叔戊醇/水两相体系脂肪酶催化合成蔗糖酯

以叔戊醇／水两相体系作为反应体系,利用脂肪酶催化的蔗糖和乙酸乙烯酯的转酯化反应来合成蔗糖酯,并考察了不同的两相体积比,温度,水相pH,酶量,糖与酰基供体比例,反应时间对蔗糖转化率的影响。结果表明这种方法是可行的．主要产物为蔗糖6-乙酸酯。发现在两相体系水体积含量为20％时．开始有蔗糖6-乙酸酯生成,然后随着水含量提高,蔗糖转化率逐渐提高,水含量在90％时,蔗糖转化率最大。但是当水含量为100％,蔗糖转化率只有2．1％。当温度为45℃,水相pH为8．0,酶量为300U。蔗糖3mmol。乙酸乙烯酯为12mmol,反应24h,蔗糖转化率达到19．8％。     

Impact of Fatty Acid Structure on CALB‐Catalyzed Esterification of Glucose

Enzymatic synthesis of fatty acid glucose esters from different fatty acyl donors are performed via enzymatic catalysis in the presence of Candida antarctica lipase B (CALB), using acetonitrile as the solvent. The acyl donor nature (fatty acid or fatty acid vinyl ester) and structure are varied. Lower reaction rates and lower conversions are obtained with fatty acids in comparison to their corresponding vinyl esters. Moreover, the acyl donor with the longest chain length gives the highest conversions. The presence of unsaturation on the acyl donor chain is also shown to be detrimental to the conversion. Practical Applications: The practical applications of the present work are related to the production of gluco‐esters that could be used as nonionic surfactants as detergents, cosmetics and food emulsifiers, emollients or conservatives, respectively. In this study, it is shown that in order to get high production yields, each reaction parameter has to be tuned properly.     

Application of lipase immobilized on nylon‐6 for the synthesis of butyl acetate by transesterification reaction in n‐heptane

A purified alkaline thermotolerant bacterial lipase from Bacillus coagulans BTS‐3 was immobilized on nylon‐6 matrix activated by glutaraldehyde. The matrix showed ～ 70% binding efficiency for lipase. The bound lipase was used to perform transesterification in n‐heptane. The reaction studied was conversion of vinyl acetate and butanol to butyl acetate and vinyl alcohol. Synthesis of butyl acetate was used as a parameter to study the transesterification reaction. The immobilized enzyme achieved ～ 75% conversion of vinyl acetate and butanol (100 mmol/L each) into butyl acetate in n‐heptane at 55°C in 12 h. When alkane of C‐chain lower or higher than n‐heptane was used as an organic solvent, the conversion of vinyl acetate and butanol to butyl acetate decreased. During the repetitive transesterification under optimal conditions, the nylon bound lipase produced 77.6 mmol/L of butyl acetate after third cycle of reuse. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

有机碱在脂肪酶Candida antarctica B催化仲醇拆分中的促进作用

以离子液体[bm im]PF6为反应溶剂,乙酸乙烯酯为酰基供体,考察了脂肪酶Candida antarcticaB催化外消旋仲醇的动力学拆分。主要考察了有机碱和无机碱对拆分反应的影响,结果发现,在反应底物中加入三乙胺或吡啶,能够极大地提高脂肪酶的活性和对映选择性,当底物邻甲氧基-α-苯乙醇和1-(2-萘基)乙醇在拆分时加入吡啶后,脂肪酶的对映选择性是未加入有机碱时的5~8倍,分别从E=65提高到E=525和从E=209提高到E=1 059,因此,可高对映选择性地获得邻甲氧基-α-苯乙醇和1-(2-萘基)乙醇的手性单体。随着吡啶加入量的增加,脂肪酶的对映选择性有所下降,因此,有机碱的最佳加入量为0.01 mol,原因可能是在该加入量下,脂肪酶达到了最佳pH,即处于活性构象,从而具有较高的活性和对映选择性。     

脂肪酶固定化研究和应用

采用硅藻土作载体,进行了脂肪酶的固定化。利用固定化酶选择性催化1-苯乙醇与乙酸乙烯酯的转酯化反应,得到R-乙酸苯乙酯,进行1-苯乙醇的拆分。实验考察了不同吸附方法固定化酶的效果,确定效果最好的固定方法为载体涂布法,并对该法的固定化条件进行了优化。制备的固定化酶的转酯比活比游离酶提高了14．3倍。固定化没有改变酶的选择性．对映体过剩值仍大于98％。初步探讨了固定化酶和游离酶的反应过程。     

2-烯丙基-4-羟基-2-环戊烯酮的脂肪酶动力学拆分

2-烯丙基-4-羟基-2-环戊烯酮是合成前列腺素PGI1的关键中间体,具有重要的药用价值。该文用改进的方法合成了(±)-2-烯丙基-4-羟基-2-环戊烯酮,总收率27%,用脂肪酶对其进行动力学拆分,得到光学纯的(S)-环戊烯酮醇。结果表明,猪胰脂肪酶具有最佳的拆分效果。以乙酸乙烯酯同时作为溶剂和酰化供体,在25℃,m(酶)∶m(醇)=1.5∶1条件下,环戊烯酮醇的转化率为50.2%,(S)-醇的ee值为58.9%。采用同样条件进行二次酶动力学拆分,得到ee值>98%的手性烯酮醇,总拆分收率23%。     

