JPS5917733B2 - Manufacturing method and use of protein-bound polyurethane foam - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of polyurethane foams within which proteins such as enzymes, antibodies or antigens are immobilized.

Binding protein-containing polyurethane foams made by any of the methods of the invention can be used in any reaction in which it is desired to bring about contact between a protein and another substance.

If the protein is an enzyme, the enzyme-containing form can be used in catalytic reactions or the like for which the enzyme is a suitable catalyst. If the protein is an antibody, the antibody-containing form can be used for the removal of the antigen from biological samples, such as human blood, and vice versa. can be used to extract antibodies from biological samples. The use of protein-containing foams to achieve contact between proteins and other substances is part of the invention. Such immobilized proteins can already be found in the prior literature.

For example, U.S. Patent No. 3,574,062
diazotizing a polyester polyurethane with a diazonium salt of an amino acid and then coupling it with a non-enzymatic animal protein to yield a diazotized polyurethane;
This book describes a method for producing binding proteins (enzymes), in which the binding proteins (enzymes) are reacted with enzymes to produce immobilized enzymes.
U.S. Pat. No. 3,705,084 discloses: (a) a macroporous reactor core; (b) a polymeric surface on the reactor core, which can be a polyurethane resin;
c) A flow-through enzyme reactor is described consisting of an enzyme adsorbed onto a polymer surface and cross-linked to the polymer surface in situ by means of a bifunctional reagent (eg polyisocyanate).

U.S. Pat. No. 3,791,927 discloses a water-insoluble binding protein that is entrapped within the cells of a self-supporting reticulated foam material (which can be a polyurethane foam) and bonded to the foam material. (enzyme).

U.S. Pat. No. 3,672,955 (195/68, Stanley et al.) discloses: (a) preparing an aqueous dispersion of an enzyme by a solution of a polyisocyanate in a volatile water-incompatible solvent (e.g. methyl chloroform); A process for the preparation of binding proteins (enzymes) is described which consists of emulsification; (b) mixing the emulsion thus obtained with a solid powder carrier; and (c) then evaporating the solvent.

The Stanley polyisocyanate can be an isocyanate capped liquid polyurethane prepolymer. No. 3,672,955 is incorporated herein by reference in its entirety.
In Example 3 of this patent, Stanley reports on the binding of an enzyme component (peroxidase) in the fermentation liquor by mixing a portion of the fermentation liquor with polyisocyanate dissolved in methylchloroform. It is noteworthy.

Under Stanley's conditions, it is likely that any other enzymes present in the fermentation liquid will become immobilized (become water-insoluble, ie, bound). U.S. Pat. No. 3,929,574 (Utsudo et al.) discloses contacting an isocyanate capped liquid polyurethane prepolymer with an aqueous dispersion of an enzyme under foam-forming conditions, thereby converting the polyurethane foam and enzyme into the resulting polyurethane foam. It describes the production of bound (immobilized) enzymes by a method in which the enzymes become bound together.

U.S. Pat. No. 3,905,923 describes an immobilized enzyme formed from an enzyme and a hydrophilic poly(ureaurethane) foam, where the foam surrounds, confines and supports the enzyme in the active configuration.

The hydrophilic foam is formed by the reaction of water with a hydrophilic isocyanate capped polyoxyalkylene prepolymer. Rich chart (T. Richa
RD) and N.F. OlsOn, 1. Fixed Enzymes in Food and Microbial Processes, Plenum Publishing, New York, NY, 1974, 35.
Pages 36 to 36 describe the production of bound (immobilized) enzymes by reacting enzymes, water, and polyisocyanate polymers.

In our German publication no. 2319706,
We describe the production of a form-bound enzyme that consists of curing and foaming the prepolymer by dispersing the enzyme in water and mixing the dispersion of the enzyme with a polyurethane prepolymer.

The present invention provides that the enzyme or other protein can be first dissolved in a prepolymer and then water can be added to the solution, and that the retention of the protein in the form thus formed is such that the protein is It is based on the discovery that it is better than when dispersed in water.

Thus, the present invention provides an immobilized or bound protein (
which may be an enzyme, antibody, antigen or other protein), which method comprises: 1. In the process of making immobilized proteins chemically bonded to polyurethane foam, an (AXI) isocyanate capped polyurethane prepolymer is mixed with the protein in the liquid phase in the absence of water to form a solution, or ( 4) mixing the polyisocyanate with the protein in the liquid phase in the absence of water and then mixing the product with the polyhydroxy compound; or (11i) mixing the polyhydroxy compound in the liquid phase in the absence of water. foaming by mixing the product with the protein and then mixing the product with the polyisocyanate; and (b) mixing the solution thus obtained with an amount of water effective to produce foam. It is a feature.

For example, 0.5 to 3 parts, preferably 0.9 parts per part of the solution consisting of liquid polyurethane prepolymer and protein.
~2 parts of water can be used. The process is carried out at a temperature at which the isocyanate capped liquid polyurethane prepolymer exists in a liquid state and below the denaturation temperature of the protein to be immobilized.

Preferably, the method includes a preliminary step of forming a prepolymer by reacting the polyhydroxy compound with the polyisocyanate, and this step is performed prior to reacting the polyhydroxy compound or polyisocyanate with its reaction partner. , is characterized by mixing proteins with polyhydroxy compounds or polyisocyanates.

We advantageously wash the foam containing bound (immobilized) protein (preferably with water) to remove all unbound protein and to hydrolyze any unreacted isocyanate groups. I acknowledge that.

The liquid polyurethane prepolymer capped with toluene diisocyanate is prepared by adding toluene diisocyanate to polyethylene glycol, particularly from about 800 to 12,000, preferably from about 10
It can be prepared by reacting with polyethylene glycol having a molecular weight (average molecular weight) of 0.00.

Alternatively, the isocyanate capped liquid polyurethane prepolymer can be prepared using toluene diisocyanate, polyoxybutylene polyol polymer, ethylene glycol, diethylene glycol, polyoxyethylene polyol polymer, pentaerythritol, glycerin, trimethylolpropane and polyoxyethylene glycol. It can be prepared by reacting a polyhydric alcohol selected from propylene polyol polymers.

The isocyanate-capped liquid polyurethane prepolymer contains toluene diisocyanate in an amount of about 800 to 1
2001 can also be prepared by reacting a suitable mixture of polyethylene glycol and trimethylolpropane, preferably having an average molecular weight of about 1000, in which case trimethylolpropane and polyethylene glycol is about 1:1~
4 and toluene diisocyanate is used in a molar ratio of about 0.85 to 1.25 (preferably 0.4 molar ratio) toluene diisocyanate per equivalent (177) of -OH provided by polyethylene glycol and trimethylolpropane.
95 to 1.1) mol.

This polyisocyanate capped liquid polyurethane prepolymer is disclosed in U.S. Pat.
According to the method described in Example 1 of No. 574 (Wood et al.),
It can be prepared from toluene diisocyanate and ethylene glycol.

