Screening of Food Grade Lipases to be Used in Esterification and Interesterification Reactions of Industrial Interest

Seven food grade commercially available lipases were immobilized by covalent binding on polysiloxane–polyvinyl alcohol (POS-PVA) hybrid composite and screened to mediate reactions of industrial interest. The synthesis of butyl butyrate and the interesterification of tripalmitin with triolein were chosen as model reactions. The highest esterification activity (240.63 μM/g min) was achieved by Candida rugosa lipase, while the highest interesterification yield (31%, in 72 h) was achieved by lipase from Rhizopus oryzae, with the production of about 15 mM of the triglycerides C₅₀ and C₅₂. This lipase also showed a good performance in butyl butyrate synthesis, with an esterification activity of 171.14 μM/g min. The results demonstrated the feasibility of using lipases from C. rugosa for esterification and R. oryzae lipase for both esterification and interesterification reactions.     

Effect of lipase immobilization on resolution of (R, S)-2-octanol in nonaqueous media using modified ultrastable-Y molecular sieve as support

The lipase from Penicillium expansum PED-03 (PEL) was immobilized onto modified ultrastable-Y (USY) molecular sieve and the resolution of (R, S)-2-octanol was carried out in a bioreactor in nonaqueous media by the immobilized lipase. It was found that the conversion rate, enantiomeric excess (ee) value, and enantioselectivity (E) value of the resolution catalyzed by PEL immobilized on modified USY molecular sieve were much higher than those of the reaction catalyzed by free PEL and PEL immobilized on other supports. Immobilized on modified USY molecular sieve, the PEL exhibited obvious activity within a wider pH range and at a much higher temperature and showed a markedly enhanced stability against thermal inactivation, by which the suitable pH of the buffer used for immobilization could be “memorized.” The conversion rate of the reaction catalyzed by PEL immobilized on modified USY molecular sieve reached 48.84%, with excellent enantio-selectivity (avarege E value of eight batches >460) in nonaqueous media at “memorial” pH 9.5, 50°C for 24 h, demonstrating a good application potential in the production of optically pure (R, S)-2-octanol.     

华根霉菌丝体结合脂肪酶催化酯合成动力学拆分2-辛醇

研究了华根霉CCTCC M201021菌丝体结合脂肪酶(RCL)在非水相中催化酯合成动力学拆分外消旋2-辛醇的能力.发现RCL对该反应具有较好的光学选择性(E>100),辛酸和异辛烷分别是最佳的酰基供体和反应溶剂,体系水活度的减少对反应的光学选择性没有明显影响,但能显著提高反应初速度.在相同转化率下,通过添加3A分子筛降低体系水含量可使反应初始速度提高7.3倍.当底物浓度提高到0.230 mol/L,反应40 h时转化率达44.4%,产物酯的ee值为94.7%.与三种商品化脂肪酶进行了比较,发现在相同条件下RCL对2-辛醇的拆分不但具有较好的光学选择性(E=103.1),而且也表现出较高的反应初速度和转化率.     

Regioselective acylation of resveratrol catalyzed by lipase under microwave

The enzymatic regioselective acylation of resveratrol was achieved in organic solvents catalyzed by the lipase from Candida sp. 99–125 (CSL) under microwave irradiation. Influences of various reaction conditions have been studied. After selecting the optimum conditions [MTBE (20?ml, a_w?=?0.38), resveratrol (0.1?mmol), vinyl acetate (1.0?mmol) and CSL (100.0?mg) under microwave irradiation (35°C, 400 W)], CSL exhibited a satisfied enzyme activity (281?±?11?μmol/g/h) and yield of 75% for 4′-O-acetyl- resveratrol could be obtained in about 4?h when performing the reaction on a 25-fold-larger scale.

Schematic illustration of the enzymatic regioselective acylation of resveratrol catalyzed by the lipase from Candida sp. 99–125 (CSL) under microwave irradiation.     

Production of glycerides from glycerol and fatty acids by native lipase ofNigella sativa seed

The possible application of native lipase ofNigella sativa seed in the esterification of fatty acids to glycerol was investigated, and the effect of process parameters and the enzyme selectivity on the reaction were determined. For this aim, the esterification of oleic acid, sunflower oil fatty acids, and coco oil fatty acids with glycerol were studied.     

