Atul M. Vaidya

Substrate specificity and kinetics of Candida rugosa lipase in organic media

The fatty acid specificity of lipase from Candida rugosa during the esterification of saturated fatty acids and sulcatol in toluene has been studied. The true kinetic parameters are obtained by fitting the experimental data to a Ping-Pong kinetic model that includes alcohol inhibition. The fitted parameter values are compared with apparent values that would be obtained from restricted data sets in which one of the substrate concentrations was kept constant. It has been found that in reactions inhibited by alcohol the true Ping-Pong parameters can be significantly different from the apparent ones. Corrections for solvation are made by using activities instead of concentrations to fit the kinetic parameters. Though activity coefficients, estimated using the UNIFAC group contribution method, vary by over 25% for changing concentrations in the same solvent, their use did not improve the fit to the data. This contrasts with what has been found in comparisons of different solvents, where the differences in activity coefficients are much larger.

Continuous in situ water activity control for organic phase biocatalysis in a packed bed hollow fiber reactor.

Packed bed hollow fiber membrane reactors were used to carry out organic phase biocatalysis at constant water activity. The performance of the device was tested by carrying out the esterification of dodecanol and decanoic acid in hexane. Lipase from Candida rugosa, immobilized on microporous polypropylene and packed in the shell space of the reactor, was used to catalyze the reaction. In situ water activity control was accomplished by pumping appropriate saturated salt solutions through the microporous hollow fiber polypropylene membranes. Water generated by reaction in the organic phase, pumped continuously through the shell of the reactor, was transferred into the bulk of the aqueous phase under the water activity gradient. The reactor performance was found to be strongly dependent on the controlling water activity. By carefully selecting this control activity it was found possible to obtain complete esterification. The water activity of the organic phase could be maintained very close to that of the saturated salt solution used. The reactor could be operated in the continuous mode for 100 h without any degradation in its performance.

Organic‐Phase Enzymatic Esterification in a Hollow Fiber Membrane Reactor with In Situ Gas‐Phase Water Activity Control

Several reactor designs have been described in the recent literature for continuous organic‐phase enzymatic esterification reactions. While these designs have excellent performance characteristics, there are operational constraints in their use. The present article describes a new reactor design, the gas‐phase hollow fiber reactor (GPHFR), which does not suffer from any such limitations. The reactor consists of, commonly available, hollow fiber dialyzer modules with enzyme immobilized on the lumen of the hollow fiber membranes by ultrafiltration. Substrate mixtures are passed through the fiber lumens and subjected to esterification with a constant humidity gas phase recirculated through the shell of the reactor, acting as the medium used to control water activity. The simplicity of the device renders it suitable for use over a wide range of water activities, and its modular nature facilitates easy scale‐up. The use of the reactor for the fixed water activity esterification of an equimolar mixture of dodecanol and decanoic acid has been described. Under optimum conditions the reactor was found to give yields of ester as high as 97%. In continuous operation the immobilized enzyme was found to have a half‐life of about 70 days.

Enhanced organic-phase enzymatic esterification with continuous water removal in a controlled air-bleed evacuated-headspace reactor

The yield of organic‐phase enzymatic esterification reactions can be improved by continuous removal of product water. When water is the only volatile component of the reaction system, this can be accomplished by carrying out the reaction under a partial vacuum. The performance in such reaction systems can be further improved by employing a controlled leak of air into the headspace of the reactor. This improvement is achieved at a lower vacuum than would be required in an ideal evacuated reactor delivering the same performance. The theory of air‐leak effects has been analyzed in this paper. Experiments done to verify this theory have also been presented. Air‐bleed evacuated‐headspace reactors (ABEHRs) can produce extremely high synthetic yields. For instance, during the lipozyme‐catalyzed esterification of a solvent‐free solketal−decanoic acid mixture, a yield of 96% ester was obtained in a reactor operated under a vacuum of 0.7 bar and a temperature of 50 °C when air at 20 °C with a relative humidity of 54% was leaked into the headspace.

