IE42608B1 - Catalyst for biochemical reaction and method of preparing it - Google Patents

Description

This invention relates to a composite catalyst for accelerating biochemical reactions and a method of preparing it.

The invention is an improvement over an 5 invention disclosed in British Patent Specification No. 1,429,711.

In this earlier patent we have described a method of making a polyurethane cross-linked foam which comprises mixing and reacting a first component comprising I an isocyanate-capped polyoxyethylene polyol having a reaction functionality greater than two, a second component which comprises water and optionally a third component comprising a cross-linking agent having a reaction functionality greater them two in which case the reaction i functionality of the capped polyol may be at least equal to about two, the ratio of moles in the second component to moles NCO groups in the first component being in the range from 6.5 to 390. In that process a large stoichiometric excess of water is used, serving as reaction medium and foaming agent and there is produced a cross-linked polyurethane foam. A third component as cross-linking agent is not essential but if it is not present the reaction functionality of the polyol must be above two. This earlier patent also claims a foamable composition comprising the above specified components and cross-linked hydrophilic foam obtained by the above-specified process.

According to our said earlier patent there may be present in the second, aqueous component an additive such as a flame retardant, pigment or dye which improves the properties of the cross-linked polyurethane foam, and in the list of twenty five possible additives is included enzymes, without however saying what is the purpose of having the enzyme present. Thus a product according to our said earlier patent may be a crosslinked polyurethane foam in which is bound an enzyme exemplified by urease.---------------------------------------- 2a 42608 We have now found that a polyurethane foam containing any one of a number of determined enzymes bound to the molecule, can be used aa a composite catalyst for the biochemical reaction to which that enzyme is specific* The enzymes which we have found to provide a valuable composite catalyst for a biochemical reaction when chemically bound to previously free ieocyanate groups in the polyurethane molecule are: cellulase, pectinase, papain, bromelain, chymotrypsin, trypsin, ficin, lysozyme, lactase, penicillin amidase, amyloglucosidase, glucose isomerase, alpha amylase, amino acid acylase, amino acid oxidase, asparaginase, glucose oxidase, invertase, peroxidase, pullulanase and rennin.

The invention also includes a method of making the composite catalyst of this invention which method comprises contacting a polyurethane prepolymer having at least two free ieocyanate groups per molecule with an aqueous dispersion of the chosen enzyme under foam-forming conditions whereby the prepolymer foams to a polyurethane foam having the enzyme bound in the molecule· t We have established that if one of the enzymes ι specified above is dispersed in the second, aqueous, reactant in the process of our earlier patent, it becomes chemically bound to the polyurethane foam, and that process is the preferred method of making the composite catalyst of the present invention.

There has previously been disclosed, in U.S. Patent . 3,672,955, a procedure for providing a composite catalyst wherein an enzyme acts as cross-linking agent for a polyurethane, so that the enzyme becomes bound in the polyurethane molecule, and the unfoamed enzyme-bound polyurethane ia oeated onto a partisuiata sarriar auflh aa oalcined rice hulls. - 3 43608 In the catalyst of the present invention certain enzymes can be included by chemical bonding in the polyurethane molecules, and the resultant enzymebound polyurethane foam can be used as a composite catalyst for the biochemical reaction to which the enzyme is the appropriate catalyst, without the need to coat the polyurethane onto carrier particles.

In the foam, there is more enzyme activity than when the same amount of enzyme is in an unfoamed polyurethane coating. The foam can suitably be shaped to fit a reaction column, or can be divided into pieces of desired size.

We have determined that one of the stated enzymes, when chemically bound in a polyurethane foam according to the present invention, retains much more of its activity than the same enzyme when bound to a polyurethane according to U.S. Patent 3,672,955, especially after wading. This is demonstrated in the final example of this specification.

