Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes.
The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such "isoelectrically immobilized' reactor, the enzyme beta-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5 beta-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3 beta-hydroxy-7,12-dioxo- 5 beta-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD+) collected, separately, into the two neighbouring chambers at the anodic side.


CONCLUSIONS
in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.
Chiari
M
M
Instituto di Chimica degli Ormoni, CNR, Milano, Italy.
Dell'Orto
N
N
Mendozza
M
M
Carrea
G
G
Righetti
P G
PG
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D002197:Capillary Action; D002855:Chromatography, Thin Layer / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004586:Electrophoresis / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D009243:NAD; D015096:3-Hydroxysteroid Dehydrogenases