Aqueous—organic membrane bioreactors. Part I. A guide to membrane selection

Abstract The influence of membrane pore structure on the ease with which an aqueous-organic interface can be maintained in the plane of the membrane of a two-phase membrane reactor is discussed. Four factors affecting the pressure required to cause breakthrough of the non-wetting phase have been identified: (i) membrane pore size, (ii) asymmetry of membrane pore structure, (iii) the placement of the wetting liquid when an asymmetric membrane is used, and (iv) a change in the wetting characteristics of the membrane as the reaction progresses. The fourth factor is particularly important since two phase biocatalytic reactions frequently involve surface active reactants and/or products. It is shown that the ideal pattern of surfactant-membrane interactions - which is reflected by the desired direction of change in the angle of contact between the wetting liquid and the membrane - depends on the second and third factors. An experiment is suggested to assess the importance of the various factors and a set of rules of thumb have been presented to assist in the selection of membrane material and type. The importance of correctly identifying the wetting liquid when an amphiphilic membrane polymer is used has been pointed out.

Stepped water activity control for efficient enzymatic interesterification

Abstract The benefits of controlling water activity, aw, during enzymatically catalysed synthesis reactions, such as reverse-hydrolytic reactions promoted by lipases, are now well recognized. Numerous techniques for controlling aw in the laboratory and their implementation in continuous reactors have been discussed in the published literature. However, in enzymatic interesterification reactions, such as acidolysis and transesterification, it is not appropriate merely to maintain the aw of the reaction system at one value since the two stages of the reaction, namely the cleavage of the original acyl bond and the formation of a new one, are best carried out at different levels of water activity – the former at a high aw and the latter at a lower one. The use of a continuous packed-bed hollow-fibre reactor has been described in this article for carrying out solvent-free acidolysis of ethyl laurate with octanoic acid with in situ aw control, using air that has been pre-equilibrated with saturated salt solutions to the desired aw. At a single optimum (aw = 0.54), the highest steady-state conversion to ethyl octanoate was 32%. However, it is possible to obtain a steady-state conversion of 46% by operating the reactor with a step change in the water activity, from an initial value of unity to 0.23.

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.

PREDICTION OF DENATURING TENDENCY OF ORGANIC SOLVENTS IN MIXTURES WITH WATER BY MEASUREMENT OF NAPHTHALENE SOLUBILITY

Addition of miscible organic solvents to water increases the solubility of naphthalene. The logarithm of the solubility is linearly dependent on the co-solvent concentration, in an intermediate range. The relative solubilising effects of different solvents correlate well with their known tendency to denature proteins (using literature data for trypsin, cytochrome c, chymotrypsinogen, chymotrypsin, laccase and myoglobin). This is expected if denaturation occurs when the hydrophobic effect has been reduced by a characteristic extent for a given protein. Naphthalene solubility predicts denaturation as well as does the denaturation capacity model.

Aqueous-organic membrane bioreactors part II. Breakthrough pressure measurement

Abstract The effect of membrane type—structure and wettability—on the operation of two-phase, aqueous-organic, membrane bioreactors has been studied. The influence of surfactants on membrane wettability is reported. A simple, but highly sensitive, technique for the measurement of breakthrough pressures is described. Experimental measurements of the variation in break through pressures as the concentration of tenside in the system was changed are reported. On the basis of the results from these measurements it is concluded that: (i) hydrophilic and, highly retentive, amphiphilic ultrafiltration membranes may be used to operate two-phase bioreactors, (ii) amphiphilic microfiltration membranes should never be used in such reactors and (iii) PTFE membranes would always be a poor choice for use in such devices because they always have a low breakthrough resistance in two-liquid systems—breakthrough pressures as low as 100 mbar were observed for the system ethyl laurate-water-PTFE, which contains no surface-active component. It is shown that these results are in general agreement with rules of thumb for the selection of membranes, presented earlier. The influence of membrane history on its wetting behavior—due to effects such as polymer surface restructuring—is highlighted. The limits on the utility of simple breakthrough pressure tests in determining suitable membranes, for use in two-phase bioreactors, owing to possible complications resulting from the exact mechanism of enzyme action is pointed out.

High specific activity of whole cells in an aqueous-organic two-phase membrane bioreactor

Abstract Whole Bacillus subtilis cells can catalyze stereoselective hydrolysis of menthyl acetate in a small-scale two-phase membrane bioreactor. A layer of cells was deposited on one side of a 0.2 μm microporous nylon membrane and then neat liquid racemic menthyl acetate was circulated past the same side. This organic phase was pressurized slightly relative to the aqueous buffer which flowed past the other face of the membrane. No breakthrough or cell detachment was observed over 24 h operation. The total reaction rate reached a maximum with a cell layer of about 40 μm, then declined for thicker layers. The specific activity (per unit biomass) at the maximum was about five times higher than with the same cells in an emulsion reactor. This probably reflects continuous good contact with both phases.