Biocatalyst, insoluble

Immobilized biocatalysts are enzymes, cells or organelles (or combinations of these) which are in a state that permits their rense (The Working Party of Immobilized Biocatalysts, 1983). Examples are insolnble enzymes, e.g. nsed in a fixed bed reactor or soluble enzymes, e.g. used in a semipermeable membrane reactor. This chapter will describe methods of industrial interest for making biocatalysts insoluble. 

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

The interest and success of the enzyme-catalyzed reactions in this kind of media is due to several advantages such as (i) solubilization of hydrophobic substrates (ii) ease of recovery of some products (iii) catalysis of reactions that are unfavorable in water (e.g. reversal of hydrolysis reactions in favor of synthesis) (iv) ease of recovery of insoluble biocatalysts (v) increased biocatalyst thermostability (vi) suppression of water-induced side reactions. Furthermore, as already said, enzyme selectivity can be markedly influenced, and even reversed, by the solvent. 

FIG. 1 Reaction systems containing organic solvent, and corresponding theoretical concentration profiles for water insoluble substrate. solid biocatalyst organic phase aqueous phase or biocatalyst dissolved in aqueous phase. 

As the majority of enzyme systems in aqueous-organic biphasic media, lipase-catalyzed hydrolysis uses substrates that are insoluble in aqueous media and yield hydrophobic products (Fig. 3). Therefore, the aqueous phase serves as a biocatalyst reservoir. 

In the early 1980 s Julia and Colonna published a series of papers which, to some extent, filled the gap left by the natural biocatalysts. The Spanish and Italian collaborators showed that a, -unsaturated ketones of type 1 underwent asymmetric oxidation to give the epoxide 2 using a three-phase system, namely aqueous hydrogen peroxide containing sodium hydroxide, an organic solvent such as tetrachloromethane and insoluble poly-(l)-alanine, (Scheme 1) [12]. The reaction takes place via a Michael-type addition of peroxide anion (the Weitz-Scheffer reaction). 

Biological catalysts in the form of enzymes, cells, organelles, or synzymes that are tethered to a fixed bed, polymer, or other insoluble carrier or entrapped by a semi-impermeable membrane . Immobilization often confers added stability, permits reuse of the biocatalyst, and allows the development of flow reactors. The mode of immobilization may produce distinct populations of biocatalyst, each exhibiting different activities within the same sample. The study of immobilized enzymes can also provide insights into the chemical basis of enzyme latency, a well-known phenomenon characterized by the limited availability of active enzyme as a consequence of immobilization and/or encapsulization. 

The use of enzymes as biocatalysts for the synthesis of water-soluble conducting polymers is simple, environmentally benign, and gives yields of over 90% due to the high efficiency of the enzyme catalyst. Since the use of an enzyme solution does not allow the recovery and reuse of the expensive enzyme, well-established strategies of enzyme immobilization onto solid supports have been applied to HRP [22-30]. A recent work reported an alternative method that allows the recycle and reuse of HRP in the biocatalytic synthesis of ICPs. The method is based on the use of a biphasic catalytic system in which the enzyme is encapsulated by simple solubilization into an IL. The main strategy consisted of encapsulating the HRP in room-temperature IPs insoluble in water, and the other components of the reaction... 

Multi-phase reactors are of interest in biocatalytic reactions if one or several components of the reaction are insoluble or insufficiently soluble in aqueous phases but if an aqueous phase has to be kept, if only for the biocatalyst. However, a two-phase system can be utilized advantageously for the shifting of an equilibrium this is demonstrated below. We analyze the simple reaction A <=> B in an organic-aqueous two-phase system with the assumption that reactant A and product B partition between the two phases. The partition coefficients Pw and Porg are de-... 

Numerous efforts have been devoted to the development of insoluble immobilized enzymes for various applications. Among the benefits of using immobilized enzymes rather than their soluble counterparts are the reusability and improved stability of heterogeneous biocatalysts with the aim of reducing the production cost by efficient recycling and control of the process [88]. 

The fluidized bed reactor has been used for phenol removal instead of fixed bed as most of the products formed are insoluble. The operation in packed bed reactors would lead to clogging phenomena and undesirable pressure drop [47, 88]. When deactivation of biocatalysts occurs and regeneration is needed, the liquid-solid circulating fluidized bed is a worthy alternative, as demonstrated for phenol polymerization [89]. The continuous enzymatic polymerization was carried out in a riser section and a downcomer was used for the regeneration of the coated immobilized particles. 

The adsorption of biomolecules onto carriers that are insoluble in water is the simplest method of immobilization. An aqueous solution of the biomolecules is contacted with the active carrier material for a defined period of time. Thereafter the molecules that are not adsorbed are removed by washing. Anionic and cationic ion exchange resins, active charcoal, silica gel, clay, aluminum oxide, porous glass, and ceramics are being currently used as active material. The carrier should exhibit high affinity and capacity for the biomolecule and the latter must remain active in the adsorbed state. The carrier should adsorb neither reaction products nor inhibitors of the biocatalyst. 

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