The effect of monoalkyl phosphates and fluoride on dissolution of hydroxyapatite, and interactions with saliva.

The aims were to investigate the effect of monoalkyl phosphates (MAPs) and fluoride on dissolution rate of native and saliva-coated hydroxyapatite (HA). Fluoride at 300 mg/l (as NaF) inhibited dissolution of native HA by 12%, while potassium and sodium dodecyl phosphates (PDP, SDP), at 0.1% or higher, inhibited dissolution by 26-34%. MAPs, but not fluoride, also showed persistence of action. MAPs at 0.5% and fluoride at 300 mg/l were then tested separately against HA pre-treated with human saliva for 2 or 18 h. Agents were applied with brushing to half the specimens, and without brushing to the other half. In control (water-treated) specimens, pre-treatment of HA with human saliva reduced dissolution rate on average by 41% (2 h) and 63% (18 h). Brushing did not have a statistically significant effect on dissolution rate of saliva-coated specimens. In brushed specimens, fluoride significantly increased the inhibition due to 2- or 18-hour saliva pre-treatment. It is hypothesised that brushing partially removes the salivary film and allows KOH-soluble calcium fluoride formation at the surfaces of HA particles. Inhibition was reduced by PDP in 2-hour/non-brushed specimens and in 18-hour/brushed specimens. PDP did not affect dissolution rates in the remaining groups and SDP did not affect dissolution rate in any group. Possible reasons for these variable results are discussed. The experiments show that pre-treatment with saliva can significantly modify results of tests on potential anti-erosive agents and it is recommended that saliva pre-treatment should be a routine part of testing such agents.

Inhibition of erosive dissolution by sodium fluoride: evidence for a dose-response.

OBJECTIVES: The aim of this study was to investigate the protective effects of sodium fluoride solutions and commercial mouthrinses on hydroxyapatite (HA) dissolution in citric acid in vitro, with and without a salivary pellicle. METHODS: A rapid-throughput HA solubility-reduction model was employed in which HA dissolution was quantified using ion chromatography. Two HA substrates were selected, a high-resolution powder and 80 µm diameter beads, and studied in the presence and absence of a salivary pellicle (pooled human saliva, 2 h). Immediately prior to acid exposure, substrates were exposed to one of a number of pre-treatments that included aqueous fluoride (F(-)) solutions and commercially available mouthrinses with F(-) concentrations of 0-450 µg/g (as NaF). Dissolution reduction was calculated relative to a deionised water negative control. RESULTS: For aqueous solutions and mouthrinses, a fluoride dose-response was observed with a plateau around 100 µg/g F(-) for both HA substrates, with or without pellicle. Concentrations as low as 10 µg/g F(-) significantly reduced HA dissolution. The HA substrate had little impact on the fluoride dose-response, and the fluoride was equally effective in the presence of a pellicle as in its absence. CONCLUSIONS: Fluoride significantly reduced HA dissolution at concentrations of 10 µg/g and higher. A fluoride dose-response was seen at low concentrations. This study illustrates the use of a powerful rapid-throughput HA solubility-reduction model for investigating HA dissolution in citric acid in the presence of dissolution inhibitors. CLINICAL SIGNIFICANCE: A single exposure to fluoride solutions with fluoride concentrations and exposure time representative of brushing or rinsing with mainstream oral care products was shown to significantly inhibit HA dissolution under conditions relevant to dental erosion. A similar efficacy was observed in the presence and absence of salivary pellicle.

Inhibition of enamel erosion and promotion of lesion rehardening by fluoride: a white light interferometry and microindentation study.

