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.     

Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was 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 was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.     

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.     

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.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

Microwave-assisted synthesis of ethyl laurate using immobilized lipase: Optimization,mechanism and thermodynamic studies

Microwave-assisted synthesis of ethyl laurate using lauric acid and ethanol catalyzed by Fermase CALB has been investigated. The effect of operating parameters like molar ratio (lauric acid: ethanol), enzyme loading, temperature, and molecular sieves was systematically studied. A maximum conversion of 98.2% was obtained in 10 ​min compared to the conventional method, where the reaction required 4 ​h to achieve 92.4% conversion. The optimum parameters for the microwave-assisted synthesis were 1:2 ​M ratio of lauric acid to ethanol, 45 ​°C temperature, 1.8% (w/w) enzyme amount, 1.5% (w/w) molecular sieves. The enzyme lipase was reused for up to seven successive cycles under microwave irradiation. The thermodynamic study was carried out to determine various thermodynamic parameters for the reaction. The esterification mechanism was proposed, and the impact of microwave irradiation on the immobilized enzyme after several reuse was studied by scanning electron microscopy.     

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design

Candida rugosa lipase was covalently immobilized on silanized controlled poresilica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized from in long-term operation. Proteins (albumin and lecithin) and organic molecules (β-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150–450 U/g of dry support) was performed by experimental design. Two 2²full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better, stabilizing effect was found when small amounts of albumin or PEG-1500, were added simul-taneou sly to the lipase on to the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-150 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).     

Production of biodiesel by immobilized Candida sp. lipase at high water content

A new process for enzymatic synthesis of biodiesel at high water content (10–20%) with 96% conversion by lipase from Candida sp. 99–125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to staturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.     

Identification of Antioxidant and Anti-α-amylase Components in Lotus (Nelumbo nucifera,Gaertn.) Seed Epicarp

Nano-sized Fe₃O₄ was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of “interface activation” by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.

     

Reaction kinetics of the esterification of lauric acid in iso-octane using an immobilized biocatalyst

The kinetics of the esterification of lauric acid with geraniol catalyzed by a commercially immobilized lipase preparation fromMucor miehei, Lipozyme, was studied in well-stirred flasks under conditions of no external mass transfer limitations. It was shown that the reaction is inhibited by lauric acid and the reaction mechanism can be described as a Ping-Pong Bi-Bi with Dead-End inhibition caused by lauric acid.     

Studies on the factors that affect stereoselective esterification of (R,S) 2-octanol with octanoic acid catalyzed by lipase in organic solvent

Stereoselective esterification of(R, S) 2-octanol with octanoic acid catalyzed byCandida Sp lipase (CSL) was carried out in cyclohexane. We have studied the effects of factors, such as temperature and the microenvironment of lipase, on this reaction. The results showed that CSL favoredR enantiomer of(R, S) 2-octanol, and that the esterification activity and stereoselectivity of the lipase were dependent on these factors. The higher the temperature, the greater the esterification activity of CSL. A slight increase in stereoselectivity can be seen with temperature decrease. The optimal range of pH value for this reaction was 4.9–6.2. When the salt concentration was between 0 and 0.05 mol/L, CSL showed high activity. The salt concentration in the reaction system and the pH value at which CSL powder was prepared from the aqueous solution had no evident effect on the stereoselectivity of CSL. The optimal range of the water content in the reaction system was 0.4–1.6%. The esterification activity and the stereoselectivity of CSL were enhanced 1.4-fold and 2.0-fold, respectively, by immediately removing the produced water. (S) 2-octanol with 95.2% enantiomeric excess (ee) was prepared. Based on these results, we have discussed why that all these factors affected this reaction.     

Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

微乳液中脂肪酶和含明胶的微乳液凝胶中固定化脂肪酶的催化特性

在双2-乙基己基琥珀酸酯磺酸钠(AOT)油包水微乳液中Calytical脂肪酶催化月桂酸和戊醇的酯化反应动力学研究表明,反应符合乒乓(BiBi)机制.表观速率常数k_m酸=0.13518mol/L,k_m醇=0.22423mol/L,最大反应速度v_max=1.3873×10^-5mol/(L·min·mg).将该脂肪酶固定于含明胶的微乳液凝胶(MBGs)中,制得固定化脂肪酶,含酶MBGs在非极性溶剂中可作为固相催化剂,并研究了其在辛烷中催化酯化的性能.所制得的含酶MBGs物理稳定性好,重复利用10次以上,其转化率仍达初始转化率的90%.     

