Method and apparatus for governing the reaction rate of enzymatic reactions

• US 4,016,044 A
• Filed: 05/29/1975
• Issued: 04/05/1977
• Est. Priority Date: 01/10/1973
• Status: Expired due to Term

• Current Assignee: Battelle Memorial Institute
• Original Assignee: Battelle Memorial Institute
• Inventors: Fresnel, Jean-Marie, Trosset, Daniele
• Primary Examiner(s): Penland, R. B.
• Application Number:
  US05/581,761
• Time in Patent Office: 677 Days
• Field of Search: 195/31, 195/63, 195/103.5, 195/68, 195/115, 195/116, 195/104, 195/108, 195/117-118, 195/DIG. 1, 195/127, 204/195 B, 204/195 T, 204/1 E, 204/1 R
• US Class Current: 205/701
• CPC Class Codes: B01J 31/003 containing enzymes
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C25B 3/00 Electrolytic production of organic compounds
• CPC Class Codes: C25B 9/40 Cells or assemblies of cells comprising electrodes made of particles; Assemblies of constructional parts thereof
• CPC Class Codes: Y10S 435/813 Continuous fermentation
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

Heterogeneous enzymatic catalysis is carried out by means of an enzymatic electrode comprising an enzyme fixed to a solid, conductive support. To govern the reaction rate during continuous transformation of a substrate, the enzymatic activity is regulated by adjusting the potential of the enzyme support so as to transform the substrate at a given rate. The electrode is provided with a particulate enzymatic mass operatively associated with an inert working electrode for contact with this mass to thereby subject the particles thereof to the adjustable potential. Continuous movement of a liquid reaction system is produced so as to provide intimate contact thereof with each particle of the mass, which may be arranged as a fixed bed, an agitated bed or a fluidized bed. The potential of the enzyme support is caused to continually alternate between two levels while adjusting the mean value thereof so as to maintain a corresponding mean enzymatic activity for transforming the substrate at the desired rate.

Claims

1. A method of governing the reaction rate of enzymatic reactions for continuously transforming a substrate by contact with an enzyme fixed to a solid carrier, comprising the steps of:

bringing an electrically conductive liquid reaction system, containing at least one substrate to be transformed, into contact with an enzymatic electrode comprising at least one enzyme fixed to an electrically conductive solid support medium; and

regulating the catalytic activity of said enzyme by applying an electric potential to said support medium while adjusting the value of said potential so as to transform said substrate at a given rate, said method further comprising the improvements consisting of;

a. providing said enzymatic electrode with a solid, inert, working electrode and with a particulate mass of discrete enzymatic particles, each formed essentially of said conductive support medium having said enzyme fixed to the surface thereof;

b. placing said enzymatic electrode in a reaction space wherein said particulate mass of enzymatic particles is arranged for electrical contact thereof with said working electrode;

c. circulating said liquid reaction system within said reaction space; and

d. applying said adjustable potential, via said working electrode, to the conductive support medium of said enzymatic particles.

2. The method of claim 1 wherein said particulate mass of enzymatic particles consists of graphite particles having a mean size lying between 0.4 and 1.5 mm, with said enzyme fixed to the surface thereof.

3. The method according to claim 1 wherein said working electrode is subjected to continuous movement in a loose bed of said enzymatic particles whereby to cause agitation of said bed to provide continually repeated direct contact of all particles therein with said working electrode.

4. A method of governing the reaction rate of enzymatic reactions for continuously transforming a substrate by contact with an enzyme fixed to a solid carrier, comprising the steps of:

bringing an electrically conductive liquid reaction system, containing at least one substrate to be transformed, into contact with an enzymatic electrode comprising at least one enzyme fixed to an electrically conductive solid support medium; and

regulating the catalytic activity of said enzyme by applying an electric potential to said support medium while adjusting the value of said potential so as to transform said substrate at a given rate, said method further comprising the improvements consisting of;

applying to said support medium a variable potential alternating between two levels, while adjusting the mean value thereof so as to provide a corresponding mean catalytic activity of said enzyme for transforming said substrate at a given rate.

5. A method of governing the reaction rate of enzymatic reactions for continuously transforming a substrate by contact with an enzyme fixed to a solid carrier, comprising the steps of:

bringing an electrically conductive liquid reaction system, containing at least one substrate to be transformed, into contact with an enzymatic electrode comprising at least one enzyme fixed to an electrically conductive solid support medium; and

regulating the catalytic activity of said enzyme by applying an electric potential to said support medium while adjusting the value of said potential so as to transform said substrate at a given rate, said method further comprising the improvements consisting of;

a. providing said enzymatic electrode with a solid inert working electrode and with a particulate mass of discrete enzymatic particles, each formed essentially of said conductive support medium having said enzyme fixed to the surface thereof;

b. placing said enzymatic electrode in a reaction space wherein said particulate mass of enzymatic particles is arranged for electrical contact thereof with said working electrode;

c. circulating said liquid reaction system within said reaction space; and

d. applying an alternating potential via said working electrode to the support medium of said enzymatic particles, while adjusting the mean value of said potential so as to provide a corresponding mean catalytic activity of said enzyme fixed to said particles, whereby to transform said substrate at a given rate.