That patent is incorporated herein by reference in its entirety. In a preferred form of the method of the invention, the protein is first mixed with a polyisocyanate or polyol;
This mixture, still in the absence of water, is then reacted with a polyol or polyisocyanate, respectively, to form a polyurethane prepolymer, which is then foamed and cured in the presence of water.

In these preferred forms of the invention, it is generally preferred to use about 2 to 500 or 50 to 100 η protein per 17 polyols.

The ratio of polyisocyanate to the sum of polyol and enzyme must be such that there is an effective amount of isocyanate groups in the polyurethane prepolymer to react with water and produce foam. Generally about 1,25 to 4,5, especially 1 per polyol plus enzyme 17
, 8 to 2.2 milliequivalents of NCO groups are preferred. In another form of the invention, a liquid polyurethane prepolymer and a substrate capable of reacting with an enzyme are first mixed, and then this first mixture is mixed with an enzyme in the absence of water to form a second mixture. A composition is formed; it is then foamed by mixing with water to provide a poly(urea urethane) foam.

If a substrate is used, it is generally about 5 to 1 per mole of enzyme.
Preference is given to using 00 mmol of substrate, especially 8 to 12 mmol.

If a substrate is used, the enzyme is penicillin amidase and the substrate is benzylpenicillin, or the enzyme is glucoamylase and the substrate is starch,
Alternatively, the enzyme is suitably an amino acid acylase and the substrate is an acylated amino acid. As well as methods for producing polyurethane foams containing proteins, the present invention also provides solutions prepared by mixing isocyanate capped liquid polyurethane prepolymers and proteins in the absence of water. do. The prepolymer in this solution can be as described above. Still other embodiments of the invention provide a hydrophilic poly(urea-urethane) having an oxyalkylene backbone containing from about 0.1 to 50% by weight on an anhydrous basis and at least 50 mole percent oxyethylene. ) a protein-containing foam consisting of a foam matrix; the hydrophilic foam is produced by reacting with water a solution consisting essentially of the protein dissolved in an isocyanate-terminated prepolymer (isocyanate-capped liquid polyurethane prepolymer). and the hydrophilic form incorporates and supports the protein in an active configuration for biological activity, the protein being an enzyme, antibody or antigen.

In the method of the invention, the isocyanate-capped polyurethane prepolymer is: (a) a solvent for dissolving the protein to be bound; and (b) reacting with the protein to bind it to the poly(urethane). - urethane)
It acts as a reactant to bind to the foam formed when the product solution is mixed with water to form the foam.

The solidification temperature of the isocyanate-capped liquid polyurethane prepolymer used in the process of the present invention is based on the molecular weight of the prepolymer and the structure of the prepolymer's backbone.

The thermal denaturation temperature of proteins is generally greater than about 35°C.

However, certain proteins are stable for relatively short periods of time at higher temperatures (eg, up to about 70° C. or somewhat higher). Generally, when producing intermediate products (enzyme solutions in the absence of water), about 2 to 10 times or more (e.g. up to 100 to 1000 times or more) the stoichiometric amount of isocyanate is used. Preference is given to using a liquid polyurethane prepolymer which has been capped.

The intermediate product can be mixed in a subsequent step with water as described above to produce a protein tightly bound to the polyurethane foam. The present invention relates to a method for immobilizing (binding) proteins to polyurethane foams in an active and usable form.

The polyurethane prepolymer is prepared by known methods with an excess of di- and tri-isocyanates and other polyisocyanates (including mixtures of polyisocyanates) and active hydrogen-containing compounds, especially glycols, polyglycols, It is produced by reaction with polyester polyols, polyether polyols, other polyols and mixtures of two or more such polyols. This reaction provides an isocyanate capped liquid polyurethane prepolymer. The protein and the prepolymer are mixed in the absence of water to form a solution (step "a"), which is then mixed with water and reacted (step "b") to produce a chemical and/or biological A poly(
Produces urea-urethane). The formation of the solution in step 'a' above takes place in the absence of water.

Said step "a" can be carried out in the presence or absence of a diluent, or in the presence of a mixture of diluents. The description herein makes it clear to those skilled in the art that proteins can be denatured or It will be obvious that no diluent can be present during the above foaming step which would prevent or substantially reduce foaming. Diluents that can be used include Stanley et al.
72955), but are not limited to those listed (... Diluents include those that are very water soluble, such as acetone; those that are moderately water soluble, such as methyl acetate, methyl ethyl ketone, etc. or water-insoluble ones, such as benzene, and the above-mentioned US Pat. No. 3,672,955 to Stanley et al.
Other diluents listed in this issue may also be used. No. 3,672,955 is incorporated herein by reference in its entirety. The diluent is (a) an isocyanate-capped liquid polyurethane prepolymer; and (b)
Helps reduce the viscosity of the resulting mixture. The foaming stage (step (b) above) can also be carried out in the absence of a diluent or in the presence of one or more such diluents (
That is, it can be carried out in the presence of the above-mentioned diluents).

However, if the foaming step is carried out in the presence of a diluent, the viscosity of the resulting solution and the mixture of water and diluent with which it is mixed to produce the foam is increased by the reaction of water and isocyanate groups. A self-supporting poly(urea-urethane) containing immobilized proteins that precludes the formation of carbon dioxide-induced foaming.
Care must be taken to avoid the presence of so much diluent as to cause insufficient foaming to occur. Emulsifiers can be used in the foaming step if the diluent is water-insoluble or substantially water-insoluble.

Stanley et al. (U.S. Pat. No. 3,672,955) describes the use of such emulsifiers. Binding (protein fixation) reactions are generally applicable to any protein, including, but not limited to, enzymes, antibodies, and antigens.

For example, the following can be combined: urease, cellulase, pectinase, papain, promelene, chymotrypsin, trypsin, phycin, lysozyme, glucose isomerase, lactase, penicillin amidase, human immunoglobulin G, invertase, asparginase, etc. We cannot find an enzyme, antibody or antigen to which we cannot bind. Protein purity is not critical. Binding (protein fixation) is: (a)
Pure crystalline protein: (b) a partially purified amorphous protein; (c) an impure dry extract having enzymatic, antibody or antigenic activity; or (d) an unpurified dry extract from a fermentation broth ( This can be achieved, for example, using an acetone-precipitated product obtained from the fermentation broth. Our experiments demonstrate that proteins of virtually any purity (including enzymes, antibodies, and antigens) can be conjugated using the methods of the invention. The aforementioned US Pat. No. 3,672,955 states that proteins (enzymes) can be attached to isocyanate-capped polyurethanes.

In the process of that patent, an isocyanate-capped polyurethane is dissolved in a water-immiscible solvent.
This solution is emulsified using an emulsifier in the presence of active enzyme dispersed in water. The method of the present invention is similar in some respects to Stanley, US Pat. No. 3,672,955.