In the process of U.S. Patent 3,672,955 an aqueous dispersion of the enzyme is mixed with an organic solution of a pdyisocyanate to form an emulsion which is coated onto the carrier particles, and although the water then reacts with the isocyanate groups to form bubbles of carbon dioxide, this gas is immediately released because - 4 '42608 of the very low viscosity of the solution, which in turn, is Caused by the use of the water-immiscible solvent.

'In the process of the present invention these gas bubbles are retained? foam-forming conditions are employed, To help retain the gas bubbles it is very desirable, that the· prepolymer be added to the dispersion of the enzyme as such, without first adding a water-immiscible solvent of diluent to the prepolymer to reduce the viscosity of the polyurethane-forming mixture..

The polyurethane prepolymer can be produced in the known way by the reaction of di- and triisocyanates and . other polyisocyanates'with compounds containing active hydrogen, particularly glycols, polyglycols, polyester polyols and polyether polyols. Preferably the prepolymer > is a polyoxyethylene polyol (a polyol which includes -OCHjCHj- units), which when capped with the isocyanate has a reaction functionality of at leaet 2, preferably more than 2. This reaction makes an isocyanate-capped polyurethane prepolymer. The enzyme preferably in an ) aqueous medium, is bound by bringing it into contact with the polyurethane prepolymer before the polyurethane is foamed· Since the polymerization reaction (e.g. of the polyol with the isocyanate) is exothermic, the temperature of the reaction mixture must self-evidently be maintained at below the temperature of thermal denaturation for the j enzyme. In the binding step, the enzyme is maintained in a native conformation by the use of the appropriate pH, ionic stsngth, presence of enzyme substrate, or necessary cations. The bound enzyme so produced is * catalytically active.

. The physical form of the enzyme is not critical. Binding has beep done using pure crystalline enzymes - 5 42608 (lysozyme, trypsin); with partially purified noncrystalline enzymes (papain, bromelain); with impure extracts containing enzyme activity (ficin, pectinase); with unseparated fermentation broths containing an extracellular enzyme without purification or concentration (cellulase) and with intracellular enzymes bound to the cell walls (glucose isomerase) * Our work indicates that our invention can be used to bind enzymes of substantially any purity. Under appropriate enzyme reaction conditions the bound enzyme of the present invention may be used for the following conversions: starch to sugars (enzyme amyloglucosidase), glucose to fructose (enzyme β glucose isomerase), lactose to glucose (enzyme = lactase) and penicillin G to 6-aminopenicillanic acid (enzyme «> penicillin amidase).

The reaction mechanism is apparently the reaction of one or more amino groups on the enzyme with one or more isocyanate groups on the polyurethane prepolymer molecule. Hence, the polyurethane prepolymer must contain at least two free isocyanate groups. Therefore in a polyisocyanate/ polyol reaction used to prepare the prepolymer, a suffiient stoichiometric excess of isocyanate groups in the polyisocyanate reactant over hydroxyl groups in the polyol reactant is required to provide an average of two or more free isocyanate groups in the resulting polyurethane prepolymer.

The term polyurethane prepolymer is used herein in a broad sense, to include prepolymers which contain a urethane group in the molecule and compounds which contains a urea or other analogous group in place of the urethane group. Further, the term polyurethane is correspondingly used in a broad sense, to include final polymers whih contain urea or other analogous linkages in place of urethane ones. Reaction of an amino group on the enzyme with an isocyanate group of the prepolymer produces a urea linkage.

Any polyurethane prepolymer which contains at least two free isocyanate groups per molecule is suitable for use in the method of this invention. We prefer that the polyurethane prepolymer contains an average of two isocyanate groups per molecule, but it may contain up to eight. Higher molar contents of isocyanate groups are operable, but offer no advantage. Xn any case, all excess isocyanate groups left in the polyurethane fdatn (after binding of the enzyme) will be destroyed by hydrolysis upon the first contact of the foam with water, for example, during a washing step before the bound enzyme is used. The water in the 6.5 - 390 moles above referred to may include that used in such subsequent washing steps to hydrolyze residual isocyanate groups in the polyurethane foam.