6. The method of claim 5 wherein said particulate mass of enzymatic particles is arranged to form a fixed porous bed in contact with said working electrode, said liquid reaction system being continuously circulated through said fixed bed within said reaction space, and said alternating potential being applied to the working electrode and thereby applied to the support medium of the enzymatic particles of said fixed bed.

7. A method of governing the reaction rate of enzymatic reactions for continuously transforming a substrate by contact with an enzyme fixed to a solid carrier, comprising the steps of:

bringing an electrically conductive liquid reaction system, containing at least one substrate to be transformed, into contact with an enzymatic electrode comprising at least one enzyme fixed to an electrically conductive solid support medium; and

regulating the catalytic activity of said enzyme by applying an electric potential to said support medium while adjusting the value of said potential so as to transform said substrate at a given rate, said method further comprising the improvements consisting of;

a. providing said enzymatic electrode with a solid, inert, working electrode and with a particulate mass of discrete enzymatic particles each formed essentially of said conductive support medium having said enzyme fixed to the surface thereof;

b. placing said enzymatic electrode in a reaction space wherein said particulate mass is arranged to provide an agitated bed of said enzymatic particles whereby to provide repeated contact thereof with said working electrode;

c. circulating said liquid rection system in said reaction space so as to provide said agitated bed of enzymatic particles in suspension in said liquid system, whereby each suspended particle in said agitated bed undergoes continually repeated impingements on said working electrode; and

d. applying said adjustable potential to said working electrode so as to thereby apply said potential to the support medium of each suspended enzymatic particle by intermittent contact thereof during said continually repeated impingements thereof on said working electrode, each said suspended particle being at a different potential level while undergoing movement within said agitated bed, between successive impingements thereof on said working electrode.

8. The method of claim 7, wherein said liquid reaction system is subjected to a circulating movement by means of a stirring member providing said agitated bed within said reaction space, said space further having an inert counter-electrode arranged therein, whereby the suspended particles in said agitated bed undergo successive impingement on said counter-electrode, alternating with said successive impingements thereof on said working electrode.

9. The method of claim 7 wherein said agitated bed is provided in the form of a fluidized bed of said enzymatic particles in suspension in an ascending stream of said liquid reaction system circulating through said reaction space.

10. An apparatus for governing the reaction rate of an enzymatic reaction for continuously transforming a substrate contained in an electrically conductive liquid system, comprising:

a. at least one enzymatic electrode having at least one enzyme fixed to the surface of a solid, electronically conductive support associated with means for applying an adjustable potential to said electrode;

b. a reaction chamber which is equipped with an enzymatic electrode and with an inert counter-electrode and is adapted to receive said liquid reaction system and to discharge the products of the enzymatic reaction; and

c. adjustable electrical supply and voltage control means connected to said enzymatic electrode and to said counterelectrode and adapted to apply between said electrodes a potential difference which is adjustable so as to regulate the catalytic activity of said enzyme;

the improvement wherein;

said enzymatic electrode comprises a particulate enzymatic mass, composed of discrete enzymatic particles, each essentially formed of said solid conductive support having said enzyme fixed thereto, and further comprises a solid, inert working electrode, operatively associated with said mass and with said electrical supply and voltage control means, so as to subject the particles of said mass to an adjustable potential;

said apparatus further comprises circulating means adapted to circulate said liquid reaction system within said reaction chamber so as to provide intimate contact thereof with each particle of said enzymatic mass, the particles of said mass being subjected to said adjustable potential during said circulation.

11. The apparatus of claim 10 wherein said electrical supply and voltage control means are arranged to apply to said working electrode a potential which varies periodically between two potential levels and to adjust the mean value of said two potential levels, so as to thereby regulate the activity of said enzyme.

12. The apparatus of claim 10 wherein said enzymatic mass constitutes a porous fixed bed of said particles arranged in contact with said working electrode.

13. The apparatus of claim 10 further comprising a stirring device, arranged to form an agitated bed of said enzymatic particles in suspension in said liquid reaction system and to thereby produce continually repeated collisions of the particles of the agitated bed with said working electrode.

14. The apparatus of claim 10 wherein the said circulating means are arranged to produce an ascending stream of said liquid reaction system passing through said reaction chamber at an adjustable speed, to allow formation of a fluidized bed of said enzymatic particles and to thereby provide continually repeated collisions of each particle of the fluidized bed with said working electrode.