Lipase-catalyzed Reaction Research Articles

Enhanced synthesis of isoamyl acetate using an ionic liquid–alcohol biphasic system at high hydrostatic pressure

Isoamyl acetate has a banana flavor that can be considered a “natural” ingredient when synthesized using a lipase-catalyzed reaction. Production of isoamyl acetate was up to 10-fold higher with free lipase versus immobilized Candida antarctica lipase B (CALB) after 3 h at 300 MPa and 80 °C in 1-butyl-3-methylimidazolium hexafluoro-phosphate, isoamyl alcohol biphasic system. Rate of catalysis by free CALB was 15-fold greater at 500 MPa, 40 °C than at 0.1 MPa, 40 °C and 14-fold at 500 MPa, 80 °C than at 0.1 MPa, 80 °C. Activation energy of free lipase calculated between 40 and 80 °C at 0.1 MPa (55.6 ± 4.2 kJ mol−1) or 300 MPa (56.2 ± 4.6 kJ mol−1) was not significantly different. Similarly, activation volume (ΔV‡) of free lipase calculated between 0.1 and 500 MPa at 40 °C (−16.1 ± 1.5 cm3 mol−1) or 80 °C (−16.7 ± 1.4 cm3 mol−1) was not significantly different. After treatment at high pressure and upon pressure release, the free lipase was temporarily suspended in a semi-solid IL phase. This study is the first to combine the use of a room temperature ionic liquid (RTIL) and high hydrostatic pressure (HHP) for enhanced enzyme catalysis.

ChemInform Abstract: Asymmetric Synthesis of Novel gem-Difluorinated Compounds Using Chemo-Enzymatic Methodology

Asymmetric synthesis of two types of partly difluorinated compounds has been accomplished based on the chemo-enzymatic reaction strategy. Optically active 1, 1-difluoro-2, 3- (bishydroxymethyl) cyclopropane has been synthesized through lipase-catalyzed reaction; prochiral diacetate of cis-1, 1-difluoro-2, 3-bis (hydroxymethyl) cyclopropane was converted to the corresponding monoacetate through Alcaligenes sp. lipase-catalyzed hydrolysis with >99% enantiomeric excess. Racemic acetate of trans-1, 1-difluoro-2, 3-bis (hydroxymethyl) cyclopropane was resolved by Pseudomonas sp. lipase-catalyzed hydrolysis successfully. Lipase-catalyzed reaction was employed to resolve bis-difluorocyclopropane derivatives; the first synthesis of optically pure (trans, trans) -2, 2, 5, 5-tetrafluoro-1, 6-bis (hydroxymethyl) bicyclopropane has thus been accomplished. Carbon radicals from allyl O-trimethylsilyl-α-bromo-α, α-difluoroacetal can cyclize onto the olefinic part regiospecifically to give γ-lactols in good yield. The lactols are then converted to the corresponding α, α-difluoro-γ-lactones. Systematic synthesis of multifluorinated-α, α-difluoro-γ-lactones has been accomplished through intramolecular radical cyclization as a key reaction. The synthesis of difluorinated analogue of an insect sex pheromone has been accomplished through intramolecular radical cyclization and lipase-catalyzed reaction.

ChemInform Abstract: Synthesis of Optically Active Partly gem-Difluorinated Allylic Alcohols via [2,3]-Wittig Rearrangements and Lipase-Catalyzed Reaction.

[2,3]-Wittig rearrangement of 1,1,2-trifluoroallylic ethers gave five types of novel 4,4,5-trifluroalk-1,5-dien-3-ols. The rearrangement reaction gave the alcohols with perfect (E)*-selection over the newly created olefin bond for two substrates. Lipase-catalyzed optical resolution of 4,4,5-trifluoroalk-1,5-dien-3-ols was successfully performed to afford optically active partly gem-difluorinated allylic alcohols for the first time.

