Salt Hydrate Pairs Research Articles

The Role of Water Activity in Terms of Enzyme Activity and Enantioselectivity during Enzymatic Esterification in Non-conventional Media

During enzymatic esterification in non-conventional media, the activity and enantioselectivity of the enzyme is significantly influenced by the water content of the reaction medium, which continuously changes as water is produced during the esterification. To provide constant reaction parameters, water activity should be kept constant. The commonly used salt hydrate pairs may be difficult to apply and often hinder enzyme activity. During the enantioselective esterification of racemic 2-bromopropionic acid in various solvents (organic solvents, ionic liquids), it was proven that the conditions related to the optimal water content required for kinetic examinations can be provided without using any salt or salt hydrate pairs. This conclusion is based on the realization that the optimal water activity can be set by first determining the initial water content that is necessary to achieve the maximum reaction rate in the given solvent.

Continuous Flow Bioamination of Ketones in Organic Solvents at Controlled Water Activity using Immobilized ω-Transaminases.

Compared with biocatalysis in aqueous media, the use of enzymes in neat organic solvents enables increased solubility of hydrophobic substrates and can lead to more favorable thermodynamic equilibria, avoidance of possible hydrolytic side reactions and easier product recovery. ω‐Transaminases from Arthrobacter sp. (AsR−ωTA) and Chromobacterium violaceum (Cv−ωTA) were immobilized on controlled porosity glass metal‐ion affinity beads (EziG) and applied in neat organic solvents for the amination of 1‐phenoxypropan‐2‐one with 2‐propylamine. The reaction system was investigated in terms of type of carrier material, organic solvents and reaction temperature. Optimal conditions were found with more hydrophobic carrier materials and toluene as reaction solvent. The system's water activity (aw) was controlled via salt hydrate pairs during both the biocatalyst immobilization step and the progress of the reaction in different non‐polar solvents. Notably, the two immobilized ωTAs displayed different optimal values of aw, namely 0.7 for EziG3−AsR−ωTA and 0.2 for EziG3−Cv−ωTA. In general, high catalytic activity was observed in various organic solvents even when a high substrate concentration (450–550 mM) and only one equivalent of 2‐propylamine were applied. Under batch conditions, a chemical turnover (TTN) above 13000 was obtained over four subsequent reaction cycles with the same batch of EziG‐immobilized ωTA. Finally, the applicability of the immobilized biocatalyst in neat organic solvents was further demonstrated in a continuous flow packed‐bed reactor. The flow reactor showed excellent performance without observable loss of enzymatic catalytic activity over several days of operation. In general, ca. 70% conversion was obtained in 72 hours using a 1.82 mL flow reactor and toluene as flow solvent, thus affording a space‐time yield of 1.99 g L−1 h−1. Conversion reached above 90% when the reaction was run up to 120 hours.

Enzyme-catalysed synthesis of plant steryl laurate in non-aqueous media using salt hydrate pairs and its characterisation

The objective of this work was to produce plant steryl esters with lipase-catalysed esterification using salt hydrate pairs in n-hexane and study its cholesterol-lowering properties. The optimum reaction conditions investigated were the lipase variety (Candida rugosa lipase), fatty acid variety (laurate acid), water activity (aw=0.764 [Na2SO4/Na2SO4·10H2O]), temperature (45°C), and enzyme load (10%). The following kinetic parameters were determined: Vmax=18.41mmol/min/mg; KA,acid=0.00407M; KB,sterol=0.00447M; KiA,acid=0.00205M; KiB,sterol=0.00865M. These data showed that the bioreaction followed the Michaelis–Menten equation with a Ping–Pong Bi–Bi mechanism. FT-IR and MS analyse were adopted to confirm the chemical structure of synthesized plant steryl laurate. Compared with plant sterols, the isolated plant steryl laurate had low melting temperature and higher solubility. The cholesterol-lowering property of plant steryl laurate was also evaluated and demonstrated to decrease serum total cholesterol and low-density lipoprotein cholesterol.

Thermodynamics of Inorganic Hydration and of Humidity Control, with an Extensive Database of Salt Hydrate Pairs

Water is ubiquitous, and its presence in the ambient humid air means that it may constitute an uncontrolled variable in chemical processes. Methods for humidity control may involve complete removal of water and its vapor by procedures such as evaporation under vacuum, use of drying agents such as silica gel, adjustment to a desired humidity by use of saturated aqueous solutions, or adjustment to a desired humidity by use of a pair of related salt hydrates (such as CuSO4·3H2O + CuSO4·5H2O). By the phase rule, the presence of three phases at a fixed temperature (in the latter two cases) ensures a constant equilibrium humidity. The thermodynamics of these chemical means of humidity control is presented, together with a database of almost 300 salt hydrate pairs which may be considered for humidity control by mixing into the reaction medium. However, the possibility of interaction of the hydrates with the reaction medium should not be neglected.