Representative examples of polyisocyanates which can be reacted with an active hydrogen containing compound (e.g. a glycol, polyol, polyglycol, polyester polyol, polyether polyol)to make an isocyanate-capped polyurethane prepolymer for use in the invention include: toluene-2,4-diisocyanate toluene-2,6-diisocyanate commercial mixtures of toluene-2,4- and 2,6diisocyanates 426θ8 ethylene· diisocyanate ethylidene diisocyanate propylene-1,2-diisocyanate cyclohexylene-1,2-diisocyanate cyclohexylene-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-hexamethylenediisocyanate 1.4- tetramethylene-diisocyanate 1,10-decamethylenediisocyanate 1.5- naphthaleriediisocyanate cumene-2,4-diisocyanate 4-methoxy-l,3-phenylenediisocyanate 4-chloro-l,3-phenylenediisocyanate 4-bromo-l,3-phenylenediisocyanate 4-ethoxy-l,3-phenylenediisocyanate 2,4'-diisocyanatodiphenylether .6- dimethyl-l,3-phenylenediisocyanate 2.4- dimethyl-l,3-phenylenediisocyanate 4,4'-diisocyanatodiphenylether benzidinediisocyanate 4.6- dimethyl-l,3-phenylencdii socyanate 9 , lO-anthrace-nediisocyanate 4,4'-diisocyanatodibenzyl 3,3'-dimethyl-4,4'-diisocyanatodiphenylmethane 2,b-dimethyl-4,4'-diisocyanatodiphenyl 2.4- diisocyanatostilbene - 8 42608 3,31-dimethyl-4,4’-diisocyanatodiphenyl 3,3 *-dimethoxy-4,4'-diisocyanatodiphenyl 1.4- anthraeenediisocyanate 2.5- fluorenediisocyanate 1,8-naphthalenediisocyanate 2.6- diisocyanatobenzfuran 2.4.6- toluenetriisocyanate, and p,p',p-triphenylmethane triisocyanate.

A useful class of polyurethane prepolymers is those derived from polyether polyols and polyester polyols. These compounds may be prepared, as well known in the art, by reacting a polyether (or polyester) polyol with a polyisocyanate, using an excess of the latter to ensure provision of free isocyanate groups in in the product. A typical, but by no means limiting example is illustrated in idealised equation form below; •H Polyether polyol + CH, CH, OCN· -NCO NCO Polyi socyanate •O.

(CO Isocyanate-capped polyurethane - 9 42608 (In the above formulae, m represents the number of tetramethyleneether repeating units. This may range, for example, about from 5 to 50.) The polyurethane prepolymer useful for the 5 purposes of the invention may be prepared by reacting any of the above-exemplified polyisocyanates with any of a wide variety of polyether polyols and polyester polyols, and representative examples of these polyols are described below.