Study of ultrasound-promoted, lipase-catalyzed synthesis of fructose ester

The effect of low energy ultrasound in biochemistry and biotechnology has attracted great attention in recent years. It can enhance substrate dissolution and improve mass transfer within and outside of a cell, both of which are beneficial to the synthesis of fructose ester. Here we describe the experimental study of the effect of ultrasounds of different intensity on the lipase-catalyzed synthesis of fructose ester in the solvent butanone. The results were compared with control reactions performed with no ultrasound. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) were used for qualitative and quantitative analyses. The results show the following: 1) the concentration of mono-ester and diester increased with the reaction time, either with or without ultrasonic irradiation. Low energy ultrasound accelerated the reaction due to the effect of ultrasonic steady cavitations, and high energy ultrasound was not beneficial to the reaction. 2) The application of ultrasound played an important role in our lipase-catalyzed reaction. It decreased reaction time as compared to a reaction without ultrasound that resulted in the same yield, increased reaction rate, and enhanced the amount of fructose ester produced. When the frequency was 10 kHz and sound intensity was 0.16 W·m−2, the concentration of ester was twofold more than without ultrasonic irradiation after a reaction time of up to 12 h. With the proper ultrasonic parameters, the overall concentration of production appeared to increase exponentially with the reaction time. 3) Ultrasound had little effect on the initial reaction rate, and continuous ultrasonic irradiation was favorable for the reaction. The longer the reaction continued, the more obvious the effects of the ultrasound became in our experiments.

Investigation of Lipase-Catalyzed Biodiesel Production Using Ionic Liquid [BMIM][PF6] as a Co-solvent in 500 mL Jacketed Conical and Shake Flask Reactors Using Triolein or Waste Canola Oil as Substrates

The production of biodiesel was investigated using a lipase-catalyzed (Novozym 435) reaction involving methyl acetate and ionic liquid [BMIM][PF6] as a co-solvent. The use of a lipase catalyst removed the need for alkali wastewater removal and treatment, and the methyl acetate and ionic liquid helped prevent deactivation and improve activity and stability of the lipase, respectively. Experiments were carried out in shake flask and jacketed conical reactors. Runs performed in shake flask reactor produced similar fatty acid methyl ester (FAME) yields to previous work using a small-scale reactor (83%), indicating that the reaction can achieve high yields provided that sufficient mixing between the oil and ionic liquid phases occur. The highest yield achieved in the conical reactor was 54% because of the relatively poor mixing in the reactor as a result of two phases present in the mixture. An additional benefit of using ionic liquid as a co-solvent in the process was for its ease of separation of products. Two distinct phases were present at the end of the reaction, with the ionic liquid phase containing the triacetylglycerol byproduct and any unreacted methyl acetate. The byproduct was then separated by washing with water, and the ionic liquid was removed by decantation and reused. FAMEs were also successfully produced from waste canola oil, with a 72% yield achieved in a small-scale reactor and a 30% yield in a 500 mL jacketed conical reactor.

A new mathematical method for determining the enantiomeric ratio in lipase-catalyzed reactions

We present a new mathematical method for determining the enantiomeric ratio ( E) during lipase-catalyzed kinetic resolutions. The method involves the fitting of a model to profiles of adimensionalized concentrations of the two enantiomers of the chiral substrate, plotted against degree of conversion. The model equations are presented for a reversible reaction involving bi-bi ping-pong kinetics in which the chiral substrate enters second and the chiral product leaves second. However, it is also shown that the method is easy to modify for analysis of resolutions involving other chiral substrate-product pairs and of resolutions in which the behavior of the system can be approximated by irreversible uni-uni kinetics. We show that our method retains several advantageous features of existing methods that help to ensure accuracy.

Production of trans‐free margarine stock by enzymatic interesterification of rice bran oil, palm stearin and coconut oil

Trans-free interesterified fat was produced for possible usage as a spreadable margarine stock. Rice bran oil, palm stearin and coconut oil were used as substrates for lipase-catalyzed reaction. After interesterification, 137-150 g kg(-1) medium-chain fatty acid was incorporated into the triacylglycerol (TAG) of the interesterified fats. Solid fat contents at 25 degrees C were 15.5-34.2%, and slip melting point ranged from 27.5 to 34.3 degrees C. POP and PPP (beta-tending TAG) in palm stearin decreased after interesterification. X-ray diffraction analysis demonstrated that the interesterified fats contained mostly beta' polymorphic forms, which is a desirable property for margarines. The interesterified fats showed desirable physical properties and suitable crystal form (beta' polymorph) for possible use as a spreadable margarine stock. Therefore, our result suggested that the interesterified fat without trans fatty acid could be used as an alternative to partially hydrogenated fat.

