Lipase made active in hydrophobic media

Enzymes are distinguished from other catalysts by their high substrate specificity. This is a great asset when one wants to apply them for syntheses of various compounds. Their usage, however, generally is limited to hydrophilic reaction media, because they usually are not soluble and active in hydrophobic media. Recently, we have been able to make various enzymes soluble and active in highly hydrophobic organic solvents. The key to this success is the chemical modification of enzymes with an amphipathic synthetic polymer, polyethylene glycol. The activated polymers can be attached to enzymes in aqueous buffer solutions, and once enzymes are modified they become soluble and active in various organic solvents such as benzene, toluene and cholorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalyzed the reverse reaction of hydrolysis in organic solvents. The modified lipase catalyzed various ester synthesis reactions. Because the reactions were conducted in the pure solvent system, it also was possible to study the kinetics and the substrate specificity for ester synthesis reaction. It also catalyzed the polymerization of a hydroxy group containing carboxylic acid due to the bifunctional nature. The modified lipase catalyzed ester exchange between an ester and an alcohol, between an ester and a carboxylic acid and between two esters in organic solvents. When the two substrates for ester exchange were liquid, the reaction could take place without organic solvents. The modified lipase catalyzed an ester exchange reaction between trilaurin and triolein when dissolved in these substrates. Dilauroyl-monooleoylglycerol and monolauroyl-dioleoyl-glycerol were formed from these two substrates in the presence of the modified lipase. The modified enzyme was extremely thermostable in its substrates. In the ester synthesis and ester exchange reactions, a trace amount of water was necessary for expression of the enzymic activity. It is suggested that the amphipathic polymer molecules retained water in close proximity to the enzyme. Presented at the symposium “The Biology, Biochemistry and Technology of Lipase” at the 78th annual meeting of the American Oil Chemists’ Society held May 17–21, 1987, in New Orleans, Louisiana.     

Lipase immobilization on ionic liquid‐modified magnetic nanoparticles: Ionic liquids controlled esters hydrolysis at oil–water interface

Ester hydrolysis at oil–water interface by lipase covalently immobilized on ionic liquid‐modified magnetic nanoparticles was investigated. Magnetic supports with a diameter of 10–15 nm were synthesized by covalent binding of ionic liquids (chain length C₄ and C₈ and anions Cl^?, BF₄^?, and PF₆^?) on the surface of Fe₃O₄ nanoparticles. Lipase was covalently immobilized on Fe₃O₄ nanoparticles using ionic liquids as the coupling reagent. Ionic liquid‐modified magnetic nanoparticle‐grafted lipase preferentially located at the oil–water interface. It has higher catalytic activity than its native counterpart. A modified Michaelis–Menten model was used to elucidate the effect of stirring rate, aqueous–organic phase ratio, total amount of enzyme, and ester chain length. The influences of these conditions on esters hydrolysis at oil–water interface were consistent with the introduction of the ionic liquids interlayer. Ionic liquids could be used to control the oil–water interfacial characteristics during lipase catalyzed hydrolysis, and thus control the behavior of immobilized lipase. © 2011 American Institute of Chemical Engineers AIChE J, 2012     

A kinetic model for enzymatic reactions in reverse micellar systems involving water‐insoluble substrates and enzyme activators

The activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase‐catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (k_D) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (K_m) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model. Copyright © 2003 Society of Chemical Industry     