OBJECTIVE: The primary aim of the present in vitro studies was to investigate fluoride as an inhibitor of citric acid-mediated demineralization of human enamel and promoter of lesion repair using a combination of white light interferometry, scanning electron microscopy, and microindentation. Secondary aims included investigation of the importance of brushing on bulk tissue loss, and comparison of the relative efficacy of commercially available toothpastes on inhibiting enamel surface softening and rehardening of incipient erosive lesions. METHODS: Resin-mounted polished enamel specimens were prepared from extracted human molars and pre-molars. Mean surface roughness (Sa) and bulk tissue loss following exposure to an erosive challenge, or an erosive challenge plus brushing were investigated using a MicroXAM ADE PhaseShift white light interferometer. Surface morphology was determined using a Zeiss Evo 50 scanning electron microscope (SEM). The utility of fluoride-based treatments to protect against subsequent acid demineralization and to promote remineralization of pre-formed incipient lesions was determined using microindentation-based enamel surface softening and enamel lesion rehardening models. RESULTS: Treating human enamel specimens with Sensodyne Pronamel conferred a clear protective benefit against a subsequent 300-second citric acid challenge as evidenced by the interferometry and SEM data. The increase in Sa and bulk tissue loss caused by an erosive challenge followed by brushing was markedly reduced by pre-treatment with sodium fluoride (NaF) in a concentration-dependent manner. Sensodyne Pronamel statistically outperformed Colgate Sensitive Enamel Protect both in the enamel surface softening model and lesion rehardening model, and conferred statistically superior enamel fluoride uptake. Treatment of erosive lesions with Sensodyne Pronamel resulted in statistically superior rehardening versus two Crest Pro-Health formulations containing stannous fluoride (SnF2) and sodium hexametaphosphate (NaHMP); the latter did not differ significantly from the fluoride-free negative control paste. Sensodyne Pronamel exhibited statistically significant superiority in a human saliva-based lesion rehardening model compared to Zendium Sensitive containing nominally comparable concentrations of NaF, as well as Colgate Sensitive and Colgate Sensitive Multi Protection containing sodium monofluorophosphate (NaMFP). CONCLUSION: The utility of NaF, whether delivered from simple solution or toothpaste, to reduce citric acid-mediated surface roughening and bulk tissue loss has been clearly demonstrated. The effectiveness of Sensodyne Pronamel as an anti-erosion toothpaste has also been demonstrated in various microhardness models. Crest Pro-Health toothpastes containing SnF2 and NaHMP were not statistically differentiable from a fluoride-free control paste in the lesion rehardening model. The latter result indicates that the benefit of fluoride to promote mineral formation is outweighed by the effect of NaHMP as a mineralization inhibitor in this model.

Fluoride penetration from toothpastes into incipient enamel erosive lesions investigated using dynamic secondary ion mass spectrometry.

OBJECTIVE: The primary aim of this study was to assess the utility of dynamic secondary ion mass spectrometry (DSIMS) as a convenient and sensitive technique for determining fluoride uptake and distribution into incipient human enamel erosive lesions in vitro. A secondary aim was to correlate the extent of lesion rehardening following treatment with a toothpaste slurry, with relative fluoride uptake determined by DSIMS. The final aim was to compare fluoride uptake by incipient lesions treated with toothpastes containing different sources of fluoride using DSIMS. METHODS: Relative fluoride uptake into the surface and body of enamel erosive lesions was monitored by DSIMS as a function of fluoride concentration in a series of formulation-matched experimental pastes. Fluoride uptake into lesions that had been subjected to treatment with different toothpaste slurries in a single-treatment enamel lesion rehardening model was also determined, and its relationship with regard to the extent of rehardening and also the fluoride source investigated. RESULTS: Fluoride uptake by incipient erosive lesions treated with toothpastes containing NaF was quantitatively compared by DSIMS and found to be directly proportional to the fluoride concentration over the studied range (0-1400 ppm). Lesion repair observed in a single-treatment lesion rehardening model was positively correlated with the extent of fluoride uptake by the treated lesions. DSIMS was also able to show differences between commercial toothpastes containing different sources of fluoride and their ability to deliver the fluoride into the body of the lesion. The detrimental effect of sodium hexametaphosphate (NaHMP) present in Crest Pro-Health formulations previously reported in the single-treatment lesion rehardening model was also evident from the DSIMS elemental line scans obtained from the lesion cross-sections. CONCLUSION: DSIMS has been shown to be a powerful selective technique for quantifying relative fluoride uptake into enamel erosive lesions, and determining the extent and depth of lesion penetration. The relative efficacy of toothpastes containing fluoride from a variety of sources in the single-treatment lesion rehardening study is positively correlated with fluoride uptake and penetration determined by DSIMS.

In vitro studies on the effect of sodium tripolyphosphate on the interactions of stain and salivary protein with hydroxyapatite.

OBJECTIVES: To study properties of sodium tripolyphosphate (STP) relevant to inhibition or removal of dental stain in vitro. METHODS: The effects of STP and other phosphates on adsorption of a dietary chromogen (black tea polyphenol) and salivary protein to hydroxyapatite (HA) powder were studied by analysing loss of protein or tea stain from solutions mixed with HA or HA pre-treated with the test agents. The effects on desorption of protein and stain from HA were studied by analysis of water or solutions of test agents mixed with HA or HA pre-treated with saliva or tea solution. RESULTS: At concentrations and pH representative of those likely to occur in the mouth, STP inhibited adsorption of salivary protein and black tea polyphenol to, and desorbed these substances from, HA surfaces. Adsorption and desorption of protein and stain were not influenced by pH of the STP solutions but adsorption varied with concentration. STP showed equivalent effectiveness with respect to salivary protein adsorption and desorption as a longer-chain condensed phosphate. The inhibitory activity of HA-bound STP on adsorption of salivary protein and stain resisted extensive washing. CONCLUSIONS: STP is likely to be an effective agent for inhibiting and removing dental stain, whether bound directly to mineralised surfaces or indirectly via salivary pellicle.