Enhancing enzymatic properties by the immobilization method

Effects of some immobilized carriers on enzymatic properties have been studied. The following results were obtained: (1) When cholinesterase was immobilized on the hydrophobic carrier with either α-naphthylamine, benzylamine, orp-methylbenzylamine groups, the affinities of immobilized cholinesterase for toxic organophosphors, GB (Isopnopy 1-methylphophonofluoridate) and Vx [o-ethyl-S-(2-diisopnoylomino-thyl) methyl phosphonothiolate], were enhanced 60–90 times and 700–1200 times, respectively, whereas the thermal stability of the immobilized cholinesterase increased to 110%. Approximately 82–88% activity of the immobilized cholinesterase remained after continuously operating for 8 h; and (2) Lipase was immobilized on the carrier that was made up of 6% polyethylenimine, 1% alginate gel, and 1% glutaraldehyde. The initial reaction rate of the esterification of lauric acid with lauric alcohol catalyzed by this kind of immobilized lipase was increased 21 times, as compared to lipase powder. About 72% esterification activity of lipase remained after continuous operating for 10 d.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.     

改性Ultrastable-Y分子筛固定化对非水相中脂肪酶拆分(R,S)-2-辛醇的影响

采用改性Ultrastable-Y分子筛固定Penicillium expansum PED-03 脂肪酶(PEL), 利用固定化PEL在非水相中对(R,S)-2-辛醇进行手性拆分, 考察了改性Ultrastable-Y分子筛固定化处理对PEL催化性能的影响. 结果表明, 与游离PEL及经其它载体固定化的PEL相比, 改性Ultrastable-Y分子筛固定的PEL所催化的拆分反应的转化率(c)和对映体过量值(ee)以及对映体选择性(E)均得到了较大提高. 经固定化处理后, PEL的最适反应温度明显升高, 适宜反应温度范围变宽, 其稳定性也得到了明显改善, 而适宜反应pH值则具有“记忆”性. 在间歇式反应器中利用Ultrastable-Y分子筛固定化PEL对(R,S)-2-辛醇进行手性拆分, 50 ℃反应24 h转化率(c)可达理论值的97.68%, 对映体过量值(ee)可达98.75%. 连续8批拆分反应的结果表明: 改性Ultrastable-Y分子筛固定化脂肪酶催化效率高、立体选择性强(平均E 值＞460), 且催化性能稳定, 显示了该固定化酶在(R,S)-2-辛醇的手性拆分方面具有良好的应用前景.     

十六烷基三甲基溴化铵/山梨醇酐硬脂酸酯微乳液凝胶固定化脂肪酶的催化活性及立体选择性

制备了能在水溶液中长时间稳定存在的十六烷基三甲基溴化铵/山梨醇酐硬脂酸酯（CTAB/Span-60）微乳液凝胶（MBG）,确定了Span-60在乳化剂EM（正丁醇与Span-60的混合物）中的质量分数范围;分别以正己酸与正己醇的酯化反应、α-单硬脂酸甘油酯的水解反应、消旋布洛芬与正辛醇的不对称酯化反应为指示反应,研究了CTAB/Span-60 MBG固定化脂肪酶的催化活性及立体选择性。 结果表明,Span-60在EM中的质量分数小于57%时可形成机械强度较好的CTAB/Span-60 MBG;其固定化脂肪酶在有机溶剂中的酯化活性随EM中Span-60含量的增加先是逐渐增大,35%时最大,后又逐渐小幅度降低,在所考察的Span-60含量范围内均比在CTAB MBG中高;在水溶液中固定化脂肪酶能顺利催化α-单硬脂酸甘油酯的水解反应,24 h后反应转化率不再随反应时间的延长而增加,其水解活性在重复使用9次后仅降低13.68%,表明CTAB/Span-60 MBG固定化脂肪酶能够顺利进行分离并重复使用;此体系的脂肪酶也选择性地催化生成S-构型布洛芬辛酯,产物对映体过量值（eee）随反应的进行缓缓下降,但降幅不大,即其立体选择性要比在CTAB MBG中高。 因此,CTAB/Span-60 MBG作为脂肪酶固定化载体既可用于有机溶剂中又可用于水溶液中的生物合成与生物转化反应,扩大了微乳液凝胶固定化脂肪酶的应用范围。     

Lipase from a Brazilian strain ofPenicillium citrinum

A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain ofPenicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40–60% fraction. The optimum temperature for enzyme activity was found in the range of 34–37°C. However, after 30 min at 60°C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28°C) for 8 mo. This result in lipase stability suggests an application of lipases fromP. citrinum in detergents and other products that require a high stability at room temperature.     