Selective Ethanolysis of Fish Oil Catalyzed by Immobilized Lipases

Selective ethanolysis of fish oil was catalyzed by immobilized lipases and their derivatives in organic media. Lipases from Candida antarctica B (CALB), Thermomyces lanuginosa (TLL) and Rhizomucor miehei (RML) were studied. The three lipases were immobilized by anion exchange and hydrophobic adsorption. The discrimination between the ethyl ester of eicosapentaenoic acid (EE-EPA) and the ethyl ester of docosahexaenoic acid (EE-DHA) depends on the lipase, the immobilization support, the physico-chemical modifications of the immobilized lipase derivatives and on the solvents used. TLL and RML were much more selective than CALB. EE-EPA is released 20-fold faster than EE-DHA when ethanolysis was catalyzed, in cyclohexane, by TLL hydrophobically adsorbed on Sepabeads C18. The selectivity and stability of the different derivatives in these polar organic solvents were further improved after physico-chemical modification. The best results for activity-selectivity-stability were obtained in cyclohexane for TLL adsorbed on Sepabeads C18 and further modified via solid-phase physical modification with a polyethylenimine polymer. In this case, the initial selectivity was higher than 20, and a 80 % of EPA was released as ethyl ester after 3 h at 25 °C. At this conversion, mixtures of ethyl esters highly enriched in the ethyl ester of EPA with less than 5 % of the EE-DHA were obtained. TLL derivatives remained fully active after incubation for 24 h in anhydrous solvents.     

Enzymatic alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

High‐pressure enzymatic hydrolysis of oil

The hydrolysis of sunflower and soybean oil, catalyzed by two enzymes, non‐immobilized Candida rugosa and immobilized Candida antarctica lipase, was performed at atmospheric and high‐pressure. The results showed that at atmospheric pressure between 40 °C and 60 °C initial reaction rates were influenced by the temperature variation, as expected. Due to favorable physico‐chemical properties of dense gases as reaction media, hydrolysis of soybean oil was performed in non‐conventional solvents: in supercritical (SC) CO₂ and near‐critical propane. In SC CO₂ the activity of non‐immobilized Candida rugosa lipase decreased while the reaction rates of hydrolysis catalyzed by immobilized Candida antarctica lipase were 1.5‐fold higher than at atmospheric pressure. However, the reaction rates for the hydrolyses catalyzed by both lipases, were much higher in propane than at atmospheric pressure.     

Polyethylene glycol modification ofCandida rugosa lipase

Polyethylene glycol (PEG) was covalently attached to lipase (EC 3.1.1.3) fromCandida rugosa yielding a modified lipase of higher specific activity in hydrolytic and synthetic reactions in organic solvents. PEG of molecular weights 5000 and 1900 solubilized the lipase in selected organic solvents, but PEG of molecular weight 750 was too small to accomplish this completely. The modified lipase was 10 times more stable in water than native lipase, but was less stable in benzene. The selectivity of the modified lipase was also altered to favor reaction with oleic versus stearic acid.     

Michaelis–Menten kinetics for enzymatic depolymerization of a linear polyester synthesized from Z‐protected glutamic acid

A biodegradable polyester resin was polymerized from N‐benzyloxycarbonyl‐L ‐glutamic acid and ethylene glycol. Rhizopus delemar lipase was used as a biocatalyst for the rupture of ester bonds during the hydrolysis studies. Depolymerization was observed to follow a Michaelis–Menten mechanism, with the maximum rate of monomer formation dP/dt_max = 1.12 × 10⁻⁸ mol/s and the rate constant K_m × 2.03 × 10⁻⁴ mol. Subject to initial conditions described by the most probable distribution and Michaelis–Menten–type depolymerization rate expressions, population density distribution dynamics of the polymeric molecules that formed the resin were explicitly described using a deterministic approach. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 514–520, 2001     

Dynamic enzymatic resolution of Naproxen methyl ester in a membrane bioreactor

A lipase‐catalyzed enantioselective continuous hydrolysis process under in situ racemization of substrate using sodium hydroxide as catalyst was developed for the production of (S)‐Naproxen from racemic Naproxen methyl ester in an aqueous–organic biphase system. Use of a tubular silicone rubber membrane in the stirred tank reactor to separate the chemical catalytic racemization and biocatalytic resolution processes, served to avoid the key problem associated with conventional dynamic resolution, viz the incompatibility of in situ chemical racemization with the presence of a biocatalyst. To overcome product inhibition and to facilitate product recovery from the aqueous–organic emulsion containing substrate and lipase, a hydrophilic porous semipermeable membrane was used in the stirred tank reactor. Greater than 60% conversion of the racemate with an enantiomeric excess of product (ee_p) greater than 96% was obtained. In addition, transformation of Candida rugosa lipase (CRL) isoenzymes was observed in the reaction process. © 2001 Society of Chemical Industry     

