Control of water activity

Figure 9.6 Control of water activity by equilibration via the gas phase or via silicone tubing. 1 gas phase 2 immobilised enzyme 3 reaction medium (substrate in organic solvent) 4 stirring bar, 5 saturated salt solution 6 salt crystals 7 silicone tubing 8 organic solvent (to keep the salt solution solvent saturated).

Wehtje, E., Svensson, I., Adlercreutz, P. and Mattiasson, B. (1993) Continuous control of water activity during biocatalysis in organic media. Biotechnol. Techniques, 7, 873-878. 

Summarizing the findings above, control of water activity aw during enzyme reactions in organic solvents is extremely important, as water activity exerts a crucial influence, and enzyme reactivity crucially depends on it. 

Control of Water Activity of Proteins, Solvents, and Substrates.198... 

CONTROL OF WATER ACTIVITY OF PROTEINS, SOLVENTS, AND SUBSTRATES... 

One of the major advantages of employing control is the improvement of precision and repeatability of kinetic results because the water content of solvents and protein preparations can vary from batch to batch and with time. In addition, control of water activity is quite simple. It is achieved by storing solvents (of low volatility), substrates, and enzymes over saturated salt solutions. The thermodynamic properties of the salt dictate the observed water activity. Several different salt types used to control for nonaqueous enzymology are listed in Table 8.5. - - - The storage of saturated salt solutions is achieved by the following procedure ... 

Using mainly water non-misdble solvents several approaches are possible. In most cases, the organic solvent has to be saturated with water in order not to remove the boundary water surrounding the enzyme, which otherwise results in deactivation. In such microaqueous systems the pH of this tiny amount of water should be carefully chosen for optimal enzyme activity. The control of water activity can be achieved by addition of salts or utilization of saturated salt solutions I81, 821. The simplest way of using an enzyme in organic solvents is to suspend the insoluble enzyme in the required solvent. This technique was first reported in 1900 [83] and has been extended over the last few years to encompass many systems (mainly proteases and lipases) [75, 84, 85L Organic solvents may be replaced by supercritical liquid carbon dioxide, which exhibits similar properties to hexane[86, 146]. 

Figure 8-5. Control of water activity by adding salt hydrates to reaction mixture. Synthesis of butyl butanoate catalyzed by Candida rugosa lipase. Control reactions with catalyst relatively wet ( ) or dry (O) initially. Reaction in the presence of Na2SC>4 plus Na2SO4.10H2O (O). Kvittingen et al. 22 ...

There have been some cases of confusion in the control of water activity between saturated salt solutions (see above) and salt hydrate pairs. These can both be useful methods, but the principles and recommended applications are quite different. Avoid phrases like control of water activity using salts , which do not make it clear which method is being used. 

The control of water activity in foods is an important tool for extending shelf life. It is responsible for the quality of foods affected by microbiological, chemical, and physical changes. The physical properties, quantity, and quality of water within the food have a strong impact on food effectiveness, quality attributes, shelf life, textural properties, and processing. 

Most of these bio conversions were carried out in conventional hollow-fiber modules, with the biocatalyst either immobilized in the membrane or freely resuspended in the aqueous phase. However, the widespread use of hydrolytic enzymes for synthesis reactions [119,202] presented a particular problem, since a shifting of the equilibrium was required to obtain a high yield of the synthesis product [119]. To effectively perform such bioconversions it was necessary both to remove water formed and to maintain water activity at the optimum level, which could be achieved by the use of salt hydrates [65]. On-line control of water activity was performed by continuously contacting the reaction medium with... 

The control of water activity is a significant technical problem when the enzyme is in nearly dry solvent. Where a lipase catalyses ester synthesis, rather than transesterification, stoichiometric amounts of water are released. 

The non situ experiment pioneered by Sass uses a preparation of an electrode in an ultrahigh vacuum through cryogenic coadsorption of known quantities of electrolyte species (i.e., solvent, ions, and neutral molecules) on a metal surface. " Such experiments serve as a simulation, or better, as a synthetic model of electrodes. The use of surface spectroscopic techniques makes it possible to determine the coverage and structure of a synthesized electrolyte. The interfacial potential (i.e., the electrode work function) is measured using the voltaic cell technique. Of course, there are reasonable objections to the UHV technique, such as too little water, too low a temperature, too small interfacial potentials, and lack of control of ionic activities. ... 

Finally, we note that all transfers to alcohol-water mixtures or additions of alcohol to crystal mother liquor involve changes in the proton activity of the solution. Care must be taken to ensure that the pH does not change too much, or the crystal may be disrupted. Worse still, the enzymatic activity may be abolished. Control of proton activity in mixed solvents is discussed in Section III,D. If dielectric effects are controlled and pH is properly adjusted, the microenvironment of a crystalline protein will correspond closely to that of aqueous solution at room temperature. Such correspondence is essential for temporal resolution of individual steps in a catalytic reaction. 

Finally, solvents can dissolve different amounts of water and thereby indirectly influence the enzyme activity. However, this effect can be compensated for by controlling the water activity (see above). 

In order to study the effects of water activity on an enzymatic reaction, there is a need for practical methods to adjust the water activity in the reaction mixture. Likewise, it is highly desirable to keep this parameter close to the optimal value during large-scale conversions. A range of water activity control methods have been developed [11], and which one to choose depends on the scale of reaction, the quantities of water to be removed or added, and availability of equipment. 

When it is important to control the water activity in a reactor, a water activity sensor is quite useful. The sensor should ideally measure the water activity in the liquid reaction medium. However, the sensors available are designed for gas phase measurements, and, provided there is effective enough equilibration between the liquid and gaseous phases, they can be used to control the water activity in the reactor. If the measured water activity is above the set point, drying is initiated, for example, by passing dry air through the reactor. On the other hand, if the water activity is too low, water can be added, either as liquid water or as humid air. Automatically controlled systems of this kind have been successfully used to monitor and control enzymatic reactions in organic media [13, 14]. 

Olsson, G. Andrews, J.F. "The dissolved oxygen profile - a valuable tool for control of the activated sludge process Water Res., 1978, 12, 985-1004. 

Originally, water activity could not be measured directly. One method was to measure the weight loss of a product held at a range of controlled relative humidities, which also has the effect of holding the product over a range of water activities. If a product is held at its own water activity it neither gains nor loses weight, and this point is described as its equilibrium water activity. 

Enzymatic reactions in organic media have been a major issue in the field of biocatalysis over the last two decades. Carboxylesterases (mostly lipases) have been used in monophasic organic solution under controlled values of water activity (ajj for catalyzing ester formation the reaction equilibrium can be shifted towards ester formation by interesterification or transesterification [1]. Direct esterification is often hampered by water formation, which may increase o , thus negatively influencing the equihbrium. 

The related achetakinins double the rate of fluid secretion by isolated Acheta Malpighian tubules (Coast, G. M., et al. J. Insect Phvsiol.. in press.)- In addition, some leucokinins stimulate fluid secretion and/or depolarize transepithelial membrane potentials in the malpighian tubules of the yellow fever mosquito, Aedes aegvpti. in a fashion similar to several uncharacterized peptides isolated from mosquito head extracts (23). Thus, the neuropeptide family of leucokinins, achetakinins, and homologs may function in the control of water and ion balance, as well as myotropic activity, in a number of insects. To determine the relationships of the C-terminal sequence of the leucokinins to myotropic activity, analogs were synthesized and tested on the isolated cockroach hindgut. 

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