Lipase-catalyzed domino Michael–aldol reaction of 2-methyl-1,3-cycloalkanedione and methyl vinyl ketone for the synthesis of bicyclic compounds

Synthesis of bicyclic compounds was achieved via a lipase-catalyzed, stereoselective, domino Michael–aldol reaction of 2-methyl-1,3-cycloalkanedione and methyl vinyl ketone. Appropriate reaction conditions, including the type of enzyme, solvent, and temperature, were determined. In addition, the effects of solvent polarity and addtives were investigated. The reaction proceeded in the presence of lipase AS in a solution of 20% acetone in dimethylsulfoxide (DMSO) at 10 °C for 8 days, followed by the addition of p-toluenesulfonic acid (TsOH) to afford bicyclic compounds in 51–83% yields with moderate stereoselectivity. Although this domino Michael–aldol reaction showed only moderate stereoselectivity, even with the acid-supported enhancement of the reaction, these results represent potential new applications for lipase.     

非水介质中脂肪酶催化亚麻酸油醇酯合成的研究

已知脂肪酶（ＥＣ３．１．１．３）在非水体系中能够催化合成脂肪族、芳香族和其它多种多样的酯类．这些反应中的大多数都是以链较长的脂肪酸为底物．其中一类反应是高级脂肪酸和脂肪醇合成酯蜡的酯化反应．合成的酯蜡用途广泛,可以用在化工、纺织、医药、日化、食品等工业中．利用传统的化学法合成酯蜡,需要在高温高压及强酸条件下进行反应,副反应多,产物的分离纯化困难,生产成本高,且对于长链脂肪酸和醇的反应难度增大．利用脂肪酶水解反应的逆反应酯化反应合成酯蜡的方法,具有反应条件温和,产物的分离纯化容易等优点,能够弥补化…     

Purification and Characterization of an Organic Solvent-Tolerant Lipase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> CS-2

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 °C and 8.0. The lipase was found to be stable at pH 4–10 and below 50 °C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca²⁺, while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.     

Sorption of aliphatic alcohols, acetates, and their mixtures by corn starch cryotextures depending on concentrations of sorbates and starch in initial sols

The sorption of a mixture ofn-hexanol,n-octanol, andn-hexyl andn-octyl acetates from aqueous solutions by corn starch cryotextures was studied using capillary gas chromatography at different initial concentrations of the sorbates (1–25 mmol L⁻¹) and corn starch (2–6%). The amounts of compounds sorbed by cryotextures are proportional to the increase in their concentration in the initial sol and the length of the alkyl substituent. Linear equations describing the concentration dependence were proposed. The sorption ofn-hexanol from a mixture of substances containingn-octanol increases as compared to that from the individual alcohol. It was shown that the degree of sorption of aroma by cryotextures was independent of the content of starch in the initial sol. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1496–1501, August, 1999.     

Lipase-catalyzed production of optically active acids via asymmetric hydrolysis of esters

It has been found that lipase fromCandida cylindracea hydrolyzes octyl R(+)- but not S(-)-2-chloropropionate. At the same time, the enzyme exhibits no appreciable stereoselectivity in the hydrolysis of the methyl ester of the same acid. Solubility determination experiments showed that at the concentrations used, methyl 2-chloropropionate was completely dissolved in water, whereas the octyl ester existed as an emulsion in water. It is therefore speculated that in order to express its stereoselectivity the lipase needs to adsorb on the substrate—water interface. R,S-2-chloropropionic acid was preparatively resolved via yeast lipase-catalyzed asymmetric hydrolysis of its octyl ester. Gram quantities of R(+)-chloropropionic acid and octyl S(-)-2-chloropropionate of high optical purity were readily prepared.     