Fatty acid vinyl esters as acylating agents: A new method for the enzymatic synthesis of monoacylglycerols

Lipase-catalyzed synthesis of monoacylglycerols (MAG) was performed by transesterification reactions between fatty acid vinyl esters and either glycerol (1) or 1,2-O-isopropylidene-rac-glycerol (2), without solvents or in the presence ofn-pentane. Vinyl decanoate, vinyl laurate, vinyl stearate and vinyl palmitate have been converted to the corresponding monoacylglycerols. As expected for the reaction with1, a mixture of mono-, di- and triacylglycerols was synthesized. The highest concentrations of MAG were achieved with vinyl stearate (30% 2-MAG and 15% 1-MAG). The reactions of fatty acid vinyl esters with the protected glycerol (2) led to the corresponding protected 3-monoacylglycerols with 100% conversion after short reaction times. The subsequent cleavage of these acetonides was performed by four different methods. The fastest cleavage was found with trifluoroacetic acid as catalyst, whereas the highest concentration of MAG (100%) was obtained for the boric acid-catalyzed hydrolysis of the acetonides.     

Improved Synthesis of Monopalmitin on a Large Scale by Two Enzymatic Methods

Monoacylglycerols (MAG) are precursors for the synthesis of symmetrical and unsymmetrical triacylglycerols (TAG). In the present study, we improved two methods for synthesizing MAG. One method involved the enzymatic transesterification of vinyl palmitate with glycerol catalyzed by Novozym 435 lipase, and the other method was an enzymatic esterification of 1,2-acetonide glycerol with palmitic acid catalyzed by Novozym 435 lipase and then the cleavage of 1,2-acetonide-3-palmitoyl glycerol in methanol catalyzed by Amberlyst-15 to produce monopalmitin. Pure monopalmitin was obtained after repeated crystallization. The main novelties of this study are twofold: Novozym 435 proved to be very effective in catalyzing the transesterification between vinyl palmitate and glycerol without absorbing glycerol onto silica gel; and the enzyme catalyzed reaction between 1,2-acetonide glycerol and palmitic acid was simpler and safer than the typical method of using 4-dimethyl aminopyridine and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride as catalysts. Our methods for the synthesis of monopalmitin are much simpler and environmentally friendlier than the reported methods, and they are economical and scalable to larger quantity production.     

Regioselective Alcoholysis of Silychristin Acetates Catalyzed by Lipases

A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22) of silychristin was accomplished by lipase PS (Pseudomonas cepacia) immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule.     

Polymeric membranes for lipase immobilization

Lipase triacylglycerol acylhydrolase, (E.C. 3.1.1.3) is an enzyme that is fully active on aggregated substrates and practically inactive on monodisperse systems. A lipase immobilized on polymeric membranes has been applied for sunflower oil hydrolysis. The influence of membrane properties on enzyme activity is studied. Membranes made of poly(vinyl chloride), collagen, cellulose acetate and polytetrafluoroethylene were used for adsorption of lipase. The porosity and hydrophobicity of membranes did not influence the lipase activity. The difference of the work of adhesion for the water/membrane system and oil/membrane system reflected the activity data, while work of adhesion for water or oil (done separately) did not. For oil hydrolysis to occur on the membrane surface, accessibility of two liquid phases is important, and the lower the difference of work of adhesion between water and oil, the greater the activity of immobilized lipase.     

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.     

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     

Experimental design of the kinetic resolution of a key precursor of high‐value bioactive myo‐inositols by an immobilized lipase

BACKGROUND: The response surface methodology was successfully applied to the optimization of the reaction variables for the kinetic resolution of a precursor of high‐value myo‐inositols, ( ± )‐1,2‐O‐isopropylidene‐3,6‐di‐O‐benzyl‐myo‐inositol (( ± )‐1), by Novozym 435. The resolutions were run separately, with two acylating agents, ethyl acetate and vinyl acetate, in a solvent‐free system. The variables analyzed were reaction temperature, substrate concentration, water concentration and enzyme activity. A statistical model was employed for the evaluation of the influence of the variables on conversion and enantiomeric excess (ee). RESULTS: The optimal conditions for this resolution using vinyl acetate as acylating agent were 45 °C, 5 mg mL^?1 of substrate, 71 U of enzyme activity and 0%w/w of water concentration. The high conversion (49.2 %) and ee (>99%) reached in the chemoenzymatic synthesis of acylated product, L‐(?)‐5‐O‐Acetyl‐3,6‐di‐O‐benzyl‐1,2‐O‐isopropylidene‐myo‐inositol, secure the efficient synthesis of the D enantiomorph present in the original racemic mixture (( ± )‐1) as well. CONCLUSIONS: The use of the experimental design strategy was productive, leading to a 14‐fold increase in the productivity of the reaction compared with the non‐optimized conditions. Both derivative L‐(?)‐2 and remaining substrate D‐(+)‐1 were obtained at high ee. © 2012 Society of Chemical Industry     

