexanol with varying z= [alcohol]/[surfactant] (6.4 or 4.8) was amplified by 25-250% with increment in surfactant tail length in comparison with widely used cationic w/o microemulsions having solution compositions (z=16). As a notable outcome of this research, we found w/o microemulsions of 25 mM tetradecyltrimethylammonium bromide/water/isooctane/n-hexanol (z=8) producing the highest ever activity of lipase in any w/o microemulsions.     

Partial transesterification of sunflower oil with ethanol by a silica fiber reinforced aerogel encapsulated lipase

A commercial lyophilized lipase of Burkholderia cepacia, from Amano, was encapsulated in silica aerogels reinforced with silica quartz fiber felt. This biocatalyst was applied in the direct transesterification of sunflower seed oil with ethanol, without any other solvent. When the molar ratio of ethanol to oil was two or three, the oil transformation kinetics was found to be very slow after the formation of 1 mole of fatty ethyl ester per mole of initial triglyceride. For a molar proportion of ethanol to oil ≈1, the recycling activity also decreased gradually in successive tests to reach an activity ≈7% of the initial activity, during the 5th test. Textural and structural analysis of the aerogels before and after catalytic tests showed that this deterioration was associated with a modification of the aerogel, by preferential adsorption of glycerol or possibly other transesterification products such as diglycerides. Besides, it is proposed that one of the cause for the aerogel loss of activity at an initial molar ratio of ethanol:oil of 3:1 was due to a progressive inhibition of the enzyme by excess adsorbed ethanol. The aerogel samples were also compared to a commercial product of lipase immobilized on polymer beads, from Fluka. The silica aerogels somewhat improved, to a limited extent, the activity during recycling.     

聚ε-己内酯的微米硅球固定化猪胰脂肪酶促合成

本文采用微米硅球固定化猪胰脂肪酶为催化剂合成聚ε-己内酯, 以期获得具有较高分子量、 良好生物相容性和使用安全性的生物可降解医用高分子材料.     

Enhancement of the Activity and Enantioselectivity of Lipase by Sol–Gel Encapsulation Immobilization onto β-cyclodextrin-Based Polymer

Candida rugosa lipase was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane and octyltriethoxysilane in the presence of β-cyclodextrin-based polymer. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yield of the encapsulated lipase was 65 U/g, which is 7.5 times higher than that of the covalently immobilized lipase. The β-cyclodextrin-based encapsulated lipases had higher conversion and enantioselectivity compared with covalently immobilized lipase. The study confirms an excellent enantioselectivity (E >300) for the encapsulated lipase with an enantiomeric excess value of 98% for S-naproxen.     

Molecular modelling studies of substrate binding to the lipase from Rhizomucor miehei

Lipase enzymes have found increasingly widespread use, especially in biotransformation reactions in organic synthesis. Due to their efficiency and high enantioselectivity, they can be employed in a variety of reactions to carry out asymmetric hydrolyses, esterifications and transesterifications. However, the reasons for their stereospecificity have not been fully correlated with the enzyme structure. Employing molecular modelling techniques and existing experimental data, a transesterification reaction using Rhizomucor miehei lipase was studied. The results indicate that the major controlling factor for this reaction is hydrophobic in nature, providing support for previous literature hypotheses. In addition, computational experiments suggest that the origin of enantioselectivity is the formation of essential hydrogen bonds in and around the catalytic triad of active site residues. Only one enantiomer of the substrate is able to form these hydrogen bonds during the formation of the first tetrahedral transition state.     

类水滑石催化酯交换反应活性、寿命及失活原因研究

研究了5种不同阳离子组合的类水滑石对碳酸二甲酯与苯酚酯交换反应的催化活性和ZnAl, MgAl水滑石的使用寿命. 结果显示, 类水滑石的催化性能与组成的阳离子性质有关: 三价阳离子的性质影响类水滑石的催化活性高低, 二价阳离子的性质则影响其使用寿命长短. 通过对失活原因的研究发现, 特征的层状结构形式是类水滑石具有活性的关键: 层状结构塌陷, 活性随之消失; 层状结构稳定, 使用寿命长. 当苯酚与碳酸二甲酯的摩尔比为2∶1, 催化剂的质量分数为原料总质量的1%, 反应时间8 h时, MgAl水滑石具有最优的催化性能: 碳酸二甲酯转化率51.8%, 酯交换产物选择性94.2%, 5次循环使用后催化活性保持稳定. 其催化性能明显优于文献报道的其它相关固体催化剂.     