Esterification activity and stability of Candida rugosa lipase immobilized into chitosan

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2–C12), and butyric acid and different aliphatic alcohols (C2–C10). After 24 h, the acids tested achieved conversions of about 40–45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system’s butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 2³ full factorial design. The range studied was 37–50°C for temperature (X₁), 1.25–2.5% (w/v) for the catalyst concentration (X₂), and 1 and 2 for the acid:alcohol molar ratio (X₃). Catalyst concentration (X₂) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37°C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.     

以三乙酸甘油酯为底物制备光学活性戊醇的探索

1前言(S)-2-甲基-1-丁醇在手性精细化学品及药物合成中有重要的作用[1]。从20世纪90年代起,国外的(R,S)-2-甲基-1-丁醇拆分方面做了一些有益的尝试。Ayter Sagiroglu等人利用固定化脂肪酶催化三丁酸甘油酯与(R,S)-2-甲基-1-丁醇发生不对称酯交换反应,可制备e.e.为98%的(S)-2-甲基-1-丁醇[2]。同时我们的研究表明,无溶剂体系中,以猪胰脂肪酶或酵母脂肪酶为催化剂,三丁酸甘油酯为底物,拆分(R,S)-2-甲基-1-丁醇,可制备e.e.值大于90%的(S)-2-甲基-1-丁醇[3]。采用三丁酸甘油酯,虽然所得产品e.e.较高,但其价格昂贵(1000元/kg)、反应周期较长…     

Stereoselectivity and competing reactions as studied by lipase-catalyzed esterifications in aqueous lecithin-based gelatin gels

 The enzyme catalyzed conversion of R/S-(±)-2-octanol with hexanoic acid to R/S-(±)-2-octyl hexanoate has been studied in different microenvironments and in the presence of the competing substrate ethanol. The reactions were performed in various gels made from aqueous gelatin solutions and liposome dispersions or isotropic liquid solutions, with or without oil and ethanol. The lipase Candida sp. (SP 525) was dissolved in the dispersions or solutions stabilized by the naturally occurring zwitterionic surfactant soybean lecithin. The sectioned porous gel was immersed in hexane containing 0.33 mol dm^-3 of racemic 2-octanol and hexanoic acid. Since ethanol acts both as a substrate and as a part of the gel it is of fundamental interest to know the phase behaviour of the used systems. Partial phase diagrams for the systems ethanol–water–soybean lecithin and ethanol/water (7:3)–oil–soybean lecithin were determined at 298.2 K. The oil was either castor oil or hexadecane. The conversion of R-2-octyl hexanoate was about 0.45 when no or small amounts of ethanol was present, but decreased considerably with high amounts of ethanol present and ethyl hexanoate became the main product. Hydrolysis of R-2-octyl hexanoate was favoured in the latter systems and hexanoic acid formed was immediately esterified to ethyl hexanoate. The conversion of R-2-octyl hexanoate and ethyl hexanoate depends only on the ethanol content present in the systems and is thus independent of the environment of the enzyme. However, the chiral esters synthesized from racemic 2-octanol and hexanoic acid showed high optical purities regardless of the ethanol content. Received: 1 July 1996 Accepted: 30 August 1996     

Esterification of caprylic acid with alcohol over nano-crystalline sulfated zirconia

The catalytic activity of nano-crystalline sulfated zirconia catalyst, prepared by sol–gel method and characterized by various analytical tools, was evaluated for the esterification of caprylic acid with different short chain alcohols. The lower concentration of catalyst (0.5 wt%) exhibited 96–98% conversion of caprylic acid with methanol and 100% selectivity for methyl caprylate at 60 °C. The conversion was decreased with increasing carbon chain of alcohols namely with ethanol, n-propanol and n-butanol at 60 °C but increased significantly (91–98%) at higher reaction temperature. The selectivity for respective alkyl caprylate was observed to be 100% irrespective of the alcohol used. The activity of the catalyst was slightly decreased with successive five reaction cycles due to the water formed during the reaction.     

Lipase catalysis in organic solvents

The conditions for esterification and transesterification catalyzed by porcine pancreatic lipase in organic media were studied. It was found that the enzyme reaction was dependent on the following factors: the pH at which the enzyme powder was prepared from its solution, the polarity of organic media, the reaction temperature, the water content in reaction system, and the substrate structures. Effects of the above factors on enzyme activity were discussed.