Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic (liquid/solid) media

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (a_w = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high a_w (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration. The mass of the enzyme preparation was shown to be a parameter affecting the capacity of the lipase to produce enough water in its immediate environment. The lack of activity observed for a small quantity of enzyme was eliminated by addition of heat-denaturated lipase.     

Intramolecular esterification by lipase powder in microaqueous benzene: effect of moisture content

In view of the biochemical reaction catalyzed by enzyme powder suspended in a water-insoluble organic solvent, an equation was derived to estimate the amount of water bound to the enzyme powder. With this equation, an apparent adsorption isotherm between free water (water freely dissolved in benzene) and bound water (water bound to crude lipase powder of Pseudomonas fluorescens) was obtained. A direct lactonization reaction (synthesis of cyclopentadenolide from 15-hydroxypen-tadecanoic acid) catalyzed by crude lipase powder of Pseudomonas fluorescens was carried out batchwise in microaqueous benzene at 40oC. A kinetic model of the enzymatic reversible lactonization reaction was derived, from which the effect of moisture content on the initial reaction rate with a fully hydrated enzyme was mathematically expressed. The observed initial reaction rate first increased, then decreased with increasing moisture content, giving rise to the maximum rate at a certain level of the moisture content. The drop in the reaction rate at lower moisture content was due to a lesser hydration of the enzyme molecule (hydration-limited) and the decrease in the reaction rate at higher moisture content was attributed to the dependence of the true initial rate of the reversible reaction on the moisture content (true reversible reaction limited), and could be simulated by the kinetic model. The equilibrium yield approached 100% at a lower moisture content.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

Modification of flavonoid using lipase in non-conventional media: effect of the water content

The enzymatic acylation of a flavonoid (naringin) was investigated in this work. This atypic substrate for a lipase was esterified very selectively by the immobilized Candida antarctica lipase: a single product was synthesized and was assumed to be the 6-O-palmitate naringin ester acylated on the glucose moiety. As lipase-catalyzed esterification reactions in organic media are greatly influenced by the water content, the effect of the initial hydration level of the reaction medium components was pointed out for naringin palmitate synthesis. 2-Methyl 2-butanol (solvent) and naringin (acyl acceptor) provided high amounts of water and when dried increased the conversion yield by 63% and the specific activity by 60%. On the contrary, the enzyme must not be dried because water is essential for the three-dimensional structure of the protein and, if absent, results in a 67% loss of activity. As water was produced in parallel to ester synthesis, the equilibrium of the reaction might be shifted by its removal. When the reaction was carried out with 100 g l(-1) molecular sieves 4A added after 24 h of reaction, a conversion yield of 43% was reached after 55 h reaction.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

Enzymatic ester synthesis with continuous measurement of water activity

Ester synthesis from aliphatic monoalcohols and organic acids was investigated by using a microbial lipase. The reaction medium only contained the substrates and the enzyme without addition of water or organic solvent. During the reaction, water was produced and the water activity (aw) increased. Batch reactors and continuous-flow reactors were used. In batch, the aw was 0.13 at the beginning of the reaction and increased to reach a plateau at 0.77, after which ester synthesis continued without modification of the aw. Different alcohols and acids were tried in solid-liquid reactors, and all cases synthesis occurred, leading to a significant increase in the water activity. For continuous-flow reactors, the use of silica beads retaining water inside the reactor where the enzymatic reaction took place resulted in some control of the enzymatic reaction by changing the aw.     

Characteristics of lipase-catalyzed hydrolysis of olive oil in AOT-isooctane reversed micelles

Candida rugosa lipase solubilized in organic solvents in the presence of both surfactant and water could catalyze the hydrolysis of triglycerides, and kinetic analysis of the lipase-catalyzed reaction was found to be possible in this system. Among eight organic solvents tested, isooctane was most effective for the hydrolysis of olive oil in reversed micelles. Temperature effect, pH profile, K(m,app) and V(max,app) were determined. Among various chemical compounds, Cu(2+), Hg(2+), and Fe(3+) inhibited lipase severely. But the enzyme activity was restorable partially by adding histidine or glycine to the system containing these metal ions. The enzyme activity was dependent on R (molar ratio of water to surfactant) and maximum activity was obtained at R = 10.5. Upon addition of glycerol to the reversed micelles, lipase activity was affected in a different fashion depending on the R values. Stability of the lipase in reversed micelles was also dependent on R, and it was most stable at R = 5.5.     

Biodiesel production using Aspergillus niger as a whole‐cell biocatalyst in a packed‐bed reactor

Enzymatic lipase transesterification of palm oil to biodiesel in a packed‐bed reactor (PBR) using a novel strain of the fungus Aspergillus niger, immobilized within polyurethane biomass support particles (BSPs), was investigated. A three‐step addition of methanol was used to reduce lipase inhibition by immiscible methanol. The influence of water content and PBR flow rate was investigated. FAME yield was enhanced with an increase of PBR flow rate in the range of 0.15–30 L h^?1, where inefficient mixing of the reaction mixture at lower flow rates resulted in low conversion rates i.e. 69% after 72‐h reaction. Adding the third mole equivalent of methanol resulted in lipase inhibition due to methanol migration into the accumulated glycerol layer. Glutaraldehyde (GA) solution (0.5 vol.%) was used to stabilize lipase activity, which led to a high FAME yield (>90%) in the PBR after 72‐h of reaction time at a flow rate of 15 L h^?1, and a water content of 15%. Moreover, a high conversion rate (>85%) was maintained after four palm oil batch conversion cycles in the PBR. In contrast, lipase activity of non‐GA‐treated cells decreased with each PBR batch cycle, where only 70% FAME was produced after the forth PBR cycle. Transesterification of palm oil in a PBR system using BSPs‐immobilized A. niger as a whole‐cell biocatalyst is a viable process for enzymatic biodiesel production.     

Lipases have similar water activity profiles in different reactions

The shape of the profiles of enzyme activity versus water activity for four different lipases were independent of the reaction used to determine the activity. The profile for each lipase (Rhizopus arrhizus, Pseudomonas sp., Candida rugosa and Lipozyme) in esterification, hydrolysis and transesterifications profiles were the same. In transesterification the yield was unaffected by the water activity but the hydrolysis yield increased with increasing aW .     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

The lipase-catalyzed hydrolysis of lutein diesters in non-aqueous media is favored at extremely low water activities

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Preparative scale enantioseparation of flurbiprofen by lipase-catalysed reaction

Effects of reaction media, alcohols and water on the enzyme activity of the immobilised Candida antarctica lipase were investigated for the separation of racemic flurbiprofen by an esterification reaction catalysed by immobilised enzyme in organic media. The S-enantiomer of flurbiprofen was directly resolved by the immobilised lipase esterification reaction in acetonitrile. Ping-Pong Bi–Bi kinetics were found to fit the initial reaction well of all the experimental runs. Model parameters for the reaction kinetics were evaluated from experiments at relatively low substrate concentrations, have shown to be applicable for preparative separation scale at high concentrations. Finally, the gram-scale production of single enantiomer with the optical purity of 93% e.e. was obtained.     

Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

微环境对脂肪酶催化拆分外消旋2－辛醇的影响

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