We can use the same isocyanate-capped polyurethane prepolymers (referred to as polyisocyanates in the patent); we can use the same polyols (referred to as polyisocyanates in the patent); ); and we can use the same enzyme. As in that patent (although we do not wish to be bound to any particular theory), the mechanism clearly suggests that one or more amine and/or hydroxyl groups on the protein and the polyurethane reaction with one or more than one isocyanate group on one molecule of the prepolymer. As in the Stanley patent, the isocyanate-capped liquid polyurethane prepolymer of the present invention combines a polyol and a polyisocyanate by eliminating the presence of free (unreacted) isocyanate groups on one molecule of the polyurethane prepolymer. It can be prepared by reaction using an excess of isocyanate to ensure.

The method of the present invention is also similar in some respects to the aforementioned US Pat. No. 3,924,574 (Utsudo et al.). We can use the same isocyanate-capped polyurethane prepolymer (referred to as isocyanate-capped polyurethane in the patent), and we can use the same protein (enzyme). I can do it. As in US Pat. No. 3,929,574, the method of the present invention is directed to the preparation of proteins bound to polyurethane foams. However, unlike the method of the US patent, we mix the prepolymer of the present invention and the enzyme under substantially water-free conditions.

The patent mixes the prepolymer with an aqueous dispersion of enzyme. Thus, whereas the Wood et al. method involves mixing and foaming in one step, there are two steps in the method of the present invention.

It consists of the following two steps. 1. A mixing step in which the protein (which can be an enzyme) and the prepolymer are mixed in the absence of water to form a product solution.

2. A foaming step in which the prepared product solution is mixed with water to produce a poly(urea-urethane)-bound protein-containing foam containing immobilized enzymes.

In our method (see Examples below), much more protein is bound to the foamed polyurethane than in the patent's method (i.e., poly(urea monomer) is bound in an active but water-insoluble form. urethane) can be present in the foam).

In other words, the method of the present invention allows much more efficient use of proteins than in the patented method. U.S. Pat. No. 3,924,574 containing at least two free isocyanate groups per molecule of prepolymer.
Including those by number.

Any liquid polyurethane prepolymer is suitable for protein attachment according to the present invention. Prepolymer one is 1
Preferably it contains on average two isocyanate groups per molecule. Higher ratios can also be used, for example 2 to 8 isocyanate groups per polyurethane molecule. Higher ratios can be used, but there is no benefit. Excess isocyanate groups remaining in the polyurethane foam (after attachment of the enzyme) are degraded by hydrolysis upon first contact of the foam with water, for example in a washing step prior to use of the attachment enzyme. The isocyanate-capped liquid polyurethane prepolymer used in the present invention has at least two isocyanate groups (reactive isocyanate groups) per molecule of the prepolymer. Isocyanate capped polyurethane prepolymer: (a) 40~
as a free-flowing liquid at 70°C; or dissolved in an inert solvent such as those described in the Stanley et al. A "liquid polyurethane prepolymer" is defined as "liquid polyurethane prepolymer" when it produces a solution containing ~50% (or 10-25%) of an isocyanate-capped polyurethane prepolymer.6 as used herein. The term isocyanate-capped liquid polyurethane prepolymer refers to liquid polyurethane or polyurea molecules containing at least about two free isocyanate groups per molecule.

Representative examples of polyisocyanates that can be reacted with active hydrogen-containing compounds (e.g., glycols, polyols, polyglycols, polyester polyols, polyether polyols, etc.) to yield isocyanate-capped polyurethanes according to the present invention include: Includes those shown in Table 1.

Table 1 Toluene-2,4-diisocyanateToluene-2,6-diisocyanateCommercial mixtures of toluene-2,4- and 2,6-diisocyanateEthylene diisocyanateEthylidene diisocyanatePropylene-1. 2-diisocyanatocyclohexylene-1,2-diisocyanatocyclohexylene-1,4-diisocyanate m-phenylene diisocyanate 3,3'-diphenyl-4,4'-biphenylene diisocyanate 4・4'-biphenylene diisocyanate 3, 3'-dichloro-4, 4'-biphenylene diisocyanate 1, 6-hexamethylene diisocyanate 1, 4-tetramethylene diisocyanate 1
・10-decamethylene diisocyanate 1,5-naphthalene diisocyanate cumene-2,4-diisocyanate 4-methoxy-1,3-phenylene diisocyanate 4
-chloro-1,3-phenylene diisocyanate 4-furomo 1,3-phenylene diisocyanate 4-ethoxy-1,3-phenylene diisocyanate 2,4'-diisocyanat diphenyl ether 5. 6-dimethyl-1
・3-phenylene diisocyanate 2,4-dimethyl-
1,3-phenylene diisocyanate 4,47-diisocyanato diphenyl ether benzine diisocyanate 4,6-dimethyl-1,3-phenylene diisocyanate 9,10-anthracene diisocyanate 4,4' −
Diisocyanatodibenzyl 3,3/-dimethyl-4,4'-diisocyanatodiphenylmethane 2,6-dimethyl-4,4'-diisocyanatodiphenyl 2,4-diisocyanatostilbene 3. P 'P゜卜.

anthracene diisocyanate fluorene diisocyanate naphthalene diisocyanate diisocyanato benzfuran 6-toluene diisocyanate p''-triphenylmethane triisocyanana Useful classes of liquid isocyanates in capped polyurethane prepolymers include polyether Derived from polyols and polyester polyols.

These compounds are, as known in the art,
Polyether (or polyester) polyols can be prepared by reacting with polyisocyanates, using an excess of the latter to ensure free isocyanate groups in the product. can.
A typical but not limiting example:
An ideal form is shown below: (In the above formula, m represents the number of tetramethylene ether repeating units.

This may range from about 5 to 50, for example. ) Compounds useful for the purposes of this invention include any of the polyisocyanates exemplified above, combined with (a) simple polyols such as those shown in Table 2 below; and (b) polyether polyols and It can be prepared by reacting with any of a wide variety of polyols, including polyester polyols.

Representative examples of these polyols are described below. Among the polyether polyols that can be used are those prepared by the reaction of alkylene oxides with active hydrogen group-containing initiators; typical examples of initiators include, for example, ethylene glycol. Polyhydric alcohols; polyamines such as ethylenediamine; phosphoric acid, and the like.

The reaction is usually carried out in the presence of either an acidic or basic catalyst. Examples of alkylene oxides that can be used in the synthesis are ethylene oxide, propylene oxide, any of the butylene oxide isomers, and two or more different alkylene oxides, such as mixtures of ethylene oxide and propylene oxide. Includes mixtures of oxides. The polyether polyol produced contains a polyether skeleton and has hydroxyl groups as terminal groups. The number of hydroxyl groups per polymer molecule depends on the functionality of the active hydrogen initiator.

