Enzyme recovery using reversed micelles

The objective of this study was to develop a liquid-liquid extraction process for the recovery of extracellular enzymes. The potentials of reaching this goal by using reversed micelles in an organic solvent have been investigated.Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. The considerable biotechnological potential of these systems is derived principally from the ability of the water droplets to dissolve enzymes without loss of activity. Enzymes can be transported from a bulk aqueous phase to a reversed micellar phase and visa versa.The distribution coefficient of an enzyme between a reversed micellar and an aqueous phase depends on the interactions which are possible between the enzyme and the reversed micelle. When ionic surfactants are used, electrostatic interactions have been shown to be the most important ones. The distribution can therefore be controlled by adjusting pH and ionic strength. The optimum pH for transfer depends on the size and titration behaviour of the enzyme. The extraction to a reversed micellar phase therefore shows enzyme selectivity.Using the possibility to vary the distribution coefficient a continuous forward and back extraction process has been developed (Chapter 2). In two mixer/settler units the enzyme α-amylase is concentrated using a recirculating reversed micellar phase of the cationic surfactant trioctylmethylammonium chloride and the cosurfactant octanol in isooctane.During the forward extraction some inactivation of the enzyme occurs by a complexation between the enzyme and the surfactant in the aqueous phase (Chapter 3). The extraction process has been modelled in terms of mass transfer and inactivation of the enzyme in all phases. As predicted by the model the extraction efficiency can be optimized by reducing the concentration of enzyme in the first aqueous phase through increasing the distribution coefficient (by the addition of a nonionic surfactant to the reversed micellar phase) and by increasing the mass transfer rate during the forward extraction. The observed enzyme recovery values correlate quite well with the values predicted by the model.An important parameter of the extractions is the mass transfer rate of the enzyme to and from the reversed micellar phase. During forward extraction the rate of mass transfer is controlled by diffusion in the aqueous phase. The back extraction rate, however, is governed by the interfacial process of coalescence of the reversed micelles with the bulk interface. This process is strongly dependent on the pH, probably due to interactions of the surfactant with charged groups on the enzyme (Chapter 4).An alternative process for the recovery of the enzyme from the reversed micellar phase uses the temperature effect on the phase behaviour of the system (Chapter 5). By increasing the temperature some aqueous phase is expelled from the organic phase, enabling the enzyme to be recovered in this phase. This phenomenon was applied successfully in an extraction process with two centrifugal extractors.The applicability of the process to fermentation broths has to be subjected to further investigation, but some established general features are discussed (Chapter 6). In conclusion it can be stated that reversed micellar extraction of enzymes is a selective process with an enormous potential to purify and concentrate proteins in one operation.The study described in this thesis was performed in a partnership between the Departments of Food Science (Food and Bioprocess Engineering Group), Biochemistry and Physical and Colloid Chemistry. The project was financed by the Netherlands Technology Foundation (STW).

Bibliographic Details

Main Author:	Dekker, M.
Other Authors:	van 't Riet, K.
Format:	Doctoral thesis biblioteca
Language:	English
Published:	Landbouwuniversiteit Wageningen
Subjects:	biochemistry, biology, biotechnology, chemical industry, enzymes, enzymology, fermentation, food biotechnology, fractionation, immobilization, purification, separation, techniques, biochemie, biologie, biotechnologie, chemische industrie, enzymen, enzymologie, fermentatie, fractionering, immobilisatie, scheiding, technieken, voedselbiotechnologie, zuiveren,
Online Access:	https://research.wur.nl/en/publications/enzyme-recovery-using-reversed-micelles-2
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