Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Reexamination Certificate
1999-09-14
2001-07-31
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
C435S041000, C435S135000, C435S182000, C435S187000, C435S188000
Reexamination Certificate
active
06268191
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is directed to physically immobilizing enzymes for use in non-aqueous enzymatic reactions.
The motivation for using enzymes in organic environments, which are completely unlike their natural uses in an aqueous environment, is predominantly for industrial applications. Pharmaceutical companies, in particular, have an interest in using enzymes in non-aqueous environments since most drug synthesis is performed in organic solvents. One particularly desirable application for the use of enyzmes in organic media is catalyzing stereospecific reactions where only one isomer is useful out of several possible isomers. Enzymes are limited in their ability to function effectively as catalysts in organic media, are generally useful in catalyzing only specific reactions, and generally are useful only at relatively low temperatures and pressures. Therefore there is a need to improve the ability to employ enzymes to conquer these problems and facilitate the use of various enzymes in organic reactions.
Immobilization of enzymes is important in industrial application. When enzymes are used in reactors they must be “immobilized” so that they can be separated from the product, recovered, and recycled. Immobilization of enzymes on supports in aqueous media is widely practiced. The literature also describes the covalent attachment of enzymes to inorganic and organic surfaces, membranes and gels. Work has also been performed on physically entrapping enzymes in membranes and gels. Successful industrial implementation of non-aqueous enzymology will require new approaches for immobilizing enzymes in organic media without compromising their activity.
Substantial literature exists relating to non-aqueous enzymology, expecially in the examination of the kinetics of anhydrous enzymes in various organic solvents under varied reactions conditions. Issues have been described such as the increased thermal stability of enzyme(s) in organic solvents which have been attributed to the high kintic barriers preventing protein unfolding, altered substrate specificity, and the ability of these enzymes to catalyze novel reactions in this media. The requirements of the enzyme for water in organic solvents has also been examined.
The recovery and reuse of nonimmobilized enzymes has been of substantial difficulty in the art to present. Using currently available techniques of enzyme recovery, either the method of removal contaminated the enzyme, which renders the enzyme useless for reuse, or the activity of the enzyme is destroyed in the recovery process. In either case, removing the biofunctional enzyme from a reaction mixture which it catalyzes has proved difficult. In addition, when using a dry enzyme in an organic solvent, not only must the difficulty of removal be confronted, but also the enzyme's tendency to agglomerate, which results in a reduction of the surface area available to catalyze the reaction, thereby significantly slowing the rate of reaction. Each of these problems must be considered when attempting to recover intact enzymes from nonaqueous environments.
Methods of enzyme immobilization which have been discussed in the literature include: covalent binding, non-covalent binding, and physical entrapment. Immobilizing enzymes by covalently bonding them to a carrier has the advantage that the enzyme is prevented from leaking from the carrier regardless of the stringency of the conditions. However, this form of immobilization has the disadvantage that it generally alters the conformational structure and reactivity of the active site. Non-covalent bonds such as hydrophobic binding, polar binding electrostatic interactions and hydrogen bridge binding (adsorption) have been used to associate the enzyme with a carrier material without forming covalent bonds. Since the binding is not as strong as covalent bonding, the conformation of the enzyme is usually not significantly altered and therefore the reactivity of the enzyme is not severely reduced. However, this weaker binding also leaves the possibility for the enzyme to leak from the carrier more easily. Entrapment of the enzyme does not involve any type of chemical binding, but instead only physically restricts the enzyme's movement within a polymer matrix. It therefore does not interfere with the enzyme conformation at all. However, depending on the method of entrapment, the enzyme may be damaged if polymer conditions are more stringent or may simply be occluded so that its reactivity is reduced.
Recent research on entrapment has been performed using kappa-carrageenan as the polymer matrix for immobilizing both cells and enzymes. &kgr;-carrageenan is a linear polysaccharide made up of alternating 1,3-linked B-D-galactose-4-sulfate and 1-4-linked 3,6-anhydr-&agr;-D-galactose as shown in figure 1.
This polymer forms a gel network in two steps. The first step involves the partial association of polymer chains into double helices. The association of helices into “domains” by the addition of a cation (usually potassium) produces the gel network. The gelation temperatures of &kgr;-carrageenan polymers are reported to be dependent on the cation concentration, and relatively independent of carrageenan concentration. The most common method used to immobilize biological cell suspensions in carrageenan gels is to prepare a carrageenan solution in the absence of cations, and when the solution has cooled to about 45° C. the cell suspension is added and the gel is configured into the desired geometry. Once the gel has cooled, it is cured with a KCI solution. Moon et al (Biotechnol. Proc., 7, 516 (1991)) also attempted to immobilize cells by diffusing them into pre-formed gels. These gels were cured with KCI solution and post-crosslinked to prevent cell leakage. However, cell viability was destroyed during this process.
SUMMARY OF THE INVENTION
This present invention provides a method for immobilizing an enzyme for use as a catalyst in organic reactions by imbibing an enzyme into a dehydrated hydrocolloid polymer gel bead. More particularly, the invention comprises the steps of (a) dehydrating a hydrocolloid gel bead having an average particle size in the range of 5 &mgr;m 150 &mgr;m in diameter, (b) imbibing into the dehydrated hydrocolloid gel bead an aqueous solution of the enzyme to form an imbibed gel bead; and (c) optionally dehydrating the imbibed gel bead, recovering a gel bead in which a catalytically effective amount of enzyme has been immobilized. The enzyme may be an oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, decarboxylase, carboxylase, aldolase, thiolase, or synthase. In a particularly preferred embodiment, the hydrocolloid is carrageenan or kappa carrageenan. One preferred enzyme is subtilisin Carlsberg. In a particularly preferred embodiment, the hydrocolloid is carrageenan and the enzyme is subtilisin Carlsberg, in which the aqueous solution of enzyme contains in the range of 0.05 to 40 wt % enzyme. A preferred method of dehydration is lyophilization. The invention also provides a gel bead in which an enzyme has been immobilized by the foregoing process.
REFERENCES:
patent: 4182655 (1980-01-01), Hartmeier
patent: 4975375 (1990-12-01), Haruta et al.
patent: 5662840 (1997-09-01), Thomas et al.
Pantiliano et al., “Large increases in General Stability for Subtilsin BPN' through Incremental Changes in The Free Energy of Unfolding”, Biochemistry vol. 28, pp. 7205-7213 (1989).
Guide to Protein Purification, “Methods in Enzymology”, vol. 182 pp. 77-78, The Academic Press, Inc. 1990.
Moon et al., “Characterization of k-Carrageenen Gels Used for Immobilization of Bacillus firmus”, Biotechnol. Prog., vol. 7 (6) pp. 518-525 (1991).
Chen et al., “Enzyme Engineering for Nonaqueous Solvents. II. Additive effects of Mutations on the Stability and Activity of Subtilsin E in Polar Organic Media”, Biotechnol Prog., vol. 7 125-129 (1991).
Enzyme Catalysis in Organic Synthesis, A comprehensive Handbook, pp. 74-87, 1993.
Volkin et al., “Enzyme Thermoinactivation in Anhydrous Organic Solvents”, Biote
Cukras Catherine-Ann
Pfeffer Henry A.
Prud'homme Robert K.
Anderson Robert L.
Baker Patrick C.
Monroe Bruce M.
Naff David M.
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