For example, dihydric alcohols such as ethylene glycol (as an active hydrogen initiator) lead to polyniether chains having two hydroxyl groups per polymer molecule. When the oxide polymerization is carried out in the presence of the trifunctional alcohol glycerin, the polyether molecules formed are 1
It has an average of three hydroxyl groups per molecule.

Even higher functionality (more hydroxyl groups) can be obtained when the oxide is polymerized in the presence of polyols such as, for example, pentaerythritol, sorbitol, sucrose, dipentaerythritol, etc. Other examples of polyhydric alcohols that can be reacted with alkylene oxides to provide useful polyether polyols include those shown in Table 2. Table 2 Propylene glycol trimethylene glycol 1,2-butylene glycol 1,3-butanediol 1,4-butanediol 1,5-pentanediol 1,2-hexylene glycol 1,10-decanediol 1,2-cyclohexane diol 2-butene-1,4-diol 3-cyclohexene-1,1-dimethanol 4-methyl-3-cyclohexene-1,1-dimethanol 3-methylene-1,5-pentanediol diethylene glycol (2-hydroxyethoxy )-1-propanol 4-(
2-hydroxyethoxy)-1-butanol 5-(2
-Hydroxypropoxy)-1-pentanol-1-(
2-Hydroxymethoxy)-2-hexanol 1-(
2-Hydroxypropoxy)-2-octanonolearyloxy2-allyloxymethyllopanediolpentanediolmethyl-1,3 [(4-pentyloxy)methyl]panediolpro3-(0-propenylphenoxy)-1 0 pandiol thiodiglycol 2-p2,2'-[thiobis(ethyleneoxy)]diethanol polyethylene ether glycol (molecular weight approximately 200)
ZU 2,2/-isopropylidene bis(p oxy)diethanol 1,2,6-hexanetriol 1,1,1-trimethylolpropane 3-(2-hydroxyethoxy)-1 pandiol phenylene 2-proethylene glycol 3-(2-hydroxypropoxy) 2,4-dimethyl-2-(2-hydroxyethoxy)methylpentanediol-1,51,1,1-tris [(
2'-hydroxyethoxy)methyl]ethane 1.1.1
-Tris[(2-hydroxypropoxy)methyl]propanetriethanolaminetriisopropanolamineresorcinolpyrogallolphloroglucinolhydroquinone 4,6-dicatecolhonoresinolmethylphloroglucinolhexylresorcinol3-hydroxy-2-naphthol2-hydroxy -1-naphthol 2,5-dihydroxy-1-naphthol tert-butylcatechol Bisphenols such as 2,2-bis(p-hydroxyphenyl)propane and bis-1(p-hydroxyphenino(ha)methane) A particularly useful class of polyether polyols is the polytetramethylene glycols.

These are prepared by ring-opening polymerization of tetrahydrofuran and have the following repeating units in the polymer skeleton.
The terminal group of the polymer chain is a hydroxyl group. Also particularly desirable are polyoxyethylene polyols HO+CH2
CH2-0+XH, where x is about 1 polyol
000 (or about 2000 or somewhat higher).

Polyester polyols that can be used as precursors can be easily prepared by condensation polymerization of polyols and polybasic acids.

The polyol and acid reactant are used in proportions such that essentially all acid groups are esterified and the resulting chain of ester units is terminated with hydroxyl groups. Typical examples of polybasic acids for producing these polymers are oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, brassic acid, and thapsin. Acid, maleic acid, fumaric acid, glutaconic acid, α-hydromuconic acid, β-hydromuconic acid, α
-butyl-α-monoethylglutanoic acid, α・β-diethylsuccinic acid, o-phthalic acid, isophthalic acid, terephthalic acid,
Hemimellitic acid, trimellitic acid, trimesic acid, merophanic acid, Prehnitic acid
, pyromellitic acid, citric acid, benzenepentacarboxylic acid, 1,4-cyclohexanedicarboxylic acid, diglycolic acid, thiodiglycolic acid, dimerized oleic acid, dimerized linoleic acid, and the like. Typical examples of polyols for producing these polymers include ethylene glycol, 1,3-propylene glycol, 1,2 propylene glycol, 1,4-butylene glycol, 1,3-butylene glycol, 1,2 -butylene glycol, butene-1,4-diol, 1,5-pentanediol, 1
・4-pentanediol, 1,3-pentanediol,
1,6-hexanediol, hexene-1,6-diol, 1,7-heptanediol, diethylene glycol, glycerin, trimethylolpropane, 1,3,6-
Hexanetriol, trimethanolamine, pentaerythritol, sorbitol, and any of the other polyols listed above in connection with the preparation of polyether polyols. By intimate contact with proteins such as enzymes, antibodies, antigens, etc., isocyanate capped polyurethane prepolymers become highly chemically active.

Some of the free isocyanate groups of the prepolymer react with the amine groups of the protein, and upon subsequent addition of water, some of the isocyanate groups react with the water to provide carbon dioxide and release onto the polyurethane foam. It is thought that amine groups are generated. These latter amine groups react with free isocyanate groups on adjacent polyurethane molecules, and this reaction (generation of urea bonds) results in the formation and growth of poly(urea-urethane) polymers and also Introducing crosslinks between polymer molecules. This growth and crosslinking is essential for good foam formation. We (although we do not wish to be bound by theory)
, the superior results of the present invention (i.e., the binding of a substantially greater proportion of added enzyme than obtained in the prior art) are due to the fact that the isocyanate groups of the prepolymer in the product solution of step (a) above are This is believed to be due to the reaction between the protein and the isocyanate groups in the isocyanate-capped liquid polyurethane prepolymer in the absence of a competitive reaction with water. In conventional methods, competitive reactions (i.e. protein and prepolymer on the one hand, water and prepolymer on the other hand) occur due to the simultaneous presence of water and unbound proteins (proteins that have not yet had a chance to react with the prepolymer). reactions) proceed simultaneously and compete with each other. As mentioned above, in the method of the invention, the protein is added before adding the water. During foaming, for example, crosslinking agents (polyamines, polythiols, polyacids),
Other additives such as surfactants, wetting agents, defoamers, dyes, antioxidants, fillers, etc. may also be present.

Of course, it is the release of carbon dioxide that provides the gas for foam production. The ratio of protein to isocyanate capped liquid polyurethane prepolymer is not critical. However, this ratio ensures that all the isocyanate groups in the prepolymer are not consumed by reaction with the protein and are therefore free of unreacted groups that can be used to react with water to generate carbon dioxide. It is important that the isocyanate group remains. It is the carbon dioxide thus liberated that causes foaming to result in the polyurethane foam (to which the protein is bound).

The ratio of water to protein and isocyanate capped liquid polyurethane prepolymer is not critical;
However, generally about 0.5 to 3 or 0.9 parts by weight per part by weight of the prepolymer and protein combined.
Preference is given to using ~2 parts of water. Conjugated enzymes prepared by the methods of the invention are useful in analytical chemistry.

