Real time measurement and control of thermodynamic water activities for enzymatic catalysis in hexane

The esterification reaction of geraniol with acetic acid catalyzed by immobilized Candida antarctica lipase B was studied in hexane using a pervaporation-assisted batch reactor. The effect of thermodynamic water activity (a(w)) on the initial reaction rate was investigated at a(w) ranging from 0.02 to 1.0. The a(w) was monitored on-line in real time. a(w) was actively controlled throughout the reaction by using highly water-selective membrane pervaporation. This novel combination of a(w) sensing and control eliminates changes in a(w) during the reaction even in the initial phase of relatively rapid water release during an esterification. No chemicals are introduced for a(w) control, and no purge gases or liquids are needed. A maximum in the initial reaction rate was found approximately at a(w)=0.1. The initial reaction rate declined quickly at higher a(w), and dropped precipitously at lower a(w).     

Computer-aided real-time estimation of reaction conversion for lipase-catalyzed esterification in solvent-free systems

Real-time conversion estimation through macroscopic balancing was investigated for enzymatic esterification reactions in a solvent-free system. In principle, the conversion of ester synthesis can be determined from the amount of water produced by the reaction because water is formed as a by-product in the same molar ratio as the product. In this study, we show that the water production rate, and thereby the reaction conversion, can be estimated on-line from measurements of the relative humidity of the inlet and outlet air and the material balances of water in the system. In order to test the performance of the real-time conversion estimation method, the lipase-catalyzed esterification reaction of n-capric acid and n-decyl alcohol in solvent-free media was conducted while controlling the water activity at various values. When the reaction conversions estimated on-line were compared with those analyzed off-line by gas chromatography, good agreement was obtained: the average mean absolute error was +/- 2.4% of the reaction conversion despite the simplicity of the method. The on-line estimation method presented here requires no expensive or complicated analytical instruments and no sampling of reaction medium. It can be used for monitoring nonaqueous enzymatic reactions where water is produced or consumed during reaction.     

A water activity control system for enzymatic reactions in organic media

A water activity control system for enzymatic synthesis in organic media, for litre-scale reactors has been constructed. Water activity, a(w), is a key factor when using enzymes in non-conventional media and the optimum value varies for different enzymes. The control system consists of a water activity sensor in the headspace of a jacketed glass reactor (equipped with narrow steel tubes to introduce air), gas-washing bottles containing blue silica gel (a(w)=0) and water (a(w)=1), a PC to monitor water activity and a programmable logic controller (PLC) to control the water activity. The system was evaluated by adjusting water activity in the medium, with a deviation from the set point of less than +/-0.05. Synthesis of cetyl palmitate, under controlled water activity and catalysed by two different lipase preparations, namely, Novozym 435 (immobilised Candida antarctica lipase B) and immobilised Candida rugosa lipase, were also performed. Novozym 435 catalyses reactions very well at extremely low water activity while C. rugosa lipase shows low activity for a(w)<0.5.     

On-line conversion estimation for solvent-free enzymatic esterification systems with water activity control

On-line conversion estimation of enzymatic esterification reactions in solvent-free media was investigated. In principle, conversion to ester can be determined from the amount of water produced by the reaction, because water is formed as a by-product in a stoichiometric manner. In this study, we estimated the water production rate only from some measurements of relative humidity and water balances without using any analytical methods. In order to test the performance of the on-line conversion estimation, the lipase-catalyzed esterification ofn-capric acid andn-decyl alcohol in solvent-free media was performed whilst controlling water activity at various values. The reaction conversions estimated on-line were similar to those determined by offline gas chromatographic analysis. However, when the water activity was controlled at higher values, discrepancies between the estimated conversion values and the measured values became significant. The deviation was found to be due to the inaccurate measurement of the water content in the reaction medium during the initial stages of the reaction. Using a digital filter, we were able to improve the accuracy of the on-line conversion estimation method considerably. Despite the simplicity of this method, the on-line estimated conversions were in good agreement with the off-line measured values.     

Prokaryotic aminopeptidase activity along a continuous salinity gradient in a hypersaline coastal lagoon (the Coorong, South Australia)

The relation between climatic parameters (relative air humidity) and the water activity of the Urmia Sea water determines the possible maximum evaporation of the lake. Using the Pitzer thermodynamic approach, the activity of the Urmia Lake water during evaporation was calculated and compared to the present relative air humidity above the water. Present climatic conditions allow the Urmia Sea water to evaporate down to water with activity of 0.55, corresponding to the lowest air humidity measured over the lake. This water activity falls in the range of halite precipitation, while carnalite precipitation starts at somewhat lower (a H₂O = 0.499) point. Our dynamic model predicts that for air humidity as low as 55% (reflecting present climate conditions), the Urmia Sea level may drop to as low as 1270 m (i. e., 1270 m above mean sea level). At that point, the lake water volume will have a volume of 11 km³. For the sake of comparison, at the beginning of 1990, the level of the lake was 1275 m, its volume was 25 km³, and its surface area was 5145 km².     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