Biocatalytic kinetic resolution of rac‐1‐phenylethanol and rac‐2‐pentanol in hexane medium: ACYL donor and water content effects

The kinetic resolutions of rac‐1‐phenylethanol and rac‐2‐pentanol by transesterification with vinyl esters catalysed by a commercial immobilised Candida antarctica lipase B were successfully carried out in hexane medium. This enzyme showed very high enantioselectivity for both substrates. The influence of the water content of the medium on the synthetic activity, selectivity and enantioselectivity of the enzyme was analysed, with the optimal amount of water about 100 ppm. Our results also showed that the activity per gram enzymatic derivate of CaLB was slightly higher with butyl butyrate as acyl donor.     

Esterification of n‐butyric acid with n‐butyl alcohol and transesterification of (R, S)‐phenylethanol by lipase immobilized on cellulose acetate–TiO2 gel fibre

Lipase (EC 3.1.1.3) was immobilized on cellulose acetate–TiO₂ gel fibre by the sol–gel method. The immobilized lipases were used for esterification of n‐butyric acid with n‐butyl alcohol and enantioselective acylation of (R, S)‐phenylethanol using vinyl acetate as an acyl donor. Compared with native lipase, the activity of the immobilized lipase was stable and relatively unaffected by the water content of the solvent and the substrate concentration. The data indicate that the lipases are immobilized on the fibre surface and that enzyme activity is influenced by bound water. However, the thermal reactivity and enantioselectivity of the immobilized lipase were less than those of native lipase. This may not reflect thermal inactivation of the enzyme but rather significant thermal contraction of the gel fibre by cellulose crystallization, resulting in liberation of bound water and a decrease in the amount of enzyme which is available for the reaction. Copyright © 2001 Society of Chemical Industry     

Enzymatic resolution of planar chiral ferrocene derivatives

Racemic 3-methyl, 2-ethyl, 2-bromo, and 2-trimethylsilyl derivatives of formylferrocene were kinetically resolved by enantioselective reduction with a fermenting baker's yeast. Moreover, 2-methyl, 3-methyl, 2-ethyl, 2-bromo, and 2-trimethylsilyl derivatives of hydroxymethylferrocene were optically resolved with lipases by transesterification using vinyl esters.     

非水相酶促不可逆转酯反应合成阿魏酸三油酸甘油酯

在有机相甲苯中通过阿魏酸乙烯酯(VF)和三油酸甘油酯(TO)的酶促转酯反应合成油脂抗氧化剂阿魏酸三油酸甘油酯。用阿魏酸乙烯酯作为底物可使转酯反应不可逆,这有利于产物产率的提高并缩短反应时间。通过核磁共振波谱和质谱对阿魏酸乙烯酯的结构进行了表征,并考察了底物摩尔比、反应时间、反应温度、酶用量、水活度对产物产率的影响。结果表明,以甲苯为溶剂,当底物n(VF)∶n(TO)=1∶3,反应时间62 h,反应温度55℃、酶用量20 g/L,水活度(aw)为0.07时,产物的产率最大,达96.73%。     

Baker's yeast reduction of α-(alkoxycarbonylamino)acetophenones and Lipase-catalysed resolution of 2-(alkoxycarbonylamino)-1-arylethanols

Enzymatic reduction of α-(alkoxycarbonylamino)acetophenones with baker's yeast afforded optically active (R)-2-(alkoxycarbonylamino)-1-arylethanols. However, the reduction of α-(benzyloxycarbonylamino)-4-methoxyacetophenone ( 3c ) with immobilized baker's yeast gave (S)-2-(benzyloxycarbonylamino)-1-(4-methoxyphenyl) ethanol. The lipase PS-catalysed transesterification of 2-(allyloxycarbonylamino)-1-arylethanols ( 5 ) using vinyl acetate as an acyl donor resulted in the formation of (S)-2-(allyloxycarbonyl-amino)-1-arylethyl acetates [( S )- 9 ] and (R)-2-(allyloxycarbonylamino)-1-aryle-thanols [( R )- 5 ].     

Immobilization of lipase on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel

Lipase from Candida rugosa was immobilized by entrapment while polymerizing a poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) [poly(VP‐co‐ST)] hydrogel using ethylene dimethacrylate (EDMA) as the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases were also determined. The activities were relatively higher when the percent compositions of VP(%):ST(%) were 50:50 and 30:70. The lipase immobilized on VP(%):ST(%) 50:50 showed the highest thermal stability, while lipase immobilized on VP(%):ST(%) 30:70 exhibited the highest solvent stability. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1404–1409, 2001     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.