Gareth D. Rees

REVERSE ENZYME SYNTHESIS IN MICROEMULSION-BASED ORGANO-GELS

Lipase from three different sources has been immobilised in microemulsion-based gels (MBGs) with retention of catalytic activity. Such lipase-containing MBGs prove to be novel solid-phase catalysts for use in apolar organic solvents such as n-heptane. Using these systems, preparative-scale synthesis of a wide variety of esters under mild conditions was possible with products easily isolated and obtained in high yield. Stereoselective esterification of octan-2-ol was observed for all three lipases with Chromobacterium viscosum (CV) lipase yielding product with an enantiomeric excess of 92%. Repeated usage of a CV lipase-containing MBG resulted in a visually unchanged gel whose activity was 75% of the initial value after 30 days. The sectioned MBGs were well suited for use in column flow reactors and were also found to be effective esterification catalysts at temperatures as low as -20 degrees C.

Biocatalysis Using Gelatin Microemulsion-based Organogels Containing Immobilized Chromobacterium viscosum Lipase

Chromobacterium viscosum (CV) lipase was immobilized in gelatin-containing Aerosol-OT (AOT) microemulsion-based organogels (MBGs). The behavior of this novel, predominantly hydrophobic matrix as an esterification catalyst has been examined. The biocatalyst was most effective when the MBG was granulated to yield gel particles of approximately 500 mum diameter, providing a total surface area of ca. 10(6) mm(2) per 10 cm(3) of gel. The gel was generally contacted with a solution of the substrate(s) in a hydrocarbon oil. Under most conditions reaction was not diffusion limited. Apparent lipase activity was influenced by certain compositional changes in the MBG, but most significantly when the R value, the mole ratio of water to surfactant, was altered. Higher activities were observed at lower R values. Although gels of lowest R value expressed the highest condensation activity, such formulations were physically unsuitable as immobilization matrices due to their proximity to the gel-solution phase boundary. MBGs of intermediate R values (between 60 and 80) were considered most suitable because they offer relatively high condensation activity and good physical stability. The gelatin concentration also exerted a small but measurable influence on the observed condensation rates. Apparent lipase activity was also influenced to some extent by the nature of the parent hydrocarbon used to prepare the MBG. Higher activities were obtained using formulations derived from isooctane and cyclohexane rather than the n-alkanes. Condensation activities expressed by CV lipase in the MBGs were broadly comparable to those expressed in the analogous parent water-in-oil (w/o) microemulsions. The MBGs functioned effectively in neat substrate solutions, but the condensation activity expressed by the MBGs in a series of successive batch syntheses was adversely affected by the formation and retention of the water coproduct. Selective removal of the water was achieved using a concentrated solution of dry reverse micelles, which resulted in recovery of lost activity. Pretreatment of lipase-containing MBGs resulted in the formation of MBGs with enhanced catalytic properties and modified composing the conventional procedure. (c) 1997 John Wiley & Sons, Inc.

Preparative-scale kinetic resolutions catalysed by microbial lipases immobilised in AOT-stabilised microemulsion-based organogels: cryoenzymology as a tool for improving enantioselectivity.

Gelatin-containing microemulsion based organogels have been used as an immobilisation matrix for lipases from a number of different sources. Kinetic resolutions of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by esterification with decanoic acid have been performed using Chromobacterium viscosum (CV) lipase. CV lipase is highly enantioselective in favour of the (R)-(-) isomer of octan-3-ol, but the enantioselectivity is both reversed and decreased by the introduction of unsaturation at the 1-position. Marked improvements in enantioselectivity were achieved by carrying out the reaction at -15 degrees C, the enantiomeric excess of the ester product increasing from 47% (E = 3) to 73% (E = 8) in the case of 1-octen-3-ol, and from 17% (E = 1.4) to 38% (E = 2.5) in the case of 1-octyn-3-ol. The enantiomeric excess was approximately 85% (E approximately 15) for octan-3-ol, and there was no marked improvement in enantioselectivity even at -15 degrees C. Apparent activation energies for the esterification using decanoic acid of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by CV lipase were 32 kJ mol-1, 31 kJ mol-1 and 41 kJ mol-1, respectively. This compares to an activation energy of 21 kJ mol-1 for the esterification of octan-1-ol with decanoic acid using CV lipase under the same conditions. Lipases from Pseudomonas (Fluka), Pseudomonas (Genzyme) and lipoprotein lipase ex Microbial (Genzyme) also selectively esterified the (R)-(-) isomer of racemic octan-3-ol, the two Pseudomonas preparations yielding product with an enantiomeric excess of 90%. Candida cylindracea lipase did not exhibit activity in gelatin-containing MBGs. Large-scale syntheses were performed in a 1 dm3 batch reactor in which 200 cm3 of pelleted MBG (containing 350 mg of CV lipase) was used repeatedly for the kinetic resolution of octan-3-ol.

