Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;...
Patent
1997-07-23
1998-11-10
Redding, David A.
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
435183, 4352852, 4352891, 435814, C12N 1100, C12M 300
Patent
active
058342729
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention refers to a process for enzyme immobilization, consisting in trapping said enzymes, while still in solution, between two isoelectric membranes encompassing the isoelectric point (pI) of the enzyme in the reactor. The invention refers also to an enzyme reactor comprising said immobilized enzymes. The reaction chamber of the enzyme reactor consists of a multicompartment electrolyzer, assembled with a set of isoelectric membranes (having pI values increasing monotonically from anode to cathode) and the reaction is preferably (but not exclusively) carried out under the influence of an electric field. In case of charged reactants (and/or products), said substances can be carried electrophoretically into the reaction chamber and/or transported out by the electric field. Also valuable cofactors (e.g., NAD.sup.+, NADH) can be moved in and out of the reaction chamber electrophoretically, regenerated outside and fed back into the enzyme reactor. In this way, instead of stoichiometric amounts, only catalytic amounts of coenzymes are required. The present invention refers also to a method for decreasing the water activity in the reaction chamber, thus stabilizing the trapped enzymes. Since catalysis occurs under focusing conditions, with concomitant evacuation of all buffering ions from the reaction chamber, this would quickly lead to enzyme inactivation. The enzyme is kept fully active by the following means: (a) "co-immobilization" of buffers in the enzyme chamber. This is achieved by using amphoteric buffers, with pI values encompassed by the pI values of the two flanking (at anodic and cathodic side, respectively) membranes. A series of such buffers exist covering the pH 3-10 scale;
(b) by addition of polyols (e.g., ethylene glycol, glycerol, erythritol, xylitol, sorbitol) or polymers (e.g., dextrans, polyethylene glycol) or carbohydrates (e.g., sucrose, lactose) to the reaction chambers. Comprised in the present invention are also different means for bringing uncharged substrates electrophoretically into the reaction chamber. Examples of such means are: (a) charging neutral sugars by forming charged complexes with borate; (b) inserting neutral substrates into negatively (e.g., sodium dodecyl sulphate) or positively (e.g., cetyl-trymethyl ammonium bromide) charged micelles. The present invention refers also to the preparation of novel isoelectric membranes by using highly stable acrylamido monomers (in general N-mono- and di-substituted compounds, such as dimethyl acrylamide and N-acryloyl amino ethoxy ethanol), thus allowing stable operation for prolonged period of time, due to their high resistance to alkaline hydrolysis. Still a further object of the present invention is also the possibility of forming highly porous isoelectric membranes (e.g., by the process of lateral aggregation during polymerization) so as to allow unhindered migration in and out of the reaction chamber of macromolecular substrates (e.g., proteins, nucleic acids, lipo- and poly-saccharides). In the present invention, the multicompartment electrolyzer, constituting the reaction chamber, comprises in general an electric field coupled to a hydraulic flow, for recycling enzymes and reactants in and out of the electric field to, e.g., a heat sink or to suitable chromatographic columns, for harvesting valuable reaction products, or to additional reaction chambers for, e.g., regenerating valuable cofactors via secondary enzymatic or chemical reactions. The present invention refers also the use of cascade reactors, where two or more enzymes are coupled serially (e.g. in the same chamber or in adjacent chambers, trapped in between suitable isoelectric membranes), for transforming different substrates (or for performing different modifications on the same substrate) in serial reactions.
BACKGROUND OF THE INVENTION
In the seventies, the field of enzymology received a new impulse due to the introduction of immobilized enzyme reactors (Messing, R. A., Immobilized Enzymes for Industrial Reactors. Academic
REFERENCES:
patent: 5082548 (1992-01-01), Faupel et al.
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