Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

Chiari, Marcella; Dell'Orto, Norma; Mendozza, Monica; Carrea, Giacomo; Righetti, Pier Giorgio

doi:10.1016/0165-022x(95)00019-n

Cited by 13 publications

(8 citation statements)

References 25 publications

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“…As the ME is an open system, it offers the opportunity of developing continuous processes. The first example of an enzyme reactor based on the ME was proposed by Chiari et al (1996): In this preliminary report, a coenzyme-dependent enzymatic transformation was studied. The enzyme ␤-hydroxysteroid dehydrogenase was trapped isoelectrically into a chamber delimited by isoelectric membranes encompassing its pI.…”

Section: Introductionmentioning

confidence: 99%

Isoelectrically trapped enzymatic bioreactors in a multimembrane cell coupled to an electric field: Theoretical modeling and experimental validation with urease

Nembri

Bossi

Ermakov

et al. 1997

Biotechnol. Bioeng.

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A novel type of immobilized enzyme reactor operating under an electric field is here reported: a multicompartment immobilized enzyme reactor (MIER). In this experimental set‐up, the enzyme and zwitterionic buffering ions are trapped in between two isoelectric membranes, having isoelectric point (pl) values so far apart as to trap the enzyme by an isoelectric mechanism, while allowing operation at pH optima, even when the latter pH value is quite removed from the enzyme pl. As an example, urease (pl 4.9) is trapped between a pl 4.0 and a pl 8.0 membranes, thus permitting operation (via suitable amphoteric ions buffering at pH 7.5) at the pH of optimum of activity (pH 7.5). The charged product (ammonium ions) quickly leaves the enzyme chamber under the influence of the electric field, thus allowing sustained activity for much longer time periods than in conventional reactors. As an example, while in a batch reactor 90% of original enzyme activity is lost in 200 min, only 2% activity is lost in the same period in the MIER reactor. As an additional bonus, the MIER reactor allows conversion rates of ∼95% in a wide range of substrate concentrations, whereas batch‐type reactors rarely achieve better than 50% conversion under comparable experimental conditions. © 1997 John Wiley & Sons, Inc.

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Section: Introductionmentioning

confidence: 99%

Isoelectrically trapped enzymatic bioreactors in a multimembrane cell coupled to an electric field: Theoretical modeling and experimental validation with urease

Nembri

Bossi

Ermakov

et al. 1997

Biotechnol. Bioeng.

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“…Thanks to these features, ME reactors integrate the bioconversion and the downstream processes simplifying the production and reducing the whole cost of the process. ME, as an enzymatic reactor, was used for the immobilization of a b-hydroxysteriod dehydrogenase [6], urease [7] trypsin [8] and, in the last few years, for the production of pure D-phenylglycine [9], 6-aminopenicillanic acid [10] and optically pure Naproxen [11].…”

Section: Introductionmentioning

confidence: 99%

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads

et al. 2003

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Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.

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“…A growing number of attempts have recently been made in coupling enzyme immobilization-stabilization and product recovery, and an increasing interest has been devoted to the application of electric fields for product recovery, such as electrodialysis (Nidetzky et al, 1996;Ishimura and Suga, 1992). Among the developments of integrated bioprocesses, a membrane immobilized enzyme reactor (MIER) was first described by us in 1996 (Chiari et al, 1996), and a mathematical model for the related transport equations was developed by Nembri et al (1997).…”

Section: Introductionmentioning

confidence: 99%

“…When exploring a variety of reactions (Chiari et al, 1996;Nembri et al;Bossi et al, 1998) in order to define the fields of advantageous applications of the MIER reactor, a few general guidelines were derived:…”

Section: Introductionmentioning

confidence: 99%

Electrically immobilized enzyme reactors: Bioconversion of a charged substrate. Hydrolysis of penicillin G by penicillin G acylase

Bossi¹,

Guerrera²,

Righetti³

1999

Biotechnol. Bioeng.

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The possibility of using the multicompartment immobilized enzyme reactor (MIER) in presence of a charged substrate is here explored. Penicillin G acylase is used to convert penicillin G (a free acid, with a pK of 2.6) into two charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6‐aminopenicillanic acid (6‐APA, a zwitterion with a pI of 3.6). The enzyme is trapped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectric membranes. Under normal operating conditions (continuous substrate feeding in the presence of an electric field), only a low substrate conversion can be achieved, due to rapid electrophoretic transport of unreacted penicillin G out of the reaction chamber towards the anode. Excellent conversion rates (>96%) are obtained under a “doubly‐discontinuous” operation mode: a time‐lapse substrate feeding, accompanied by short times (4–8 min) of electric field interruption. The product of interest (6‐APA, a precursor of semisynthetic penicillins), by virtue of its amphoteric nature, is trapped in a chamber delimited by a pI 3.5 membrane and a pI 5.5 membrane, adjacent to the reaction chamber on its anodic side. The other contaminant product (PAA) first accumulates in the same chamber and then progressively vacates it to collect in the anodic reservoir, leaving behind a pure 6‐APA solution. In this operation mode, vanishing amounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers. A novel class of zwitterionic buffers is additionally reported, able to cover very thoroughly any pH value along the pH 3–10 interval: polymeric, zwitterionic buffers, synthesized with the principle of the Immobiline (acrylamido weak acids and bases) chemicals. Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 383‐391, 1999.

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Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

Cited by 13 publications

References 25 publications

Isoelectrically trapped enzymatic bioreactors in a multimembrane cell coupled to an electric field: Theoretical modeling and experimental validation with urease

Isoelectrically trapped enzymatic bioreactors in a multimembrane cell coupled to an electric field: Theoretical modeling and experimental validation with urease

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads

Electrically immobilized enzyme reactors: Bioconversion of a charged substrate. Hydrolysis of penicillin G by penicillin G acylase

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