Among the polyether polyols which may be so used are those prepared by reaction of an alkylene oxide with an initiator containing active hydrogen groups, a typical example of the initiator being a polyhydric alcohol such as ethylene glycol; a polyamine such as ethylene diamine; phosphoric acid, etc. The reaction is usually carried out in the presence of either an acidic or basic catalyst. Examples of alkylene oxides which may be employed in the synthesis include ethylene oxide, propylene oxide, any of the isomeric butylene oxides, and mixtures of two or more different alkylene oxides such as mixtures of ethylene and propylene oxides. The resulting polymers contain a polyether 10 4.2 6 0 8 backbone and are terminated by hydroxyl groups. The number of hydroxyl groups per polymer molecule is determined by the functionality of the active hydrogen initiator. For example, a difunctional alcohol such as ethylene glycpl (as the active hydrogen initiator) leads to polyether chains in which there are two hydroxyl groups per polymer molecule. When polymerisation of the oxide is carried out in the presence of glycerol, a trifunctional alcohol, the resulting polyether molecules contain an average of three hydroxyl groups per molecule. Even higher functionality - more hydroxyl groups - is obtained when the oxide is polymerised in the presence of such polyols as pentaerythritol, sorbitol, sucrose or dipentaerythritol. In addition to those listed above, other examples of poly15 hydric alcohols which may be reacted with alkylene oxides to produce useful polyether polyols include: 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-cyclohexanediol 2- butene-l,4-diol 3- cyclohexane-l,1-dimethanol 4- methyl-3-cyclohexene-l,1-dimethanol 3-methylene-l,5-pentanediol diethylene glycol (2-hydroxye thoxy)-1-propano1 4- (2-hydroxyethoxy)-1-butanol - (2-hydroxypropoxy)-1-pentanol 5 1-(2-hydroxymethoxy)-2-hexanol 1- (2-hydroxypropoxy)-2-octanol 3-allyloxy-l,5-pentanediol 2- allyloxymethyl-2-methyl-l,3-propanediol [(4-pentyloxy)methyl]-1,3-propanediol 3-(o-propenylphenoxy)-1,2-propane diol j thiadiglycol ι 2,2*-[thiobis(ethyleneoxy)]diethanol polyethyleneether glycol (molecular weight about 200) 2,2’-isopropylidenebis(p-phenyleneoxy) diethanol 1,2,6-hexanetriol 1.1.1- trimethylolpropane 3- (2-hydroxyethoxy)-1,2-propanediol 3-(2-hydroxypropoxy)-1,2-propanediol 2,4-<3imethyl-2- (2-hydroxyethoxy)methylpentanediol-1,5 1,1,1-tris[(2-hydroxyethoxy)methyl)ethane 1.1.1- tris[(2-hydroxypropoxy)methyl]propane triethanolamine triisopropanolamine resorcinol pyrogallol phloroglucinol hydroquinone 4,6-di-tertiarybutyl catechol catechol orcinol _ 12 methylphloroglucinol hexylresorcinol 3-hydroxy-2-naphthol 2-hydroxy-l-naphthol 2,5-dihydroxy-l-naphthol bis-phenols such as 2,2-bis(p-hydroxyphenyl)propane and bis-(p-hydroxyphenyl)methahe 1.1.2- tris-(hydroxyphenyl)ethane 1.1.3- tris-(hydroxyphenyl)propane An especially useful category of polyether polyols are the polytetramethylene glycols. They are prepared by the ring-opening polymerisation of tetrahydrofuran, and contain the repeating unit -CH;;-CH^-CH^ CH^—‘O in the polymer backbone. Termination of the polymer chains is by hydroxyl groups.

Also especially desirable are the polyoxyethylene polyols HO-4CH2CH2-O-)^.H, because of their tolerance for and compatibility with aqueous solutions Of enzymes.

The polyester polyols which may be employed as precursors of the prepolymers are most readily prepared by condensation polymerisation of a polyol with a polybasic acid. The polyol and acid reactants are used in such proportions that essentially all the acid groups are esterified and the resulting chain of ester units is terminated by hydroxyl groups. Representative 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.brassylic acid, thapsic acid, maleic acid, fumaric - 13 42608 acid, glutaconic acid, α-hydromuconic acid, β-hydromuconic acid, α-butyl-a-ethylglutaric acid, a,β-diethylsuccinie acid, o-phthalic acid, isophthalic acid, terephthalic acid, hemimellitic acid, trimellitic acid, trimesic acid, mellophanic acid, prehnitic acid, pyronrellitic acid, citric acid, benzenepentacarboxylic acid, 1,4-cyclohexane dicarboxylic acid, diglycollic acid, thiodiglycollic acid, dimerised oleic acid and dimerized linoleic acid. Representative examples of polyols for forming 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-pentane diol, 1,4-pentane diol, 1,3-pentane diol, 1,6-hexane diol, hexene-1,6-diol, 1,7-heptane diol, diethylene glycol, glycerine, trimethylol propane» 1,3,6-hexanetriol, trimethanolamine, pentaerythritol, sorbitol, and any of the other polyols listed hereinabove in connection with the preparation of polyether polyols. The water-soluble (polyethylene glyaol based) polyurethanes represent a a preferred class for this process.