Enrichment of pinolenic acid at the sn-2 position of triacylglycerol molecules through lipase-catalyzed reaction

Reports have shown that ∆-5 polyunsaturated fatty acids (∆-5 PUFA) are enriched at sn-1,3 positions of triacylglycerols (TAG) in pine (Pinus koraiensis) nut oil (Pn). As a major ∆-5 PUFA, pinolenic acid (Pi) is about 14.2% in the oil, while the percentage of Pi at the sn-1 and/or sn-3 positions in TAG was found more than 20%. In this current study, the enhancement of Pi at the sn-2 position has been achieved by acyl migration during the lipase-catalyzed inter-esterification between Pn and palm stearin (Ps). After reaction, the proportion of Pi increased at sn-2 positional fatty acid even is similar to that in total fatty acid; for example, in the inter-esterified product of 50:50 (Pn:Ps), the same amount of Pi (7.1%) present was detected both at the sn-2 and sn-1,3 positions. However, the reduction of phytosterols and tocopherols are observed in the inter-esterified products.

Study on acyl migration kinetics of partial glycerides: Dependence on temperature and water activity

Acyl migration phenomenon was often observed during 1,3-positional specificity lipase-catalyzed reactions from triglycerides and partial glycerides, including acyl migration of 1,2-diglyceride (1,2-DG) to 1,3-diglyceride (1,3-DG) and 2-monoglyceride (2-MG) to 1-monoglyceride (1-MG). However, the acyl migration mechanism and kinetics were seldom studied despite of numerous researches on process optimization of 1,3-positional specificity lipase-catalyzed reaction. In this paper, the influence of related factors on acyl migration process as well as their influencing mechanism was further studied. It was found that temperature and water activity were two crucial factors that would influence acyl migration kinetics. Determination of the kinetic parameters under different temperatures revealed that the acyl migration reaction rates were greatly promoted by the increasing of temperature. The acyl migration rates of 1,2-diglyceride and 2-monoglyceride were quite different from each other, which was found to be due to the different activation energies. Further study of how would water influence the acyl migration process showed that water activity rather than water content was a key factor that influenced acyl migration and the acyl migration rate would decrease with the increase of water activity. It was further revealed that water activity influenced the charge dispersion of the transition state, which ultimately influenced the reaction activation energy and then influenced the acyl migration rate.

대두유와 Glyceryl Monooleate의 효소적 반응을 이용한 Diacylglycerol 함유 유지의 생산

Scaled-up production of oil containing diacylglycerol (DAG), so called diacylglycerol-oil, was produced by lipase-catalyzed reaction. Mixture of soybean oil and glyceryl monooleate with 1:2 molar ratio was esterified with Lipozyme RMIM in a batch-type reactor at 55 o C and 300 rpm during 6 hr. After short-path distillation for removal of monoacylglycerol and free fatty acid as reaction by-products, diacylglycerol-oil mainly consisted of DAG (29 area%) and TAG (71 area%). The major compositional fatty acids in diacylglycerol-oil were oleic (44.36 wt%), and linoleic acids (37.36 wt%). Acid value and iodine value of diacylglycerol-oil were 0.13 and 112.6, respectively. Solid fat content (SFC) of diacylglycerol-oil was observed after differential scanning calorimetry (DSC) analysis in which three melting peaks at -25.0, 0.1, and 11.2 o C were shown.

Transesterification of oil mixtures catalyzed by microencapsulated cutinase in reversed micelles

Recombinant cutinase from Fusarium solani pisi was used to catalyze the transesterification reaction between a mixture of triglycerides (oils) and methanol in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the purposes of producing biodiesel. The use of a bi-phase lipase-catalyzed system brings advantages in terms of catalyst re-use and the control of water activity in the medium and around the enzyme micro-environment. Small-scale batch studies were performed to study the influence of the initial enzyme and alcohol concentrations, and the substrates molar ratio. Conversions in excess of 75 were obtained with reaction times under 24 h, which makes this enzymatic process highly competitive when compared to similar lipase catalyzed reactions for biodiesel production using methanol.

In situ monitoring of turbid immobilized lipase-catalyzed esterification of oleic acid using fiber-optic Raman spectroscopy

Raman spectroscopy with a fiber-optic probe was used to monitor the immobilized Candida antarctica lipase B (Novozym 435)-catalyzed esterification of oleic acid with ethanol in iso-octane as an organic solvent. The effects of reaction temperature and molar ratio of substrates were studied. An optimal reaction rate was found at 60 °C, beyond which deactivation due to denaturing of the lipase was observed. The variation in molar ratio of substrates suggests that the esterification of oleic acid and ethanol proceeds via a Ping-Pong Bi-Bi mechanism with ethanol exhibiting an inhibitory effect. A good fit was obtained between the experimental results and the best-fit Ping-Pong Bi-Bi mechanism. This current work shows that fiber-optic Raman spectroscopy is indeed a suitable instrument to monitor immobilized lipase-catalyzed reaction in turbid organic systems in situ. Since this approach is reliable, simple to use, allows automatic data acquisition, and accurate, it should be applicable to the detailed kinetic analysis on other immobilized enzymatic reactions as well.