Enzymatic synthesis and application of fatty acid ascorbyl esters

Fatty acid ascorbyl esters are liposoluble substances that possess good antioxidative properties. These compounds could be synthesized by using various acyl donors for acylation of vitamin C in reaction catalyzed by chemical means or lipases. Enzymatic process is preferred since it is regioselective, performed under mild reaction conditions, with the obtained product being environmentally friendly. Polar organic solvents, ionic liquids, and supercritical fluids has been successfully used as a reaction medium, since commonly used solvents with high Log P values are inapplicable due to ascorbic acid high polarity. Acylation of vitamin C using fatty acids, their methyl-, ethyl-, and vinyl esters, as well as triglycerides has been performed, whereas application of the activated acyl donors enabled higher molar conversions. In each case, majority of authors reported that using excessive amount of the acyl donor had positive effect on yield of product. Furthermore, several strategies have been employed for shifting the equilibrium towards the product by water content control. These include adjusting the initial water activity by pre-equilibration of reaction mixture, enzyme preparation with water vapor of saturated salt solutions, and the removal of formed water by the addition of molecular sieves or salt hydrate pairs. The aim of this article is to provide a brief overview of the procedures described so far for the lipase-catalyzed synthesis of fatty acid ascorbyl esters with emphasis on the potential application in food, cosmetics, and pharmaceutics. Furthermore, it has been pointed out that the main obstacles for process commercialization are long reaction times, lack of adequate purification methods, and high costs of lipases. Thus, future challenges in this area are testing new catalysts, developing continuous processes for esters production, finding cheaper acyl donors and reaction mediums, as well as identifying standard procedures for purification of products which will not require consumption of large amounts of non-biocompatible organic solvents.

Control of water activity in lipase catalysed esterification of chiral alkanoic acids

Candida rugosa lipase (CRL) catalysed enantioselective esterification of racemic 2-methylhexanoic acid was performed with 1-decanol in organic solvent at constant water activity ( a w). The a w was maintained either by a salt hydrate pair added in the reaction mixture or by performing the reaction in an air tight desiccator over an aqueous saturated salt solution. It was found that the enatiomeric ratio and average reaction rate profiles were similar when controlling the water activity for the esterification reaction with these two different methods. But, in some cases the average rate of the reaction and the enantioselectivity of CRL was affected when ions from the salt hydrate pairs were present in the reaction mixture.

Improving ascorbyl oleate synthesis catalyzed by Candida antarctica lipase B in ionic liquids and water activity control by salt hydrates

Lipase catalyzed-synthesis of ascorbyl oleate was performed in ionic liquids with addition of salt hydrate pairs for water activity control. The highest yield in the synthesis of ascorbyl oleate (72%) was obtained in [BMIM][BF 4] in the presence of the salt pair NaI 2/0, which corresponds to a water activity of 0.3. Purity and chemical identity of 6- O-ascorbyl oleate was confirmed by 1H and 13C NMR analysis.

Effect of zeolites on lipase catalyzed esterification in nonaqueous media

We report on the effect of salt hydrate pairs and of zeolite NaA on the Novozym catalyzed esterification of geraniol with acetic acid in an organic solvent and in supercritical fluids. The zeolite was pre-equilibrated to the water activity ( a W) of the experiment to avoid drying effects. Medium conditions were selected to minimize changes in a W during the course of initial rate measurements. Highest enzyme activity was obtained in the presence of zeolite NaA at initial a W = 0.2. An organosoluble chromoionophoric indicator revealed that the salt hydrate pairs used to set initial a W = 0.1 or 0.7 were highly acidic when compared to the salt hydrate pair used to set a W = 0.2, and that zeolite NaA was even more basic than the latter, and able to noticeably make the medium less acidic in the presence of acetic acid. The impact of zeolite NaA is attributed to its ability to promote the formation of a more basic form of enzyme through ion exchange and thus facilitate the establishment of a formal negative charge on the enzyme active site, as required by the catalytic mechanism. This work highlights the dual role that zeolites may play in tuning a W and the ionization state of enzymes in nonaqueous media, also in esterification reactions.