On being intimately contacted with the aqueous enzyme dispersion, the free isocyanate group-containing polyurethane prepolymer becomes chemically very active.

Some of its free isocyanate groups react with the amine groups of the enzyme, and some react with water to give carbon dioxide and to form amine groups on the polyurethane molecule. These latter amine groups react with free isocyanate groups on neighbouring polyurethane molecules, and this reaction (forming a urea linkage) will cause further growth of the polyurethane and will also introduce - 14 4.2608 cross-links between the polyurethane molecules. This further preliminary growth and cross linking is essential for the formation of a good polyurethane foam. We have found that this growth must occur during contact with the enzyme for binding of the enzyme to take place. If the growth and foaming reactions are permitted to go to completion in the absence of the enzyme, the enzyme Will not bind to the finished foamed polyurethane.

Other additives such as crosslinking agents (polyamines, polythiols, polyacids) or antioxidants, fillers, etc. may also be present during foaming.

The bound enzyme product of this invention can he used in either a broth or continuous process. In either case it can be re-used indefinitely. For example, bound enzyme was used in a batch process; after reaction was complete it was removed by filtering, centrifuging or just lifting out if it was in a single piece. The bound enzyme was then placed in another substrate reactor and was still fully active. Bound urease was placed in column and substrate passed through in continuous fashion. The enzyme was still active after six months of operation.

The following Examples illustrate without limiting the invention. 43608 iExample 1 Ethylene glycol (100 gm) and toluene diisocyanate (282 gm) were mixed in a constant temperature bath at 65°C. After the material became water clear, it was cooled to 4®C. and 100 ml of a fermentation broth containing cellulase activity was added with constant stirring at 4°C. After polyurethane foam formation was complete, about 15 minutes, the foam was washed with water and assayed for cellulase activity using carboxy10 methyl cellulose as substrate and found to be active.

Example 2 The procedure of Example 1 was followed except that 100 ml of a 10% solution of pectinase in a 0.1 M phosphate buffer of pH of 5 was used instead of broth. After poly15 urethane foam formation was complete, the foam was washed with water, cut into small pieces, and placed into a column. Freshly prepared apple juice containing 0.1% sodium benzoate was passed through the column at a rate of 1 liter per hour. The column clarified the apple juice as it passed through it, and the product had no changed flavor characteristics. Example 3 A prepolymer was prepared in which 690 g. polyethylene glycol of molecular weight 1000 containing 310 g. pentaerythritol was reacted with 1830 g. of toluene diisocyanate.

(Pentaerythritol contributes cross-linking and improves heat-stability.) The polyurethane prepolymer (8.0 gm) was cooled to 4eC. and then added to a 4°C. solution containing 1.0 gm trypsin dissolved in 5.0 ml of a 0.1 M trishydroxyethylaminoethane buffer of pH 8.0 containing 0.01 M calcium chloride. The mixture was stirred and maintained at 4°C. until it began to foam. After polyurethane foam formation 8 was complete (about 15 minutes) the foam was washed in the above buffer-salt solution until the wash water showed no ultraviolet absorption. The washed polyurethane foam was assayed for trypsin activity by placing it in a buffered solution of 1% casein and measuring the increase of trichloroacetic acid soluble absorption at 280 pm as a function of time, and found to be active.

Example 4 Propylene glycol (10 g) was added to toluene diisocyanate (10 g) in a metal dish. The dish was placed on a hot plate and the compounds stirred until a homogeneous mixture was obtained, care being taken that no boiling or vaporization of the liquid took place.