Hydroxy functional acrylate and methacrylate monomers prepared via lipase—catalyzed transacylation reactions

Candida antarctica lipase B (CAL-B, Novozyme 435) catalyzes the transacylation of methyl acrylate and methyl methacrylate with diols and triols in 2-methyl-2-butanol at 50 °C. Under the experimental conditions, up to 70 mol% of the acyl donor methyl acrylate was converted. Methyl methacrylate is the less efficient acyl donor (up to 60 mol%) due to the higher sterical hindrance in the enzymatic transacylation. Under the reaction conditions high yields of the mono-acylated products are obtained, which contain minor amounts of bis(meth)acrylates. In addition it was observed that Novozyme 435 catalyzes regioselectively the acylation of the primary hydroxyl groups. In comparison with the chemical catalyzed route no selectivity was observed for unsubstituted diols. For substituted diols more mono-acylated product was formed in the lipase-catalyzed reaction than in the chemical catalyzed reaction.

Separation of citalopram intermediate from its acetate by solvent extraction

A new method to separate citalopram intermediate (diol), one of chiral aromatic amino-alcohols, from its acetate after lipase-catalyzed kinetic resolution using liquid–liquid extraction was developed. Diol was firstly derivatized with anhydrides and then extracted with different solvent systems. The effects of pH and initial diol acetate concentration on the total distribution coefficients of diol acetate were investigated. It was found that succinic anhydride was the best derivative agent and water/toluene was the optimal extraction system. The extraction conditions with initial diol acetate concentration of 10 mmol/L, pH 8.0 were applied to the practical extraction process of the mixture of diol and diol acetate obtained by lipase-catalyzed reaction. After three extraction stages at 25 °C, the purity of diol was 99.5% and the yield achieved 86.2%.

Practical synthesis of 1,3-oleoyl 2-docosahexaenoylglycerol by lipase-catalyzed reactions: An evaluation of different reaction routes

Three new synthetic routes were critically evaluated for the lipase-catalyzed production of 1,3-oleoyl-2 docosahexaenoylglycerol (ODO) in relatively large-scale (approximately 200 g). First, the production of 1,3-diolein by the reaction of glycerol and oleic acid followed by incorporation of docosahexaenoic (DHA) ethyl ester at the sn-2 position was studied. 1,3-Diolein was produced in 68.3% and 84.6% yield when stoichiometric amounts of the substrates were reacted at 25 °C for 8 h in the presence of 10% Novozym 435 and Lipozyme RM IM, respectively. Further increase in reaction temperature and time led to decrease in the 1,3-diolein yield. However, only a 9.4% yield of triacylglycerol was obtained in the subsequent reaction step when the 1,3-diolein was reacted with DHA ethyl ester in the presence of Novozym 435. Secondly, the feasibility of direct acidolysis was studied. Acidolysis of single cell oil (SCO) in excess oleic acid using Novozym 435 as the catalyst occurred twice as fast in solvent (tert-butanol) compared to a solvent-free system, and 63% oleic acid was incorporated into SCO. However, the regio-isomeric purity of the product was poor. Finally, the ethanolysis of SCO to produce DHA-enriched 2-monoacylglycerol followed by esterification with oleic acid or ethyl oleate was investigated. ODO was obtained in 50.9% regio-purity by Lipozyme RM IM-catalyzed esterification. The latter method was the most feasible for preparing ODO in large-scale. This synthetic route could be adapted for related triacylglycerols containing highly polyunsaturated when their productions in large-scale and high regio-purity are required.

A Versatile GC Method for the Analysis of Alkylglycerols and Other Neutral Lipid Classes

The present study focuses on the GC analysis of alkylglycerols and other neutral lipid classes. For that matter, a simple and rapid method based on the direct on-column injection has been developed for the simultaneous analysis of numerous fatty samples from different sources. Chemical transformation or treatment of the sample before the injection of samples is not required. In addition, quantification of different lipid classes was also evaluated with satisfactory results. Excellent results regarding reproducibility and resolution were observed. This method can be utilized for multiple purposes such as analyses of edible oils, shark liver oils, deodorizer distillates, time course analyses of lipase-catalyzed reactions, etc.