Synthesis of fatty esters by polyethylene glycol-modified lipase

Monomethoxypolyethylene glycols (PEG) of molecular masses 1900 and 5000 were activated using p-nitrophenyl chloroformate to form PEG–nitrophenyl carbonates (activated PEG) with high yield (96–98%). The activated PEG was covalently attached to Candida rugosa lipase. Increasing the molar ratio of activated PEG to the enzyme increased the degree of lipase modification. These modified lipases exhibited specific ester synthesis activities on organic solvents compared with native lipase. The degree of activity enhancement depended on the size of activated PEG used and the degree of modification of the enzyme. Maximal activity was attained after exhaustive of modification. The effects of different solvents, reaction temperature, and fatty acids on the esterification activity and the stability of the modified enzyme were investigated. The optimum esterification temperature (40° C) and preference of fatty acids as acyl donors of the modified lipase were very similar to those of the native enzyme. The modified lipase exhibited higher activity non-polar solvents than in polar solvents, and showed higher temperature, solvent and storage stability then the native lipase.     

Double‐hydrophilic polyether antiscalant used as a crystal growth modifier of calcium scales in cooling‐water systems

In this study, we synthesized a novel double‐hydrophilic poly(ethylene glycol) (PEG)‐based crystal growth modifier polyethylene glycol double‐ester of maleic anhydride/ acrylic acid named PEGDMA/AA, whose structure was still linear but also had some differences from a traditional chelating linear polymer, in which the PEG segment was incorporated. The scale inhibition behavior of PEGDMA/AA was evaluated by means of a static scale inhibition method. As the polymerization degree of PEG_nDMA was 8 (n = 8), the maximum inhibitory toward calcium carbonate (CaCO₃) and calcium sulfate (CaSO₄) were 89.0 and 98.8% at dosage levels of 12 and 3 mg/L, respectively. Comparisons with other inhibitors were also carried out. Characterization of the CaCO₃ and CaSO₄ scales with scanning electron microscopy and transmission electron microscopy proved that great changes in the size and morphology of the calcium scales took place under the influence of PEG₈DMA/AA. X‐ray diffraction and diffraction patterns further confirmed the impact of PEG₈DMA/AA as a crystal growth modifier. The three supposed mechanisms, (1) chelating solubilization, (2) multilayer type of adsorption, and (3) electrostatic repulsion function, are also described in detail. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39792.     

Increased activity of Chromobacterium viscosum lipase in AOT reverse micelles in the presence of short chain methoxypolyethylene glycol

The activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane and AOT/Tween 85/isooctane reverse micelles was significantly increased by the addition of short chain methoxypolyethylene glycols (MPEGs), taking the hydrolysis of olive oil as a model reaction. The molecular weight of MPEG had a strong effect on the lipase activity, and MPEG of nominal molecular weight 550 was found to be the most effective. To optimize the factors affecting enzymatic hydrolysis of olive oil in reverse micellar systems containing MPEG 550, the effect of various parameters, such as W_o (molar ratio of water to surfactant), pH, ionic strength, surfactant concentration and temperature were investigated. A kinetic model considering the substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was also successfully used to understand the enzyme activity in the presence of MPEG 550. Both the Michaelis constant and the substrate adsorption equilibrium constant were obviously reduced as compared with those obtained in the simple AOT reverse micellar system. © 2001 Society of Chemical Industry     

A kinetic model for enzymatic reactions in a reverse micellar system,involving water-insoluble substrate