Cutinase activity in supercritical and organic media: water activity, solvation and acid–base effects

We performed a comparative study on the activity of Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY, in n-hexane, acetonitrile, supercritical ethane (sc-ethane) and sc-CO 2, at two different water activity ( a W) values set by salt hydrate pairs in situ and at acid–base conditions fixed with solid-state buffers of aqueous p K a between 4.3 and 10.6. The reaction studied was the transesterification of vinyl butyrate by ( R,S)-2-phenyl-1-propanol. The transesterification activity of cutinase was highest and similar in sc-ethane and in n-hexane, about one order of magnitude lower in acetonitrile and even lower in sc-CO 2. Activity coefficients ( γ) generated for the two substrates indicated that they were better solvated in acetonitrile and thus less available for binding at the active site than in the other three solvents. γ data also suggested higher reaction rates in sc-ethane than in n-hexane, as observed, and provided evidence for a direct negative effect of sc-CO 2 on enzyme activity. Manipulation of the acid–base conditions of the media did not afford any improvement of the initial rates of transesterification relative to the blanks (no added acid–base buffer, only salt hydrate pair), except in the case of cutinase immobilized on zeolite NaA in sc-ethane at a W = 0.7. The poor performance of the blank in this case and the great improvement observed in the presence of a basic buffer suggest a deleterious acidic effect in the medium which, an experiment without additives confirmed, was not due to the known acidic character of the salt hydrate pair used to set a W = 0.7. In acetonitrile, increasing a W was accompanied by a decrease in initial rates of transesterification, unlike in the other solvents. There was considerable hydrolysis in acetonitrile, where initial rates of hydrolysis increased about 20-fold from a W = 0.2 to 0.7. Hydrolysis was less pronounced in sc-ethane and in n-hexane, and only at a W = 0.7, and in sc-CO 2 butyric acid was detected only at very long reaction times, in agreement with a generally low catalytic activity. Cutinase enantio-selectivity towards the alcohol substrate was low and unaffected by any manipulation of medium conditions.

Use of salt hydrate pairs to control water activity for enzyme catalysis in ionic liquids.

Salt hydrate pairs were used to control water activity in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate. It was shown that salt hydrate pairs behave essentially the same in ionic liquids as they do in organic solvents as long as they do not dissolve. Initial rate-water activity profiles were prepared for the immobilized Candida antarctica lipase catalyzed synthesis of 2-ethylhexyl methacrylate. The ability to use salt hydrate pairs for the control of water activity in ionic liquids should allow for improved comparison of enzyme activity and specificity in ionic liquids and conventional solvents.

Salt hydrates for in situ water activity control have acid-base effects on enzymes in nonaqueous media.

Salt hydrates very frequently are utilized as in situ water activity buffers in reaction mixtures of enzymes in nonaqueous media. In addition to buffering water activity, there is evidence that salt hydrates also often affect initial rates in other ways. This has been generally overlooked or thought to be related to water transfer effects. Here we show that salt hydrates can have important acid-base effects on enzymes in nonaqueous media. We performed transesterification reactions in n-hexane and in supercritical ethane catalyzed by cross-linked crystals of subtilisin, differing in the method used to set a(W), and confirmed that the presence of salt hydrate pairs significantly affected the catalytic performance of the enzyme. However, in the presence of a solid-state acid-base buffer, salt hydrates had no effect on enzymatic activity. Direct evidence for the acid-base effects of salt hydrates was obtained by testing their effect on the protonation state of an organo-soluble H(+)/Na(+) indicator. The four salt hydrate pairs tested affected the indicator to very different extents. By promoting the exchange of H(+) for Na(+), salt hydrates will tend to affect the ionization state of acidic residues in the protein and, hence, enzymatic activity. In fact, salt hydrates were able to affect the pH memory of subtilisin lyophilized from different aqueous pHs, bringing about up to 20-fold enhancements and up to 5-fold decreases in catalytic activity. The possibility of such acid-base effects need to be considered in all experiments using salt hydrates to control water activity.

Water Activity Effects on Geranyl Acetate Synthesis Catalyzed by Novozym in Supercritical Ethane and in Supercritical Carbon Dioxide

The esterification reaction of geraniol with acetic acid catalyzed by Novozym was studied in supercritical ethane (sc-ethane) and in supercritical carbon dioxide (sc-CO(2)). Water activity (a(W)) had a very strong effect on enzyme activity, with reaction rates increasing up to a(W) = 0.25 and then decreasing for higher a(W). Salt hydrate pairs could not prevent changes in a(W) during the course of reaction but were able to control a(W) to some extent and had a beneficial effect on both initial rates of esterification and conversion in sc-ethane. The enzyme was more active in sc-ethane than in sc-CO(2), confirming the deleterious effect of the latter already observed with some enzymes. Temperatures between 40 and 60 degrees C did not have a strong effect on initial rates of esterification, although reaction progress declined considerably in that temperature range. For the mixture of 50 mM acetic acid plus 200 mM geraniol, 100% conversion was achieved at a reaction time of 10 h at 40 degrees C, 100 bar, an a(W) of incubation of 0.25, and a Novozym concentration of 0.55 mg cm(-)(3) in sc-ethane. Conversion was below 50% in sc-CO(2) at otherwise identical conditions. With an equimolar mixture of the two substrates (100 mM), 98% conversion was reached at 10 h of reaction in sc-ethane (73% conversion in sc-CO(2)).