Salivary proteins interact with dietary constituents to modulate tooth staining.

Dietary components rich in polyphenols-for example, tea and red wine-are thought to cause tooth staining. In the present study, hydroxyapatite was used as a model of enamel for study of the influence of salivary proteins on the binding of different polyphenols to hydroxyapatite in vitro. Neither salivary protein pellicles nor salivary proteins in solution significantly altered the binding of the small polyphenol epigallocatechin to hydroxyapatite. However, hydroxyapatite binding of anthocyanin, a small grape-skin-derived polyphenol, or the larger polyphenols of black tea was increased by the presence of salivary proteins, either as a pellicle or in solution. Proline-rich proteins were enriched from parotid saliva and found to increase binding of anthocyanin and black tea polyphenols to hydroxyapatite, while enriched histatins did not increase binding. It is concluded that some salivary proteins, including proline-rich protein, can mediate increased staining of enamel by red-wine- and black-tea-derived polyphenols.

Real-time monitoring of stain formation and removal on calcium hydroxyapatite surfaces using quartz crystal sensor technology.

Stain formation, stain inhibition and stain removal may be monitored in real-time using a novel method employing a quartz crystal resonance sensor (QCR), based upon quartz crystal microbalance (QCM) technologies. Crystalline hydroxyapatite (HA) surfaces were prepared on phosphate-terminated, polymer-modified gold surfaces of quartz crystal transducers. The resulting sensors were placed in a specially constructed flow cell, and the interaction of adsorbates from the tea stain solution monitored as a function of time. The ability of sodium tripolyphosphate (STP) to remove extrinsic stain and also to inhibit its formation was examined. The adsorption of material from the staining solution passed over the sensor was clearly observable, and once bound, the crystal based real-time data suggest that tea adsorbates were not removed in the absence of an active under conditions of continuous flow. STP was shown to rapidly remove existing stain, and exhibited a clear inhibitory action on stain formation irrespective of whether the HA had been previously exposed to tea chromogens. The continuous data generated by the QCR technique were in good agreement with the results obtained using a discontinuous spectrophotometric method. The presently described quartz crystal model for extrinsic dental stain should provide a valuable tool to aid understanding of the interactions of staining agents with a crystalline HA surface as a model tooth surface, and to evaluate the efficacy and mode of action of STP and putative stain removal agents.

A direct-staining method to evaluate the mucoadhesion of polymers from aqueous dispersion.

A novel technique to evaluate polymer adhesion to human buccal cells following exposure to aqueous polymer dispersion, both in vitro and in vivo, is described. Adhering polymer has been visualised by staining with 0.1% (w/v) of either Alcian blue (60 min) or Eosin (10 min) solution, uncomplexed dye being removed by 0.25 M sucrose washings. The extent of polymer adhesion was quantified by measuring the relative staining intensity of control and polymer-treated cells by image analysis. In vitro, Carbopol 974P, polycarbophil (Noveon AA-1) and chitosan (CL 113) were found to adhere to human buccal cells from 0.10% (w/w) aqueous dispersions of these polymers. Following in vivo administration as a mouthwash, these polymers persisted upon the human buccal mucosa for at least 1 h.

Microemulsion-based media as novel drug delivery systems.

Microemulsions are clear, stable, isotropic mixtures of oil, water and surfactant, frequently in combination with a cosurfactant. These systems are currently of interest to the pharmaceutical scientist because of their considerable potential to act as drug delivery vehicles by incorporating a wide range of drug molecules. In order to appreciate the potential of microemulsions as delivery vehicles, this review gives an overview of the formation and phase behaviour and characterization of microemulsions. The use of microemulsions and closely related microemulsion-based systems as drug delivery vehicles is reviewed, with particular emphasis being placed on recent developments and future directions.

Gelatin-stabilised microemulsion-based organogels: rheology and application in iontophoretic transdermal drug delivery.

Gelatin-containing microemulsion-based organogels (MBGs) have been formulated using pharmaceutically acceptable surfactants and oils such as Tween 85 and isopropyl myristate. MBG formulations were subject to rheological study and their utility in transdermal drug delivery examined. Unlike most organogels, MBGs are electrically conducting and have been successfully employed in this study for the iontophoretic delivery of a model drug through excised pig skin. Iontophoresis using MBGs gave substantially higher release rates for sodium salicylate compared to passive diffusion, and fluxes were proportional to the drug loading and the current density. MBGs provide a convenient means of immobilising the drug and are rheologically similar to their hydrogel counterparts at comparable gelatin concentrations. MBGs also appear to offer improved microbial resistance in comparison to aqueous solution or hydrogels.