A kinetic model was proposed for enzymatic reactions in a reverse micellar system, involving a water-insoluble substrate. Though surfactant is one of the main structural components of reverse micelles, an increase in the surfactant concentrations affects the enzyme activity remarkably. A relationship between the enzyme activity and the surfactant concentration is discussed. In this study it was assumed that free substrate in the organic phase was in adsorption equilibrium with the surface of the micellar surfactant, and that the adsorption coefficient and the true K_m value (Michaelis constant) were independent of the surfactant concentration. The validity of this model was verified by data on the hydrolysis of olive oil, catalyzed by Chromobacterium viscosum lipase (Glycerol-ester hydrolase; EC 3.1.1.3) in an AOT/isooctane reverse micellar system. The activity value predicted by the model equation agreed well with the experimental data.     

Imprinted polymer/organo-smectite nanocomposites for paraoxon hydrolysis

We have used the imprinting technique to create a polymer/organo-smectite nanocomposite catalyst. The aim was preparing a synthetic enzyme which shows high selectivity for paraoxon hydrolysis. Paraoxon imprinted polymer/organo-smectite composites (PIPO) and non-imprinted polymer/organo-smectite composites (NIPO) were prepared using methacryloylhistidine-Cu²⁺ (MAH-Cu(II)) and [2-methacryloyloxy)ethyl]dimethylhexadecylammonium bromide] (MOEDMAC₁₆) modified smectite. These nanocomposites were applied as a catalyst in the hydrolysis of paraoxon which is an organophosphate ester and used as a pesticide. The catalytic activity of these composites was evaluated according to the enzyme kinetics model of Michaelis–Menten. PIPO showed better catalytic activity than NIPO. In addition, the paraoxon imprinted nanocomposite had greater catalytic activity (based on k_cat/K_m values) for paraoxon hydrolysis by a factor of 391 compared to that of parathion hydrolysis.     

Extraction of Candida rugosa lipase from aqueous solutions into organic solvents by forming an ion‐paired complex with AOT

Candida rugosa lipase was extracted from aqueous solutions into organic solvents by forming an ion‐paired complex with sodium bis(2‐ethylhexyl)sulfosuccinate (AOT). The optimal aqueous pH for lipase recovery was 4.5 and the optimal CaCl₂ concentration was 10 mmol dm^?3. The lipase recovery decreased with increasing aqueous enzyme concentration but increased with increasing AOT concentration in the organic phase. The presence of polar co‐solvents in the aqueous phase did not obviously improve the lipase recovery, which was also little influenced by the type of hydrophobic organic solvent used for solubilising AOT. Surprisingly, no detectable activity of the ion‐paired C. rugosa lipase was observed for both the esterification of lauric acid with 1‐propanol in isooctane and the hydrolysis of olive oil in isooctane containing an appropriate amount of water. The ion‐paired C. rugosa lipase mediated the enantioselective crystallisation of racemic ketoprofen in isooctane, indicating the feasibility of using it as a chiral mediator for the enantioseparation of hydrophobic racemic compounds in organic systems. Copyright © 2006 Society of Chemical Industry     

Immobilization of lipase onto metal–organic frameworks for enantioselective hydrolysis and transesterification

Enzyme immobilization enhances the catalytic activity and stability of the enzyme, and also improves reusability. Metal–organic frameworks (MOFs), which possess diversified structures and porosity, have been used as excellent carriers for enzyme immobilization. Pseudomonas fluorescens lipase (PFL) has been successfully immobilized onto MOFs by covalent cross-linking to obtain a series of immobilized lipase (PFL@MOFs). PFL@MOFs are used for catalytic enantioselective hydrolysis of 2-(4-hydroxyphenyl) propionic acid ethyl ester enantiomers (2-HPPAEE) in aqueous medium and transesterification of 4-methoxymandelic acid enantiomers (4-MMA) in organic medium. The experimental results indicated that PFL@Uio-66(Zr) exhibits excellent enzymatic catalysis performances and high enantioselectives. In addition, to improve catalytic activity and reusability, PFL is modified by the polyethylene glycol (PEG) to prepare PEG-modified lipase (PFL-PEG), then PFL-PEG is immobilized onto Uio-66(Zr) to prepare PFL-PEG@Uio-66(Zr), demonstrating better reusability and catalytic activity compared with PFL@Uio-66(Zr).     

