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Fundamental phenomenological description and experimental optimization of gel stabilized biocatalysts in a two-phase system

  • Douméche, Bastien
Publication Date
Jan 01, 2003
Publikationsserver der RWTH Aachen University
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The lipase from Candida rugosa is immobilized in calcium-alginate hydrogel before been placed in hexane in order to catalyse the esterification of butanol and propionic acid. The consequence of the acidification of the aqueous phase lead to the evolution of the calcium-alginate network to an alginic acid one with a size reduction of the spheres and a release of 10-15 % of water (w/w) according to the calcium-alginate preparation. The control of the pH can be only achieved by of amines in the reaction media, leading to the formation of an ion pair with propionic acid. Ion pair of propionic acid and butylamine could be re-suspended in hexane by the co-substrate (butanol) with an alcohol: ion pair ratio of 2:1. The ion pair is not dissociated by the butanol but re-suspended as observed by infrared analysis. pH-optima of Candida rugosa lipase (Crl) in presence of butylamine increase with the acidity of the media. The basic lipase of Humicola lanuginosa (HLL) present the same pH profile displaced to basic values. A mechanism based on the ionisation of the active site, supported by molecular modelling study, is proposed. About 80 % of the activity disappear when butylamine is used with Crl and HLL. Exchanging butylamine by tert-butylamine allow to recover the activity for HLL but not for Crl. The stucture of the oxyanion hole of the active centre is proposed to be involved in this phenomena. Localisation of the enzyme active sites in the hydrogel is performed via the synthesis of an amphiphilic phosphonate inhibitor (n-dodecyl, n-paranitrophenyl, n-hexylphosphonate) leading to limit the enzymatic activity at the alginate/hexane interface. Dissociation of agglomerate of 100-140 µm beads in hexane could be prevented by the use of cationic surfactants (n-cetyl, n-,n-,n-, trimetylammonium chloride, CTAC). Enzyme inactivation (30 %) is probably due to denaturation of lipase molecules located at the interface. Lyotrophic mixtures are formed and are found to be caused by the simultaneous presence of CTAC and calcium ions. A fluidised-bed reactor could be obtained by combinaison of water-immiscible solvent of high density. Crl isoforms are purified in high purity (1646 time) with a new methodology presenting fewer steps than describe in the literature.

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