For example, by passing a solution of urea through a column packed with urease bound to a polyurethane foam, the urea is quantitatively converted to ammonia, which is determined by titration or colorimetric methods. can be quantified. Invertase attached to polyurethane foam can be used to convert sucrose to invert sugar.

Lipase bound to polyurethane foam can be packed into the column.

By passing a mixture of water and ester through this column, the organic ester can be hydrolyzed (converted back to alcohol and organic acid). The acid and alcohol components can then be separated and recovered by conventional methods. This method can also be used for the analysis of mixtures of esters. Many other uses for conjugated enzymes prepared by the methods of the invention will be apparent to those skilled in the art. Bound antigens prepared by the methods of the invention are useful for the removal of antibodies from biological samples.

For example, as described below, conjugated human immunoglobulin G (IgG) antigens are useful for the removal of rheumatoid arthritis factors (antibodies) from human blood. Binding antibodies prepared by the methods of the invention are useful for removing antigens from biological samples.

For example, hepatitis antibodies can be conjugated to polyurethane foam according to the methods of the invention, and the conjugated antibodies thus obtained can be used to remove hepatitis antigens from blood (eg, blood in a blood bank). I can do it.

The binding proteins of the invention can be used in batch or continuous mode.

One method of operating in batch mode is illustrated by a method for the hydrolysis of urea present in an aqueous system. In this method, a piece or more than one piece of foamed polyurethane (i.e., the self-supporting poly(urea-urethane) foam of the invention) with a binding enzyme is added to a batch of an aqueous solution of urea to be hydrolyzed to ammonia. insert. When the hydrolysis is complete, the particles of bound enzyme are removed from the hydrolyzed sample, optionally washed with water, and placed into a separate batch of aqueous solution of the urea to be hydrolyzed. However, we prefer to use the binding protein of the invention in such a way that the form to which it is bound is loaded into a column and the solution to be treated is passed through the column.

This can be carried out in a completely continuous manner, for example with respect to the hydrolysis (conversion) of the sucrose solution by bound invertase. This can be done, for example, by hydrolyzing a number of urea solutions to ammonia with bound urease and analyzing the hydrolyzed samples (the solution coming out of the packed column) for ammonia to determine the amount of urea in each sample. It can also be carried out batchwise, as in the case of quantifying urea by measurement. When using this method, after passing each sample through a packed column, the column is washed with water, and the washing water and a hydrolyzed urea solution (ammonia solution) are mixed together, and the solution and Determine the ammonia content in the wash water. The binding proteins of the invention have a long shelf life.

For example:1. If the binding protein is an enzyme, it does not lose its activity even after several hundred hours of use.

2. If the binding protein is an antigen (useful for removing antibodies from aqueous systems), it becomes depleted (saturated) when it absorbs an equivalent amount of antibody.

It is then necessary to regenerate the binding protein (ie remove the antibody from it). This can be done by running an aqueous regeneration solution through the packed column and washing the regeneration solution out of the regenerated column.
Aqueous solution of glycine hydrochloride compound (e.g. 0.15-3
molarity, preferably about 0.5 molar) is an excellent regeneration solution. Such a glycine hydrochloride oxide solution is
It has a pH of about 2.5. 3. If the binding protein is an antibody (useful for removing antigen from aqueous systems), it becomes exhausted (saturated) when it absorbs an equivalent amount of antibody.
do.

It is then necessary to regenerate the bound protein (ie, remove the antigen from it). This can be done, for example, by running an aqueous regeneration solution, such as glycine hydrochloride as described above, through the packed column and washing the regeneration column as described above. All kinds of enzymes can be bound by the method of the invention.

Such enzymes include: oxidoreductases transferase hydrolytic enzyme lyase isomerase Ligase.

Examples of specific enzymes that can be immobilized (ie, bound) by the methods of the invention are shown in Table 3.

Table 3: Urease, trypsin, lactase, glucose oxidase, chymotrifcine ribonuclease, peroxidase, pepsin, rennin invertase, papain, asparaginase, pectinase, pectin esterase, penicillin amidase, glucose isomerase, lysozyme, amino acid acylase, flonase, alcohol dehydrogenase, alpha-amylase, beta-amylase, subtilisin, amino acid oxidase, catalase, tannase, phenol oxidase glucoamylase fluranase cellulase phycine fromeline pancreatine isoamylase lipase malate dehydrogenase hexokinase lactate dehydrogenase adenosine deaminase urigase galactose oxidase diaphorase cholinesterase aldolase pyruvate carboxylase phosphalylase cephalosporin amidase isocitrate dehydrogenase alpha -glycerophosphate dehydrogenase glyceraldehyde -3-phosphate dehydrogenase malate enzyme glucose-6 monophosphate dehydrogenase 5-dehydroxymate reductase glutathione reductase glycolate oxidase yeast cytochrome C reductase luciferase nitrite reductase glutamyl Transferase glutathione synthetase glucodiamine phosphokinase hippurate synthetase aldehyde oxidase succinate dehydrogenase nitrate reductase xanthine oxidase lipol dehydrogenase flavin peroxidase glycine oxidase carboxylase alpha-keto acid dehydrogenase transketolase.

The invention may be better understood through the following specific but non-limiting examples and procedures.

It is to be understood that the invention is not limited by these examples and procedures, which are given by way of example only; changes may be made without departing from the spirit and scope of the invention. It should also be understood that it can be done. These examples are experiments that were actually conducted.

The procedures, although not performed, are illustrative of some embodiments of the invention. Example 1 2 milliequivalents (17) of polyethylene glycol with an average molecular weight of 1000 were added to 2.63 milliequivalents (0.2297).
An isocyanate capped liquid polyurethane prepolymer was prepared by reacting with toluene diisocyanate.

The thus obtained prepolymer 1 was named ``Prepolymer 1 No. 1''. The above procedure was repeated except that 20 meq of polyethylene glycol and 26.3 meq of toluene diisocyanate were used.

The isocyanate-capped liquid polyurethane prepolymer thus obtained was named Prepolymer No. 1-A. Example 2 A mixture was prepared by mixing Prepolymer No. 12 No. 17 described in Example 9 below and 100 milligrams of human immunoglobulin GOgG).

The IgG was dissolved and the resulting solution was stirred for 15 minutes in a dry atmosphere at about 25°C. Then, while stirring the solution,
At 25° C., 27 grams of water was added thereto. The water-containing system thus obtained began to foam and foam formation was completed within 10 minutes. The foam thus obtained was thoroughly washed with water. Wash water was collected and analyzed for IgG by UV spectroscopy. It was observed that less than 1% of the initially charged IgG was washed out of the foam by the wash water. The change was that 99% of the IgG originally used was bound to the polyurethane foam. The washed form with bound IgG was named "form A". Example 3 Form A was packed into a column.