Wax ester-synthesizing activity of lipases

The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, was ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium.     

Reaction engineering with enzymes: A relatively uncharted territory

This article discusses the history of enzyme kinetics developed by Michaelis and Menten, and recent work extending kinetics for enzyme‐catalyzed reactions in organic solvents. Based on kinetic studies of the transesterification of vinyl methacrylate with 2‐hydroxyethyl acrylate catalyzed by Candida antarctica lipase B, a new model is proposed that resembles the kinetic model of controlled/living polymerizations governed by dynamic equilibrium of active and dormant species. Experimental data indicates that by judicious selection of reaction conditions steady‐state conditions can be achieved and very clean products with quantitative conversion can be produced. © 2016 American Institute of Chemical Engineers AIChE J, 63: 266–272, 2017     

Solvent effect on the lower critical solution temperature of biodegradable thermosensitive poly(organophosphazenes)

Summary The solvent effect on the lower critical solution temperature (LCST) of poly(organophosphazenes) with methoxy-poly(ethylene glycol) (MPEG) and amino acid esters as side groups was examined in terms of the structure of polyphosphazenes in aqueous solutions containing one of the organic solvents selected from monoalcohols, ethylene glycol derivatives, alkylamines, and other common solvents. When such a solvent was added to the aqueous solutions of the polymers, their LCST was found to be mainly dependent on the hydrophobic and hydrophilic properties of the solvents. Most of the alcohols and amines with shorter alkyl chains increased the LCST of the polymers but those with longer chains decreased the LCST. Trifluoroethanol (TFE) showed a strong LCST decreasing effect in spite of its short chain, which seems to be due to its strong hydrophobicity. Temperature-induced molecular weight fractionation of the polymer bearing MPEG350 (M _w= 350) and L-aspartic acid ethyl ester as a side group was carried out by using the LCST decreasing effect of TFE, and the fractionated samples were characterized by gel permeation chromatography (GPC) and ¹H- and ³¹P NMR spectroscopies. Thus it has been shown that a polymer may be fractionated to the higher and lower molecular weight fractions with smaller polydispersity indices (PDI): the polymer with the weight-average molecular weight (M _w) of 73,500 with PDI of 5.56 was fractionated to those of 106,000 with PDI of 4.37 and 11,000 with PDI of 1.86. Received: 8 September 2000/Revised version: 6 November 2000/Accepted: 9 November 2000     

Fabrication of amphiphilic copolymeric gels with enhanced activity of immobilized enzymes in organic media

Novel amphiphilic copolymeric gels were developed to immobilize lipase. NIPA‐co‐PEGMEA gels were prepared by copolymerizing N‐isopropylacrylamide (NIPA) as a thermosensitive and amphiphilic component and poly(ethylene glycol) methyl ether acrylate (PEGMEA) as a hydrophilic component in aqueous media. The gels can absorb organic solvents at temperatures higher than the lower critical solution temperature owing to the thermosensitive and amphiphilic properties of poly(NIPA). The lipase immobilized within the NIPA‐co‐PEGMEA gel, which had a NIPA : PEGMEA composition of 950 : 50 mol/m³, successfully catalyzed the esterification of oleic acid and ethanol without loss of activity during repeated use within 20–40°C. The activity of the immobilized lipase was considerably higher than that of free lipase. The NIPA‐co‐PEGMEA gels provide a structure that allows the immobilized lipase to work actively in an aqueous environment and with the dispersed state of the lipase in the gels. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41905.