Carbon-carbon bonds by hydrolytic enzymes

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases.     

Immobilization of Candida cylindracea Lipase by Covalent Attachment on Glu-Modified Bentonite

Alkaline Ca-bentonite, obtained upon acid activation and base load of natural bentonite, has a good anion exchange capability. Glu-modified alkaline Ca-bentonites were further prepared by covalent binding with glutamic acid for the immobilization of lipase OF from Candida cylindracea. The obtained immobilized lipase demonstrated a significantly higher catalytic activity than that of unmodified alkaline Ca-bentonite, giving a specific activity of 62.1 U mg⁻¹ protein, twice that of the unmodified carrier, and a total activity of 391.2 U g⁻¹ support, retaining ~ 82.3% of the activity after being reused five times for olive oil emulsion hydrolysis. X-ray diffraction and Fourier transform infrared spectroscopy assays demonstrated the successful immobilization of the lipase on the surface of the bentonite. Upon immobilization, the thermostability of the lipase improved remarkably. At 50 °C, free lipase retained only 6.0% of its initial activity at 6 h, in comparison with 15% for Ca-Bent-lipase and 50% for Glu-Ca-Bent-lipase after 8 h. The Glu-Ca-Bent-lipase is proved as an effective biocatalyst for the biodiesel preparation, improving the transesterification reaction conversion from 52.8% in the condition of free lipase to 99.9% and keeping at 56.2% after being reused five times, while the free lipase was inactive upon two reuses. The above results provide a new route in the use of inexpensive bentonite for the enzyme immobilization.

     

聚β-羟基丁酸酯和聚ε-己内酯的酯交换反应

以辛酸亚锡为催化剂 ,研究了聚 β 羟基丁酸酯 (PHB)与聚ε 己内酯 (PCL)在液相条件下的酯交换反应 .讨论了反应时间 ,反应温度和催化剂浓度对酯交换反应的影响 .采用1 3C NMR ,FTIR ,DSC ,WAXD和TGA等方法对PHB和PCL共聚酯 (PHB co PCL)的结构进行了表征 ,并对其结晶行为、晶体结构和热稳定性进行了研究 .结果表明 ,通过酯交换反应 ,所得到的共聚酯为嵌段共聚物 .提高反应温度和延长反应时间有利于酯交换反应的发生 .随着酯交换量的增加 ,PHB co PCL的结晶行为发生很大的变化 .但是 ,PHB co PCL晶体结构并没有因为PCL链段的引入而发生变化 ,而且它的热稳定性在空气气氛中略有提高     

Preparation of New Calix[4]arene-Immobilized Biopolymers for Enhancing Catalytic Properties of Candida rugosa Lipase by Sol–Gel Encapsulation

The article describes preparation of new calixarene biopolymers consisting of the immobilization of convenience calixarene derivative onto cellulose and chitosan biopolymers, and the encapsulation of these calixarene biopolymers with Candida rugosa lipase within a chemical inert sol–gel supported by polycondensation with tetraethoxysilane and octyltriethoxysilane. The catalytic properties of immobilized lipase were evaluated into model reactions employing the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of naproxen methyl esters from racemic prodrugs in aqueous buffer solution/isooctane reaction system. The resolution studies using sol–gel support have observed more improvement in the enantioselectivity of naproxen E?=?300 with Cel-Calix-E than with encapsulated lipase without calixarene-based materials. Furthermore, the encapsulated lipase (Cel-Calix-E) was still retained about 39 % of their conversion ratios after the fifth reuse in the enantioselective reaction.     

Amberlyst-15型离子交换树脂催化γ-丁内酯酯交换反应的研究

从反应温度、时间、催化剂的用量、催化剂的重复使用、产物的分离等方面系统研究了γ-丁内酯与甲醇的酯交换反应,得到了优化的反应条件,即,反应温度为28℃,反应时间为10h,γ-丁内酯/甲醇/Amberlyst-15为0.9∶12.5∶12.5(V/V/m)。在该反应条件下,酯交换产物的分离产率达到77%。同时考察了γ-丁内酯与其它6种醇的酯交换反应,证明了在较高反应温度下,该树脂对其它醇仍有较好的催化活性(产物的质量百分数为50%～60%)。然而,随着醇体积的增大,反应的转化率逐渐下降。