有机相中脂肪酶L—1754促有机硅烷醇的酯化

在有机介质中脂肪酶Ｌ－１７５４催化非天然有机硅化合物三甲基硅烷醇酯化反应的速度高于Ｌｉｐｏｚｙｍｅ。Ｌ－１７５４对脂肪酸链长有很强的特异性且不同于Ｌｉｐｏｚｙｍｅ。     

Water activity control: a way to improve the efficiency of continuous lipase esterification

During continuous lipase-catalyzed oleic acid esterification by ethanol in n-hexane, the oleic acid conversion, initially at 95%, decreases to 20% after 2 h. This decrease is caused by the accumulation of the water produced in the course of the reaction in the packed-bed reactor (PBR). In order to improve the PBR efficiency, it is necessary to evacuate the water produced. In this study, different approaches have been tested to control the water content in the PBR during continuous esterification. The first approach consisted in improving the water solubility by increasing the reaction medium polarity. The addition of polar additives to n-hexane, the use of more polar solvents, and the use of solvent-free reaction medium were tested as a means to favor the water evacuation from the PBR. First of all, the use ofn-hexane supplemented with acetone (3 M) or 2-methyl-2-propanol (1 M) enabled the conversion to be maintained at higher values than those obtained in pure n-hexane. The replacement of n-hexane by a more polar solvent, like the 5-methyl-2-hexanone, resulted in the same effect. In all cases, conversions at steady-state were always less than 95%, as obtained in pure n-hexane. This is explained by a decrease in the enzyme activity due to the increase in the medium polarity. Nevertheless, an increase in enzyme quantity allowed 90% conversion to be maintained during 1 week using 3 M acetone amended n-hexane. Good results (a steady-state conversion of about 80%) were obtained when esterification was carried out in a solvent-free reaction medium containing 2 M 2-methyl-2-propanol as a polar additive. The second approach consisted in the evaporation of the accumulated water by use of an intermittent airflow. Although this process did not enable constant esterification rate to be maintained, it did enable the initial conversion (95%) to be restored intermittently.     

Continuous control of water activity during biocatalysis in organic media

Summary This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Water activity dependence of performance of surface-displayed lipase in yeast cells: a unique water requirement for enzymatic synthetic reaction in organic media

Water activity (a(w)) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the a(w) dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing a(w) of the salt solution, to give the best performance at a(w) of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique a(w) profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher a(w) resulted in a higher reaction rate. Moreover, increasing a(w) was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique a(w) dependence probably because of the variation in cell surface characteristics.     

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.     

Enantioselective esterification of butanol-2 in an enzyme membrane reactor

The enzymatic esterification of octanoic acid with racemic butanol-2 was investigated. Esterifications of the acid were performed in a forced flow enzyme membrane reactor. The used membrane was prepared by a phase inversion process in polyamide-6 solution followed by the chemical immobilization of a lipase-catalyst. Influences of water content and pH were estimated. Their optimum values are equal to 0.5% w/w and pH 8. The reaction rate (at 303 K) of 5.1 × 10^?5 mol/h·cm² of the membrane area, and at least 85% enantiomeric excess in the produced ester mixture were obtained. The activity of immobilized lipase in the membrane process is about two times higher than that of the native lipase in the esterification performed in a tank reactor.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

The control of lipase-catalysed transesterification and esterification reaction rates. Effects of substrate polarity, water activity and water molecules on enzyme activity

The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of water-saturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.     

Effect of agitation rate and impeller design on oxygen transfer coefficients in small bioreactors using surface aeration

The effects of agitation rate and impeller type on the combined oxygen mass-transfer coefficient (kL a) in four different benchtop bioreactors have been examined. Surface oxygenation of a cell culture medium supplemented with fetal bovine serum and distilled deionized water has been studied by passing air through the bioreactor headspace at approximately one headspace volume per minute. A new ribbon-type impeller design using strips of Teflon has been shown to be superior to conventional impeller designs for oxygen transfer.     

Enzymatic Synthesis of Ethyl-glucoside Monooleate with Lipase in Solvent-free Medium

Ethylglucoside monooleate was synthesized by esterification between ethylglucoside and oleic acid with immobilized lipase from Candida antarctica in a solvent-free system. It was shown that a stirred tank reactor was suitable for the enzymatic reaction process involving substrates with low miscibility, in which the biocatalyst was recycled five times without significant activity loss. Removal of the co-product, water, from the reaction medium by carrying out the reaction under reduced pressure benefited the esterification reaction and increased the monooleate yield up to 97% within 8 hours.     

有机相中酶促有机硅烷醇的转酯

在有机介质中,脂肪酶L-1754可以催化非天然有机硅烷醇与脂肪酸酯的转酯反应.反应介质、反应体系水活度和底物的结构对酶促转酯反应有显著的影响.适宜的反应介质为正辛烷,反应体系最适水活度为0.55.