Silk-Fiber Immobilized Lipase-Catalyzed Hydrolysis of Emulsified Sunflower Oil

Lipase was immobilized in silk fibers through glutaraldehyde cross-linking to a maximum loading of 59 U/g silk-fiber and the immobilized lipase was utilized for the hydrolysis of sunflower oil (Helianthus annuus). The hydrolytic activity of the lipase, which was poor in biphasic oil in water system, was increased significantly when the sunflower oil was emulsified in aqueous medium. The hydrolytic activities of the immobilized lipase were 48.73 ± 1.26 U, 36.11 ± 0.96 U, and nil when the substrate sunflower oil was used as emulsion created by a rhamnolipid biosurfactant, Triton X100, and ultrasonication, respectively. Although the efficiency of the immobilized lipase was less than 12% than the corresponding free lipase, the immobilized lipase could be reused for the biosurfactant-mediated hydrolysis of sunflower oil up to third cycle of the reaction. The yield of the fatty acids in the second, third, and fourth cycles were 49.45%, 22.91%, and 5.09%, respectively, of the yield obtained in the first cycle.     

有机相中固定化脂肪酶催化合成植物甾醇酯

酶法合成植物甾醇酯具有反应条件温和、产物纯度和产量高等优点,但非水相酶催化的活性和稳定性普遍较低.本文以大孔树脂固定化脂肪酶为催化剂,并在催化过程中添加乳糖的类似物,构建了有机相高效合成植物甾醇酯的工艺过程.以酯化率为考察指标,对脂肪酶和反应溶剂进行筛选,对酯化条件进行优化,同时考察了糖的种类及添加量对酶催化性能的影响.结果表明,大孔树脂NKA吸附固定化的褶皱假丝酵母(Candida rugosa)脂肪酶(NKA-CRL)为最适宜的催化剂,以正己烷为反应介质,在酸醇摩尔比为2和添加酶蛋白质量7.5%的海藻糖的条件下,40°C反应10 h,酯化率达到96.6%.连续6次催化后,植物甾醇的酯化率仍维持在85.0%以上.     

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。     

Concentration,Partial Characterization,and Immobilization of Lipase Extract from <Emphasis Type="Italic">P. brevicompactum</Emphasis> by Solid-State Fermentation of Babassu Cake and Castor Bean Cake

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and −10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.     

One-pot sequences of reactions with sol-gel entrapped opposing reagents: an enzyme and metal-complex catalysts

We extend our sol-gel methodology of one-pot sequences of reactions with opposing reagents to an enzyme/metal-complex pair. Sol-gel entrapped lipase and sol-gel entrapped RhCl[P(C(6)H(5))(3)](3) or Rh(2)Co(2)(CO)(12) were used for one-pot esterification and C-C double bond hydrogenation reactions, leading to saturated esters in good yields. When only the enzyme is entrapped, the homogeneous catalysts quench its activity and poison it. Thus, when 10-undecenoic acid and 1-pentanol were subjected in one pot to the entrapped lipase and to homogeneously dissolved RhCl[P(C(6)H(5))(3)](3) under hydrogen pressure, only 7% of the saturated 1-pentyl undecanoate was obtained. The yield jumped 6.5-fold when both the enzyme and the catalyst were immobilized separately in silica sol-gel matrixes. Similar one-pot esterifications and hydrogenations by sol-gel entrapped lipase and heterogenized rhodium complexes were carried out successfully with the saturated nonoic, undecanoic, and lauric acids together with several saturated and unsaturated alcohols. The use of (S)-(-)-2-methylbutanol afforded an optically pure ester. The heterogenized lipase is capable of inducing asymmetry during esterification with a prochiral alcohol. Both the entrapped lipase and the immobilized rhodium catalysts can be recovered simply by filtration and recycled in further runs without loss of catalytic activity.     

Covalent immobilization of Candida antarctica lipase B on nanopolystyrene and its application to microwave-assisted esterification

Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10³ mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process.     

Enzymatic synthesis of monolaurin

The aim of this study was to produce monolaurin utilizing a commercial immobilized lipase (Lipozyme IM-20; Novo Nordisk, Bagsvaerd, Denmark) through the direct esterification of lauric acid and glycerol in a solvent-free system. The influence of fatty acid/glycerol molar ratio, temperature, and Lipozyme (IM-20) concentration on the molar fraction of monolaurin were determined using an experimental design. The best conditions employed were 55°C, lauric acid/glycerol molar ratio of 1.0, and 3.0% (w/w) enzyme concentration. The final product, obtained after 6 h of reaction, was 45.5% monolaurin, 26.8% dilaurin, 3.1% trilaurin, and 24.6% lauric acid. The reusability of the enzyme was also studied.