イオン液体を反応媒体とする酵素反応の新展開

The use of ionic liquids (ILs) to replace organic or aqueous solvents in biocatalysis processes has recently gained much attention and great progress has been accomplished in this area. This paper reviews our recent developments in the application of ionic liquids as solvents in enzymatic reactions. (1) The lipase recyclable use system has been accomplished using an ionic liquid solvent system. However, the reaction rate was gradually dropped with reputation of the process due to inhibitory action of acetaldehyde oligomer accumulated in the IL when vinyl acetate was used as acyl donor. This problem was solved when the reaction was carried out under reduced pressure conditions using methyl phenylthioacetate as acyl donor; there was no drop in the reaction rate despite five repetitions of the process in the [bmim][PF6] solvent system. We next attempted to evaluate the imidazolium salts again to see if they were suitable for recycling use of the enzyme using vinyl acetate as acyl donor. 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate ([bdmim][BF4]) was thus found to be an excellent solvent to realize a perfect lipase-recycling system: no accumulation of an acetaldehyde oligomer was observed in this solvent system and it was possible to use the lipase repeatedly ten times while still maintaining perfect enantioselectivity and high reactivity. (2) Lipase PS from Burkholderia cepacia is one of the most widely used enzymes applicable for various substrates, however, the activity in non-aqueous media is reduced and it has been reported that the enantioselectivity is significantly dependent on the solvent system. A remarkable acceleration was accomplished by the alkylPEG sulfate imidazolium salt (IL1) coating with lipase PS in an i-Pr2O solvent system while maintaining excellent enantioselectivity: ca. 500- to 1,000-fold acceleration was recorded for some substrates with excellent enantioselectivity. Furthermore, we discovered that (R)-pyrrolidine-substituted imidazolium cetyl-PEG10-sulfate (D-ProMe) derived from D-proline worked as more excellent activating agent of lipase PS. (3) It is generally recognized that lipase-catalyzed reaction in an ionic liquid solvent proceeded slower than those in many conventional organic solvents. To overcome this problem, attempts have been made to evolve ionic liquids appropriate for lipase-catalyzed reaction. Very rapid lipase PS-catalyzed transesterification of secondary alcohols was achieved using 2-methoxyethyl(tri-n-butyl)phosphonium bis(trifluoromethanesulfonyl)imide (TU-P1) as solvent and afforded the first example of a reaction rate superior to that in diisopropyl ether.

Continuous syntheses of ferulic acid esters by lipase at elevated temperatures

Ionic liquids (ILs) - as environmentally friendly “green” solvents- have been progressively used in various reactions as reagents, solvents and co-solvents including lipase-catalysed reactions. In fact, lipase-catalysed reactions in ILs are considered as a “green”-reaction and are more advantageous than chemical methods owing to the easy recovery of the product and the mild conditions of the reactions. The use of lipase in ILs provides many technological advantages compared to conventionally used solvents, such as selectivity enhancement, enzyme stability improvement, higher conversion rate, and better recyclability and recovery system. Nevertheless, in some cases, especially in hydrophilic ILs, lipase exhibits activity reduction when compared with organic solvents. Currently, various attempts have been made to enhance the performance of lipases in ILs. The main objective of this review is to demonstrate recent developments in the technology of using ILs as reaction media for lipase. It is expected that this review might be an inspiration in ILs assisted lipase-catalysed reactions to produce value-added materials including biofuels as well as biodiesel.

Synthesis of a novel fatty amide derivative of 7,10-dihydroxy-8(E)-octadecenoic acid by lipase-catalyzed amidation reaction

Synthesis of a novel fatty amide derivative of 7,10-dihydroxy-8(E)-octadecenoic acid by lipase-catalyzed amidation reaction

Lipase-catalyzed reactions at interfaces of two-phase systems and microemulsions.

This work describes the influence of two polar lipids, Sn-1/3 and Sn-2 monopalmitin, on the activity of lipase in biphasic systems and in microemulsions. In previous communications, we have shown that Sn-2 monoglycerides can replace Sn-1,3 regiospecific lipases at the oil–water interface, causing a drastically reduced rate of lipolysis. We here demonstrate that even if the lipase is expelled from the interface, it can catalyze esterification of the Sn-2 monoglyceride with fatty acids in both macroscopic oil–water systems and in microemulsions, leading to formation of di- and triglycerides.