Buffer polymers, co-immobile buffer and enzyme polymers and...

Details Buffer polymers, co-immobile buffer and enzyme polymers and... Buffer polymers, co-immobile buffer and enzyme polymers and...

2002-08-08

2004-07-13

Gorr, Rachel (Department: 1711)

Synthetic resins or natural rubbers -- part of the class 520 ser

Synthetic resins

Cellular products or processes of preparing a cellular...

C435S182000

Reexamination Certificate

active

06762213

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to immobile buffer polymers, co-immobile buffer and enzyme polymers (or polymers in which an enzyme and a buffer are immobilized) and to methods of synthesis of polymers including immobilized buffer and polymers including immobilized enzyme and immobilized buffer.
In general, the function of an enzyme is to catalyze chemical reactions. Enzymes have a wide range of applications. For example, industrial applications of enzymes include, but are not limited to, fermenting wine, leavening bread, curdling cheese, and brewing beer. Medical applications of enzymes include, but are not limited to, killing disease-causing microorganisms, promoting wound healing, and diagnosing certain diseases.
In general, six classes of enzymes are recognized, which include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Oxidoreductases catalyze oxidation or reduction reactions and are referred to as Enzyme Class 1 enzymes (EC1). Transferases catalyze the transfer of specific radicals or groups and are referred to EC 2 enzymes. Hydrolases catalyze hydrolysis reactions and are referred to as EC 3 enzymes. Lyases catalyze removal from or addition to substrate specific chemical groups and are referred to as EC 4 enzymes. Isomerases catalyze isomeration reactions and are referred to as EC 5 enzymes. Ligases catalyze reactions which combine or bind together substrate units and are referred to as EC 6 enzymes. These classifications cover generally all enzymes used in industrial, medical, and other applications.
The biocatalytic activity of enzymes in industrial, medical or other applications occurs within a range of environmental conditions (for example, pH, temperature, pressure, and ionic strength) similar to the typical biological environment of the enzymes. Each enzyme within the six enzyme classifications has, for example, an optimal pH range, in which the rate of enzyme catalyzed reaction is the fastest. This optimal pH range (as well as optimal ranges for other environmental conditions) can be narrow or broad, depending on the enzyme. Typically, plant-based enzymes have broader pH ranges than animal-based enzymes.
One method of maintaining optimal pH is to neutralize acid or base in solution. Buffer salts, for example, can be added to “initialize” the pH or add buffer capacity to solution. In fact, addition of buffer is the most common method for maintaining high catalytic rates for laboratory and industrial enzymatic reactions. Hydrolase enzymes (EC 3) are of special interest because they produce acid or base as a byproduct and, thus, can alter the pH of an unbuffered solution during the course of a reaction. Examples of such hydrolases includes pectinase, protease, urease, and organophosphorous hydrolase (OPH). Pectinases break down cell walls to clarify fruit juices. Proteases in detergents break down protein-based stains. Urease breaks down urea in urine into carbon dixoide and ammonia. OPH degrades organophosphates into byproducts. Pectinases, proteases, and OPH create an acid (H
3
O
+
) as byproduct. Urease creates a lewis base (NH
4
+
) as byproduct.
Over time, the tertiary or three-dimensional structure of an enzyme may erode and the enzyme (and its active site) may correspondingly lose integrity, especially at high temperatures. This process is called enzyme denaturation. The enzyme and its active site are in proper conformation when ideal physiological conditions of, for example, moderate temperature, pH, and ionic strength are present. Seemingly minor changes in these conditions may cause changes in protein folding, resulting in catalytic activity loss and permanent denaturation. To protect against denaturation, enzymes can be immobilized on or within a solid polymer to rigidify and stabilize enzyme chemical structure. Through chemical modification, immobilization masks sensitive residues in the enzyme's protein structure. Immobilized enzymes maintain their activity over a broader set of environmental conditions than native enzymes, including residence time in solution and temperature. By attaching an enzyme within a polymeric matrix as described, for example, in LeJeune, K. E., Mesiano, A. J., Bower, S. B., Grimsley, J. K., Wild, J. R., Russell, A. J., Biotechnol. Bioeng. 54, 105 (1997) (sometimes referred to herein as “LeJeune et al. (1997)”), enzyme stability is greatly enhanced.
Immobilization does not, however, protect enzymes against activity loss associated with extreme pH environments (that is, environments in which the pH is outside of the optimal pH range or well outside the optimal pH range). Both native and immobilized enzymes lose activity when placed in extreme pH environments or when significant quantities of acidic or basic byproducts are produced. For example, immobilized urease will continue to degrade urea into ammonium ion byproduct in distilled water, but when sufficient quantities of byproduct are generated, the pH rises. Immobilized urease loses activity as pH levels rise above the optimal pH and loses all activity at around pH 10. A similar effect is observed with OPH, which will degrade methyl parathion and generate acid byproduct in an unbuffered environment until reaching pH 4.
In general, immobilized enzymes are of limited use in catalyzing reactions in environments in which pH is outside of the optimal pH range of the enzyme. Moreover, immobilized hydrolases (EC 3) are of limited use in catalyzing reactions in unbuffered environments. Thus, immobilized enzymes require an added buffer to neutralize acid or base when placed in a solution with a pH outside the active range or the optimal pH range of the immobilized enzyme. Immobilized hydrolases require an added buffer to neutralize acid or base byproducts, which alter the pH to a pH outside of the active range or the optimal range of the immobilized hydrolase enzyme. However, it may not be practical or possible to add buffer to an environment in which an immobilized enzyme is to be used. For example, in field use for decontamination of a toxic agent or agents in which a large area must be decontaminated, addition of buffer may not be practical and/or buffer may not be available. Currently available immobilized enzymes cannot, therefore, be used to their full advantage in certain environments.
It is desirable, therefore, to develop immobilized enzyme systems and methods that reduce or, preferably, eliminate the above and other problems.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a polyurethane polymer including at least one buffer selected to adjust pH to a pH within a desired range. The buffer compound is immobilized within the polymer, and the polymer has a buffer capacity in excess of 3 micromoles of acid or base per gram polymer. Preferably, the polymer has a buffer capacity in excess of 60 micromoles acid or base per gram polymer. More preferably, the polyurethane polymer has a buffer capacity in excess of 100 micromoles acid or base per gram polymer. Even more preferably, the polyurethane polymer has a buffer capacity in excess of 200 micromoles acid or base per gram polymer. As described further below, attempts to incorporate significant buffer capacity in polyurethane polymers have been unsuccessful. The immobile buffer polyurethanes of the present invention provide polymers having significant buffer capacity over a wide range of polymer physical characteristics (for example, density and pore size).
In another aspect, the present invention provides a method for preparing a polyurethane polymer immobilizing at least one buffer comprising the steps: reacting a buffer compound with a multifunctional precursor for the polyurethane polymer to produce a modified precursor, the buffer compound having at least one functional group for reacting with the precursor and at least one buffering group that remains functional as a buffer after the buffer compound is reacted with the precursor; and subsequent to reacting the buffer compound with the precursor, polymerizing the modified precursor to f

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