Human blood containing 100 η of rheumatoid arthritis factor was allowed to flow slowly into the packed column.

Red blood cells were not degraded. Analysis of the blood exiting the column showed that virtually all rheumatoid arthritis factors were removed from the blood. A second portion of human blood containing about 100 η rheumatoid arthritis factor,
It was gradually run into the same column.

This blood sample showed that only a small portion of the rheumatoid arthritis factor was removed, indicating that the activity of the binding protein was depleted. The binding protein was reactivated by passing approximately 100 ml of glycine hydrochloride solution (approximately 0.5 molar with a pH of 2.5) through the packed column. The packed column was washed with water until no glycine hydrochloride oxide was present. A third blood portion containing about 100 rheumatoid arthritis factors was allowed to flow slowly into the packed column. Analysis of the blood flowing out of the column showed that all rheumatoid arthritis factors were removed from the blood. This confirms that bound 1gG was reactivated by treatment with glycine hydrochloride. bond 1g
G can be reactivated for use in a large number of experiments (15-20 or more).

Example 4 (Comparative Experiment) This experiment was conducted to demonstrate the superiority of the method of the present invention over conventional methods for preparing polyurethane-bound proteins.

Isocyanate-capped liquid polyurethane prepolymer No. 1 (Prepolymer No. 2) prepared in Example 9
17 parts of was mixed with a composition prepared by mixing 100 m9 of human IgG with 27 parts of water. The mixture of prepolymer, IgG and water thus obtained was stirred for about 10 minutes. Foaming was completed within a further 5 minutes. The foam was thoroughly washed with water and the wash water was analyzed as before. It was observed that 35% of the IgG used was present in the wash water. The conjugated 1gG prepared in this example was effective in removing rheumatoid arthritis factor from human blood without degrading red blood cells.

However, only about 65% of the amount removed by the conjugated 1gG prepared in Example 2 above was removed before requiring regeneration by treatment with glycine hydrochloride solution. Example 5 A polyurethane binding protein was prepared according to the general method of Example 2.

However, in this case the general method of Example 2 was used except that the enzyme invertase was used instead of IgG and Prepolymer No. 13 prepared in Example 12 was used instead of Prepolymer No. 12. Invertase was coupled to polyurethane. Enzyme (invertase) 9
1% polyurethane foam (poly(urine purple-urethane))
form).

The conjugated enzyme thus obtained was highly effective for the conversion of sucrose solutions. This enzyme activity lasted for many (1440) hours when the sucrose solution was continuously passed through the column packed with bound invertase. At the end of this time, the bound enzyme still retained all of its initial activity. Example 6 (Comparative Experiment) When the method of Example 5 was modified to mix the enzyme (invertase) with water and then add this enzyme-water mixture to the prepolymer, 68% of the charged enzyme was converted to polyurethane. It was just a combination.

The remainder of the charged enzyme (32% of it) was found in the wash water obtained by washing the foamed polyurethane with bound enzyme with water. Example 7 Asparaginase was attached to polyurethane foam using the general method of Example 5 and using asparaginase in place of invertase.

In this case, 75% of the enzyme (asparaginase) was bound to the foam. The bound enzyme was found to be highly active, and it was tested in 12 batches with a 1 hour wash between runs when used to convert asparagine (amide) to aspartic acid. while,
It retained its activity. Example 8 (Comparative Experiment) When repeating the method of Example 7 except changing the enzyme (asparaginase) to mix it with water before mixing it with the isocyanate capped polyurethane prepolymer, the production The enzyme did not bind to any form.

All of the enzyme was washed away from the foam by the wash water, and the washed foam showed no enzyme activity. Thus, when comparing Example 7 with Example 8 above, the superiority of the method of the invention is clear.

Example 9 2 moles of polyethylene glycol (PEGlOOOO) having an average molecular weight of 100-1 are mixed with 1 mole of trimethylolpropane (TMP).
It was dried at 10°C.

A total of 7 moles (1197) of reactive terminal hydroxyl groups (-0H
) was slowly added to a reactor containing 6.65 moles of toluene diisocyanate (TDI) while stirring the toluene diisocyanate and the product mixture (approximately 1 It took time). The toluene diisocyanate and product mixture in the reactor are 6
It was kept at 0°C. After all of the PEGlOOO-trimethylolpropane mixture had been added to the reactor, the resulting mixture was stirred for 3 hours while being maintained at about 60°C. An additional 1.05 moles of toluene diisocyanate were then added and stirring continued for an additional hour while maintaining the temperature of the mixture at approximately 60°C. Thus PEGlOOOO
- A 10 mole percent excess of toluene diisocyanate was added to the trimethylolpropane mixture. This means that all the hydroxyl groups in the polyol (PEGlOOOO plus trimethylolpropane) are capped by isocyanate and that some chain extension occurs due to crosslinking of the polyol with excess toluene diisocyanate. , made sure. Thus obtained,
The resulting liquid isocyanate-capped polyurethane prepolymer was named 6Fre Polymer No. 2゜゛. Prepolymer No. 12 can be used for protein binding according to the method of the invention. The physical properties of the foam with the binding protein can be varied by varying the proportions of polyols used to prepare the isocyanate-capped liquid polyurethane prepolymers used in the method of the invention.

The physical properties can also be changed by using other polyols, such as those selected from the table of polyols above. Example 10 A mixture was prepared using 17 Prepolymer No. 12 and Fungal Lactase No. 5 at about 25°C.

The first mixture thus obtained was stirred for 15 minutes at about 25° C. in a dry atmosphere. 27°C of water to the first mixture, producing a second mixture at about 25°C.
was added while stirring. The second mixture thus obtained completed foam formation within 10 minutes after starting foaming. The foam thus obtained was thoroughly washed with water at about 25°C. Wash water was collected and analyzed for lactase by ultraviolet spectroscopy. Less than 1% of the initially charged lactase was washed out of the foam by the wash water. In other words, 99% of the initially charged lactase was bound to the polyurethane foam. Example 1
The washed conjugated (immobilized) enzyme prepared in Example 10 using 15 instant lactase was assayed using a buffered lactose solution.

The following method was used: Experiment 1: All of the bound enzyme was placed in a flask containing a predetermined amount of buffered lactose solution;
The rate of glucose production as a function of time was measured.

Experiment 2: A similar test was performed at substantially the same time using 5η free (unconjugated) enzyme from the same rod used for the preparation of the conjugated enzyme in place of the conjugated enzyme.

The rate of hydrolysis was the same in Experiment 1 and Experiment 2, and the bound (immobilized) lactase retained its full activity, ie, the activity of the enzyme was not lost upon binding of the enzyme.

The foam (bound enzyme) was taken out from Furaneko and washed with water.

In a repeat of experiment 1, using washed foam, and in a repeat of experiment 2, using 5 immediately free (unbound) enzyme from the same rod used in the original experiment 2. Experiments 1 and 2 above were repeated.
The results obtained in these experiments (repeat experiments 1 and 2) were identical to those obtained in the first experiments 1 and 2.