Effect of salt hydrate pairs for water activity control on lipase-catalyzed synthesis of lysophospholipids in a solvent-free system

For the synthesis of biologically useful lysophospholipids with a desirable fatty acid, an easy and simple synthetic approach was developed by lipase-catalyzed esterification of the glycerophosphoryl group with free fatty acid in a solvent-free system. Among the various parameters which govern the lysophospholipid synthesis, water activity (a w) was found to be an important factor. Salt hydrate pairs could act to buffer the optimal water level during the reaction. We investigated the effect of various salt hydrate pairs which were added directly into the reaction medium or put in the headspace of the reactor on the lysophospholipid synthesis in the solvent-free system. Optimal a w values for lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), and lysophosphatidylcholine (LPC) were 0.18, 0.37, and 0.60, respectively, and these were controlled by the pairs of NaI (2 H 2O-anhydrous), CH 3COONa (3 H 2O-anhydrous), Na 4P 2O 7 (10 H 2O-anhydrous), respectively. Control of a w was especially critical for LPE synthesis. When the a w of the reaction system was not controlled, LPE was hardly formed. The yield of LPA, LPE, and LPC at their optimal a w values after 60 h were 45.3, 22.9, and 36.2%, respectively.

Effect of salt hydrate pair on lipase-catalyzed regioselective monoacylation of sucrose

Sucrose monoesters of a fatty acid were synthesized by using lipase in a solvent-free system. When lipase from Mucor miehei was used as a catalyst with capric acid as the donor and sugar as the acceptor, sucrose 6-monocaprate was predominantly produced in a yield of 25.3%. The yield of product was significantly increased by the direct addition of a suitable pair of solid salt hydrates to the reaction mixture to control the water activity (aw). Among the salt hydrate pairs investigated, the barium hydroxide, 8/1H2O pair resulted in the highest yield of the product. This salt addition method was also successfully employed for acylation of primary hydroxyl groups in various unprotected mono- and disaccharides such as glucose, galactose, fructose, trehalose, mannose, maltose, and lactose.

Selection of salt hydrate pairs for use in water control in enzyme catalysis in organic solvents

The water activities (a(w)) of 13 salt hydrate pairs were determined from vapor pressure measurements; a(w) values for a subset were also estimated from a study of water transfer to isopropylether. The application of salt hydrates as water buffers was investigated in two models: (i) effect of hydration on the initial rate of subtilisincatalyzed transesterification of the nitrophenol ester of CBZ-alanine with butanol; and (ii) effect of hydrates on the equilibrium concentrations of reactants in the esterification of dodecanol and decanoic acid, catalyzed by lipase. Transfer of ions from salt to enzyme particles was also demonstrated. The implications of the results for the successful use of salt hydrates as water buffers are discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 367-374, 1997.

Selection of salt hydrate pairs for use in water control in enzyme catalysis in organic solvents

Selection of salt hydrate pairs for use in water control in enzyme catalysis in organic solvents

Twin-core packed-bed reactors for organic-phase enzymatic esterification with water activity control

A method for the removal of water and the control of water activity, aw, during enzymatic esterification is the use of salt hydrate pairs. When this technique is used on a laboratory scale, the recovery and reuse of the salt are not critical. Potential problems, such as the reactivity of some salts, can also be overcome simply by substituting another salt. However, if this technique is to be used on a larger scale, economic constraints would require salt recovery and restric the range of salts that could be used. In this article a twin-core packed-bed reactor — used for the esterification of an equimolar mixture of decanoic acid and dodecanol catalysed by lipase from Candida rugosa — which facilitates salt recovery and permits aw control without direct contact between immobilized enzyme and salt, has been described. aw control was maintained by using suitable salt hydrate mixtures in the inner core of the reactor. The substrate mixture was esterified by pumping it through the outer core of the reactor, which contained enzyme immobilized on a macroporous polypropylene support. Complete conversion, albeit at different rates, was obtained with aw buffering at 0.48 and 0.8 by using hydrates of Na4P2O7 and Na2HPO4.