The multicomponent automated dissolution system: an alternative in the development and pharmaceutical analysis of generic polydrugs.

The necessity of assuring the quality of polydrugs, especially those with low aqueous solubility and in vivo absorption, has led to the development and evaluation of new techniques that can reduce the time and cost of analysis. This study examines the efficiency and accuracy of an automated dissolution system, fitted with an integrated multicomponent detector, for analysis of generic polydrugs using multiple linear regression (MLR). Trimethoprim and sulphamethoxazole were chosen as model drugs for this study and comparison was made with a conventional analysis based on HPLC. Both analytical systems under study gave reproducible and accurate results. Analysis of variance showed that there was no significant statistical difference between the methods of analysis, nor any statistical difference between the measured amounts of drug in the three different formulations. We have demonstrated that low cost instrumentation coupled with MLR data processing provides entirely satisfactory drug analysis to standard at least as good as that achieved using HPLC and provides an opportunity to reduce the time and analysis cost of other generic formulations.

Kinetic studies of Chromobacterium viscosum lipase in AOT water in oil microemulsions and gelatin microemulsion-based organogels.

Kinetic studies have shown that octyl decanoate synthesis by Chromobacterium viscosum (CV) lipase in sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping-pong) bi bi mechanism. There was evidence of single substrate inhibition by decanoic acid at high concentrations. Initial rate data yielded estimates for acid and alcohol Michaelis constants of ca. 10(-1) mol dm(-3) and a maximum rate under saturation conditions of ca. 10(-3) mol dm(-3) s(-1) for a lipase concentration of 0.36 mg cm(-3). CV lipase immobilized in AOT microemulsion-based organogels (MBGs) was also found to catalyze the synthesis of octyl decanoate according to the ping-pong bi bi mechanism. Reaction rates were similar in the free and immobilized systems under comparable conditions. Initial rates at saturating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions and the MBG/oil systems. Gradients yielded an apparent k(cat) = 4.4 x 10(-4) mol g(-1) s(-1) in the case of w/o microemulsions, and 6.1 x 10(-4) mol g(-1) s(-1) for CV lipase immobilized in the MBGs. A third system comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, provided a useful link between the conventional liquid microemulsion medium and the solid organogels. The nongelation of these intermediate systems stems from the early inclusion of substrate during a modified preparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a prerequisite for MBG formation. FT-NMR was employed as a semicontinuous in situ assay procedure. The apparent activity expressed by CV lipase in compositionally equivalent liquid and solid phase gelatin-containing systems was similar. An apparent activation energy of 24 +/- 2 kJ mol(-1) was determined by (1)H-NMR for esterification in gelatin-containing w/o microemulsions. This value agrees with previous determinations for CV lipase-catalyzed synthesis of octyl decanoate in "conventional" w/o microemulsions and MBG/oil systems. The similarities in lipase behavior are consistent with the claim, based largely on structural measurements, that the physico-chemical properties of the lipase-containing w/o microemulsion are to a large extent preserved on transformation to the daughter organogel. The close agreement of apparrent activation energies suggests that substrate mass transfer is not rate determining in the three studied systems.

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.

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity.

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value.

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: I. Effect of pH and water content on reaction kinetics.

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies.

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%.

Esterification reactions catalyzed by Chromobacterium viscosum lipase in CTAB-based microemulsion systems.

Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been used for multigram-scale ester synthesis, including the kinetic resolution of a secondary alcohol. The stability of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of ethylhexadecanoate as a model reaction. Under pseudo-first-order conditions, the synthesis rates were linearlydependent on the enzyme and fatty acid concentrations and the R dependence shows the characteristic bell-shaped curve (where R = [H(2)O]/[surfactant]). The dependence of enzyme activity toward octyldecanoate synthesis on the pH of the dispersed buffer phase is in marked contrast to that observed for the pH dependence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the former case, the pH-activity profile is approximately sigmoidal, which may reflect the ionization state of the fatty acid substrate. In the latter case, the pH dependence is minimal at both R = 10 and R = 50, suggesting the enzyme does not experience a changed pH environment. Inclusion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected buffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the esterification reactions, thereby providing an additional means of determining initial rates. The method may be of general value for lipase assays in microemulsions since it may provide, at the same time, information regarding enzyme regioselectivity. (c) 1995 John Wiley & Sons, Inc.

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.