Example 12 Oxidation of glycerin 317 in an amount to form an intermediate (hydroxyl capped polyether) 5007 having an equivalent weight of about 500 (i.e. containing about 17y -0H groups per 5007 intermediate) An isocyanate-capped liquid polyurethane prepolymer was prepared by reaction with ethylene.

This intermediate was reacted with commercially available toluene diisocyanate using 1.05 moles of toluene diisocyanate per 5007. The isocyanate-capped liquid polyurethane prepolymer No. 1 thus obtained was named "Prepolymer No. 13". Example 13 Part 37 of the above Prepolymer No. 12 was mixed with 2 ml of benzene and mixed for 5 minutes.

The first mixture thus obtained was then mixed with 10 grams of jack bean urease to form a second mixture, which was stirred for 15 minutes at about 25°C and then mixed with water to produce foam. . This mixture was stirred for approximately 5 minutes. After foam production was complete, the self-supporting foam was cut into small pieces (approximately 25 cubic meters each).

Pieces of these foams containing immobilized urease were washed for 18 hours. The initial rate of ammonia production obtained when a sample of a small piece of washed foam containing immobilized urease is hydrolyzed by a portion of the standard urea solution by the immobilized urease is expressed as: It was assayed for urease activity by comparing the initial rate of ammonia production obtained when hydrolyzed with a defined amount of free (unbound) jack bean urease.

The results of such an assay showed that 11% of the initially charged urease was present in the foam as active immobilized urease.

Example 14 The general procedure of Example 13 was repeated, but this time ethylene glycol was used instead of benzene.

In this case, assaying the foam containing immobilized urease according to the general method of Example 13 showed that 3.5% of the initially charged urease was present in the foam in the active state.

Example 15 The general procedure of Example 2 was repeated.

However, in this case: (a) 10W19 urease is used instead of IgG; and (b) 2 ml of ethylene glycol is added to it after stirring the mixture of Prepolymer No. 12 and urease for 15 minutes, and After stirring the resulting composition for about 5 minutes, 27 of water was added to it to give a self-supporting foam containing the immobilized (bound) protein (in this case the enzyme urease). Assaying a self-supporting foam containing immobilized urease following the general method of Example 13 shows that 21% of the initially loaded urease is present in the form as active immobilized (bound) urease. showed that.

Example 16 The general procedure of Example 15 was repeated.

However, in this case, the method was modified by (a) mixing urease (10 TI19) with 2 ml of ethylene glycol; and (b) 3 Ii! for the urease-ethylene glycol mixture. Prepolymer No. 12 was added to form a composition consisting of urease, ethylene glycol, and Prepolymer No. 1. The composition was then mixed with water as in Example 15 to produce a bound (immobilized) urease-containing foam.
Assay of the foam according to the general method used in Example 13 showed that 3.7% of the initially charged urease was present in the foam as active immobilized urease.

The diluent used in Examples 13-16 lowered the viscosity of the isocyanate capped liquid polyurethane prepolymer.

The amount of diluent used is not critical, and a reduction in the viscosity of the prepolymer is often used, since a diluted (lower viscosity) prepolymer is easier to pour and stir.
Makes operation easier. However, the viscosity must not be reduced to such an extent that the prepolymer plus diluent plus protein cannot provide a self-supporting foam when mixed with water to produce foam. Example 17 We have shown that certain enzymes (such as penicillin amidase, glucoamylase, glucose isomerase, and amino acid acylase) that can be immobilized (conjugated) by the method of the invention are It is recognized that it exhibits lower enzyme activity (based on the amount of enzyme actually present) than in the free (non-immobilized) form.

Although we do not wish to be bound by theory, we believe that this is because these enzymes have primary or secondary amino groups in their active sites. and that such amino groups of some of the bound (immobilized) enzyme molecules to the poly(urea-urethane) forms of the present invention, including bound (immobilized) enzymes, are reacts with the isocyanate-capped liquid polyurethane prepolymer of the present invention to form a urea moiety, thereby
It is thought that the active site consisting of such an amino group may be deactivated.

We believe that such a reduction in the activity of an enzyme is caused by the presence of so-called "substrates" (substances that act on the specific enzyme (e.g. benzylpenicillin in the case of penicillin amidase, starch in the case of glucoamylase, glucose isomerase). glucose in the case of amino acid acylases, and acylated amino acids such as acetylated glycine in the case of amino acid acylases) are added to the isocyanate-capped liquid polyurethane prepolymer prior to mixing the prepolymer with the enzyme to be immobilized. By mixing, it can be significantly reduced.

Experiment 1 below, compared with Experiment 2 below, illustrates the advantages of this method.

Experiment 1 The general procedure of Example 2 was repeated.

However, in this case: (a) before adding the protein to Prepolymer No. 1 (Prepolymer No. 12 described in Example 9), 50 to 50 benzylpenicillin is mixed with Prepolymer No. 1, and (b) the protein is I used in Example 2
Not gG, but 50Tf! 9 penicillin amidase. 65% of the penicillin amidase charge was bound to the poly(urea-urethane) form (i.e., 65% of the penicillin amidase charge was immobilized), and 42% of the immobilized penicillin amidase maintained its enzymatic activity. It was approved to do so.

Experiment 2 The general procedure of Experiment 1 was repeated.

However, in this case the addition of benzylpenicillin to the prepolymer was omitted. Although 66% of the charged penicillin amidase was bound (immobilized), only 14% of the immobilized penicillin amidase was found to maintain its enzymatic activity.

In Experiment 1 of Example 17, the amount of enzyme washed out from the foam containing the immobilized enzyme was quantified by spectrophotometry.

The difference between this value and the amount of loaded enzyme indicated the amount of bound (immobilized) enzyme present in the foam. The amount of immobilized enzyme that maintained enzymatic activity was determined by the general method used for quantifying the activity of immobilized urease in Example 3.

However, in this case, a standard benzylpenicillin solution was used instead of a standard urea solution, and free, unimmobilized penicillin amidase was used instead of free, unimmobilized urease. The same general method was used to measure: (a) binding in Experiment 2 of Example 17 (
(b) the enzymatic activity of the immobilized penicillin amidase in Experiment 2.

Step 1 Isocyanate capped liquid polyurethane prepolymer 1 (this is called ``prepolymer 1P-1'') is added to 1
007 ethylene glycol and 5617 toluene diisocyanate and the resulting mixture at about 65°C.
It can be prepared by keeping it at for about 1/2 hour.

Procedure 2 The general method of Example 2 can be repeated, except that Prepolymer No. 12 used in Example 2 is replaced by Prepolymer 1 P-1 described above.

The results appear to be substantially the same as those obtained in Example 2, except that the wash form with bound IgG is designated as "Form A-P."

