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

Nembri, Francesca; Bossi, Alessandra Maria; Ermakov, Sergey V.; Righetti, Pier Giorgio

doi:10.1002/(sici)1097-0290(19970105)53:1<110::aid-bit14>3.0.co;2-s

Cited by 17 publications

(3 citation statements)

References 24 publications

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“…It is also possible that, upon crossing the pH 11 membrane, it will become rapidly deprotonated, so that, ultimately, its transport into the cathodic compartment will be substantially diffusion-driven. Nevertheless, in a urease reactor based on the MCE principle, the passage of ammonium ions through a pH 11 membrane and its accumulation into the cathodic compartment has been well documented, both theoretically and experimentally . In addition, a longer residence time of the ammonium ion in this chamber would hardly affect the carbamylation event, were it to occur.…”

Section: Resultsmentioning

confidence: 99%

“…Nevertheless, in a urease reactor based on the MCE principle, the passage of ammonium ions through a pH 11 membrane and its accumulation into the cathodic compartment has been well documented, both theoretically and experimentally. 15 In addition, a longer residence time of the ammonium ion in this chamber would hardly affect the carbamylation event, were it to occur.…”

Section: Mce Fractionationmentioning

confidence: 99%

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Carbamylation of Proteins in 2-D ElectrophoresisMyth or Reality?

et al. 2003

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Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

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Section: Resultsmentioning

confidence: 99%

Section: Mce Fractionationmentioning

confidence: 99%

Carbamylation of Proteins in 2-D ElectrophoresisMyth or Reality?

et al. 2003

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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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Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase

Bossi

Cretich

Righetti

1998

Biotechnol. Bioeng.

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Penicillin G acylase (PGA) is exploited for producing pure D‐phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl‐4‐hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4‐hydroxyphenylacetyl‐(L)phenyl glycine, upon consumption of L‐phenylglycine, is converted by the enzyme back into free substrate and 4‐hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4‐hydroxyphenylacetyl‐(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4‐hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme‐driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D‐phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L‐form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 454–461, 1998.

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

Cited by 17 publications

References 24 publications

Carbamylation of Proteins in 2-D ElectrophoresisMyth or Reality?

Carbamylation of Proteins in 2-D ElectrophoresisMyth or Reality?

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

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase

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