Macrocyclic lactone synthesis by lipases in water-in-oil microemulsions

Five microbial lipases from Chromobacterium viscosum, Candida cylindracea, Pseudomonas (source Fluka), Pseudomonas (source Genzyme) and lipoprotein lipase ex Microbial (Genzyme) have been screened for lactonisation activity towards 16-hydroxyhexadecanoic acid (HHA) in a variety of different w/o microemulsion systems. With the exception of Candida cylindracea (CC), all the lipases exhibited lactonisation activity although they were inherently more active in microemulsion systems based on the anionic surfactant sodium bis(2-ethylhexyl)sulphosuccinate (AOT) than in those based on the cationic surfactant cetyltrimethylammonium bromide (CTAB). Lactone yields are typically 50-60% and are markedly better than those reported previously using microemulsions in combination with chemical catalysts. Lipase stability is superior in the CTAB microemulsion systems, while lipase stability in the low water content AOT microemulsion systems was still good with the exception of CC lipase, which is rapidly inactivated. Buffering the water pools of AOT microemulsions using diglycine buffer at pH 8.0 improved biocatalyst stability. The lactonisation activity of lipases in CTAB w/o microemulsion systems compares favourably with that obtained using the same preparations as a solid suspension in the corresponding water-saturated organic solvent. In addition, the unusual solubility properties of microemulsions allowed the use of considerably higher concentrations of substrate in the microemulsion systems as compared to water-saturated organic solvents such as n-heptane. Lactone yields obtained at equivalent concentrations in the corresponding organic solvents containing conventional condensation catalysts were consistently measured at approx. 10%.

Phospholipid-stabilised water-in-oil microemulsions: A study of ester synthesis by Humicola lanuginosa lipase

In a model reaction, octyl decanoate was synthesised from n-octanol and decanoic acid using Humicola lanuginosa lipase entrapped in hydrated lecithin reversed micelles in n-heptane. The surfactants used were pure phosphatidylcholine (PC) from egg yolk, a partially purified lecithin preparation from dried egg yolk (EY-PC) containing about 60% PC, and two from soybean (SB-PC), one containing about 40% PC the other containing about 48% PC. Reaction took place readily in all systems. The initial rate of reaction in the different phospholipid systems was compared to a similar system stabilised by sodium bis (2-ethylhexyl) sulphosuccinate (AOT) and was superior in every case. The rate of ester formation was higher in the systems stabilised by soybean lecithin compared to the system stabilised by pure phosphatidylcholine. For the system stabilised by pure PC containing 700 mM water, phase separation into an emulsion occurred at an ester concentration of around 75 mM using initial substrte concentrations of 100 mM. In contrast, the systems stabilised by the other lecithin preparations were stable microemulsions at reaction equilibrium (about 95% conversion). The effects of enzyme concentration, initial water concentration, water activity, surfactant concentration, initial substrate concentration and temperature were studied in a system stabilised by soybean lecithin. The initial rate of reaction was proportional to the enzyme concentration, and was broadly independent of the surfactant concentration. Esterification activity tended to zero at very low water activity (aw) and water content. Maximal esterification activity was expressed above ω0 ≈ 10 (ω0 = [water][PC]) where aw≈0.8. An apparent activation energy of 20 ± 2 kJ mol−1 was determined over the temperature range 10–60°C, which is consistent with previous determinations for lipases obtained using alternative microemulsion systems stabilised by synthetic surfactants such as AOT.

LIPASE-CATALYSED INTERFACIAL REACTIONS IN REVERSE MICELLAR SYSTEMS : ROLE OF WATER AND MICROENVIRONMENT IN DETERMINING ENZYME ACTIVITY OR DORMANCY

Studies of the effect of water content on the equilibrium position of a lipase-catalysed hydrolysis/esterification reaction in an AOT reverse micellar system show that the extent of reaction is apparently independent of ωo, the mole ratio of water to surfactant, over the studied range (2–40). Near-IR spectroscopy is shown to be a useful technique for the in situ monitoring of hydrolysis and synthesis reactions in reverse micelle–water-in-oil (w/o) microemulsion systems, and the methods applied are applicable to other low water systems. The hydrolysis and esterification activity of lipases tends to zero at low ωo where the thermodynamic water activity 1. It is shown that a state of reversible enzyme dormancy can be induced by dehydrating the enzyme-containing w/o microemulsion. Molecular sieve beads were considered unsuitable for this purpose because in addition to water, AOT and lipase were also adsorbed from the microemulsion. Molecular sieve powder was more effective, rapidly dehydrating w/o microemulsions whilst leaving AOT and most of the lipase in solution. Drying through vapour-phase equilibration of the microemulsion with a saturated salt solution in a closed system allowed the water activity to be precisely controlled. Although relatively slow, vapour phase drying is ‘clean’ in that it is specific only to water. AOT reverse micellar media containing dormant Chromobacterium viscosum lipase can be stored for at least a month with the lipase activity immediately regenerated on rehydration, with good recovery of catalytic activity. This phenomenon may have application in the food industry for the instant biogeneration of aroma and flavour compounds.

A near infrared assay for lipolysis in lecithin-stabilised water-in-oil microemulsions

A near infrared (N-IR) spectroscopic assay has been developed for in situ monitoring of lipolysis in water-in-oil (w/o) microemulsions stabilised by soybean lecithin. Water, fatty acid and partial glyceride concentrations may be individually determined. N-IR has general utility in the study of hydrolysis/condensation reactions in low-water organic media.