Step 3 The general method of Example 3 can be repeated with the modification that Form A-P is used instead of Form A.

The results appear to be substantially the same as those obtained in Example 3.

Step 4 The general method of Example 5 can be repeated, except that Prepolymer No. 1-1-P is used instead of Prepolymer No. 12.

The results appear to be substantially the same as those obtained in Example 5.

Procedure 5 In the absence of water, the first product can be generated by mixing 67 toluene diisocyanate and 100T19 urease.

A second product can be produced by mixing the first product and 17 ethylene glycols in the absence of water. The foam containing the enzymatically active immobilized urease is combined with a second product and an amount of water (e.g., the second product 1) effective to produce foam.
7 parts water). The thus obtained poly(urea-urethane) foam containing bound (immobilized) urease exhibits enzymatic activity; i.e., urea in aqueous solution (e.g.
Addition of this foam to 0.3 moles of urea causes hydrolysis of the urea to produce ammonia and carbon dioxide. Step 6 In place of the ethylene glycol used in Step 5, an equivalent amount (based on -0H groups) of polyethylene glycol having an average molecular weight of 1000 (e.g. PEGlOOOO)
can be used.

In this case, the result would be substantially the same as that obtained in Step 5 [i.e., an aqueous urea solution (e.g. 0.03 mole urea per 11) would not result in hydrolysis of urea. Dew]. Procedure 7 The first product can be formed by mixing 17 ethylene glycol and 100T19 urease in the absence of water.

A second product can be formed by mixing the first product and 67 toluene diisocyanate in the absence of water. A foam containing immobilized enzymatically active urease is prepared by mixing a second product and an amount of water effective to produce foam (e.g., 17 parts water to 17 parts second product). can be generated. The poly(urea-urethane) foam containing bound (immobilized) urease thus obtained exhibits enzymatic activity; i.e., addition of this foam to an aqueous urea solution (e.g. 0.03 moles of urea per 1 f) inhibits the hydration of urea. Decomposition will result in the production of ammonia and carbon dioxide. Procedure 8 Substitute the equivalent amount of ethylene glycol used in Example 7 with an average molecular weight of 100
0 polyethylene glycol (e.g. PEGlOO
O) can be used.

The results in this case are virtually identical to those obtained in Procedure 7 [i.e., addition of this form to an aqueous urea solution (e.g. 0.03 moles of urea per f) results in hydrolysis of the urea. Will〕. As used herein, the term "polyisocyanate" includes diisocyanates.

The isocyanate-capped liquid polyurethane prepolymer used in the present invention contains at least two isocyanate groups (reactive isocyanate groups) per molecule of the prepolymer.

Claims (1)

[Scope of Claims] 1. A process for producing immobilized proteins chemically bonded to polyurethane foam, which comprises: (a) (i) mixing an isocyanate-capped polyurethane prepolymer with a protein in the absence of water in a liquid phase; to form a solution, or (i
i) the polyisocyanate is mixed with the protein in the liquid phase in the absence of water and then the product is mixed with the polyhydroxy compound; or (iii) the polyhydroxy compound is mixed in the liquid phase in the absence of water. foaming by mixing the product with the protein and then mixing the product with the polyisocyanate; and (b) mixing the solution thus obtained with an amount of water effective to produce foam. Characteristic method for producing immobilized proteins. 2. A method according to claim 1, characterized in that after step (b), the immobilized protein is washed to remove unbound protein and to hydrolyze any unreacted isocyanate groups. 3. The isocyanate-capped liquid polyurethane prepolymer is prepared by reacting toluene diisocyanate and polyethylene glycol, preferably having a molecular weight of about 800 to 1200. Or the method described in Section 2. 4. The isocyanate-capped liquid polyurethane prepolymer contains toluene diisocyanate from about 800 to
Prepared by reacting a mixture of polyethylene glycol and trimethylolpropane having a molecular weight of 1200, trimethylolpropane and polyethylene glycol are used in a molar ratio of about 1:1 to 4 and toluene diisocyanate is mixed with polyethylene glycol. and trimethylolpropane in a proportion of about 0.85 to 1.25 moles per equivalent of --OH provided by trimethylolpropane. 5. Isocyanate-capped liquid polyurethane prepolymers include toluene diisocyanates selected from polyoxybutylene polyol polymers, ethylene glycol, diethylene glycol, polyoxyethylene polyols, pentaerythritol, glycerin, trimethylolpropane, and polyoxypropylene polyols. The method according to claim 1 or 2, characterized in that it is prepared by reacting with a polyalcohol. 6. The method according to any one of claims 1 to 5, wherein the protein is an enzyme, an antibody, or an antigen. 7. The method according to claim 6, wherein the protein is human immunoglobulin G. 8 Proteins are enzymes and enzymes are urease trypsin lactase glucose oxidase chymotrypsin ribonuclease peroxidase pepsin rennin invertase papain asparaginase pectinase pectin esterase penicillin amidase glucose isomerase lysozyme amino acid acylase pronase alcohol dehydrogenase α-amylase β-amylase subtilisin amino acid oxidase catalase tannase Phenol oxidase glucoamylase pullulanase cellulase phycin bromelene pancreatine isoamylase lipase malate dehydrogenase hexokinase lactate dehydrogenase adenosine deaminase uricase galactose oxidase diaphorase cholinesterase aldolase pyruvate carboxylase phosphalylase cephalosporin amidase isocitrate dehydrogenase alpha -glycerophosphate dehydrogenase glyceraldehyde -3-phosphate dehydrogenase malate enzyme glucose-6-phosphate dehydrogenase 5-dehydroxyquimate reductase glutathione reductase glycolate oxidase yeast cytochrome C reductase luciferase nitrite reductase glutamyl transferase glutathione synthetase glucosyamine phosphokinase hippurate synthetase aldehyde oxidase succinate dehydrogenase nitrate reductase xanthine oxidase lipoyl dehydrogenase flavin peroxidase glycine oxidase carboxylase α-keto acid dehydrogenase or transketolase The method described in scope item 6. 9 (a) (i) the isocyanate-capped polyurethane prepolymer is mixed with the protein in the liquid phase in the absence of water to form a solution; or (ii) the polyisocyanate is mixed with the protein in the liquid phase in the absence of water. or (iii) mixing the polyhydroxy compound in the liquid phase with the protein in the absence of water and then mixing the product with the polyisocyanate. and (b) causing foaming by mixing the solution thus obtained with an amount of water effective to produce foaming. 1. A method for producing a polyurethane prepolymer and a protein, which comprises incorporating a substrate capable of reacting with the protein into the mixture before adding water to the mixture. 10. The method according to claim 9, wherein the protein is an enzyme. 11 The enzyme is penicillin amidase and the substrate is benzylpenicillin, or the enzyme is glucoamylase and the substrate is starch, or the enzyme is amino acid acylase and the substrate is an acylated amino acid. The method according to claim 10.