The solution was removed from the heat and 1.0 ml of a solution containing 10% amyloglucosidase in 0.1 M phosphate buffer, pH 7.0, was added. The mixture was stirred rapidly until it became viscous (approximately 10 to 15 minutes). The material was then allowed to stand overnight at room temperature to finish polyurethane foam formation. The product was then submerged for 12 hours in water to remove any excess NCO groups. . The resultant product was a crystalline polyurethane foam which was washed and shown to be active in hydrolyzing a 1% starch solution.

Example 5 An elastomeric foam which contained bound amyloglucosidase was prepared in the manner of Example 4, except the amount of propylene glycol used was 20 g.

Example 6 A commercial polyisocyanate containing 9.5% free NCO and having 7 repeating butoxy groups (10 g) was - 17 42608 added to 1.0 ml of an enzyme solution (same as Example 4).

The mixture was stirred until a viscous foam was obtained (5 to 10 minutes). The material was then allowed to stand overnight at room temperature to finish foam formation. The product was submerged for 12 hours in water to remove excess NCO groups. The resultant foam bound enzyme was washed and was enzymatically active.

Examples 7 and 8 Example 3 was repeated except that the pentaerythritol was consecutively replaced first hy glycerol (Example 7) and second by trimethylol propane (Example 8). Similar results were obtained.

Example 9 Polyethylene glycol of molecular weight 1000 was dried by heating for two hours at 110°C under nitrogen at reduced pressure. Toluene diisocyanate (1.48 moles per mole of hydroxyl) was added gradually with stirring while the temperature wae maintained at 30°C in a cooling bath. After addition was complete, the temperature was raised to 60°C until reaction was complete (about two hours). This prepolymer (10 gm) was added to a solution of 10% lactase enzyme in a 4% lactose solution (10 gm). The reactants were stirred until foaming began. The resultant foam was washed in water to remove the lactose, and then placed in a lactose solution and glucose was liberated.

Example 10 A prepolymer was prepared in which polyethylene glycol of molecular weight 1000 containing 33% (wt/wt) - 18 8 glycerol was reacted with 2.63 meq. of toluene diisoeyanate. The prepolymer was added to an equal weight of the supernate after centrifugation of a fermentation broth of a glucose isomerase producing organism whose cells had been previously disrupted by sonication (ultra-sonic vibrations). The resultant foam was washed thoroughly and was placed in a 30% glucose solution containing 0.2 M MgSO^. After stirring for 24 hours at 70°, the resulting solution contained approximately 15% glucose and 15 fructose. In this example, a triol (glycerol) was used to produce crosslinking in a foam to add to the heat stability of the foam carrier.

Example 11 Conversion of penicillin G to 6-aminopenicillanic acid Penicillin amidase was extracted from Escherichia coli ATCC 9637 grown on phenylacetic acid and com steep liquor, by ammonium sulphate precipitation (D.A. Self, G. Kay and M. D. Lilly, BIOTECHNOLOGY AND BIOENGINEERING, Vol. XI, pg. 337-348 [1969]). A water solution of the enzyme (40 mg/ml) was added to an equal weight of the prepolymer described in Example 10. The temperature of the reaction mixture was maintained at 25° or below with stirring. After foam formation was complete (about 10 minutes), the resultant foam Was cut into small pieces (8 mm ) and Washed thoroughly in water. The washed foam was placed into a 10% solution of penicillin G and maintained at a pH of 8.0 and 37°C. Aliquots were removed vzith time and assayed for 6-aminopenicillanic acid using p-dimethylaminobenzaldehyde (Joseph Bomstein and William G. Evans, ANALYTICAL CHEMISTRY, Vol. 37, pg. 576-578 [1965]). After all the penicillin had been converted, the foam was removed. The foam was washed with water and placed onto another sample of substrate. The time required for complete conversion of the second sample was equal to the first, showing the enzyme was still present in the foam.

Example 12 A test was conducted to compare (1) the activity and retention of one of the chosen enzymes in a foam according to this invention, with (2) the activity and retention of the same enzyme when bound to a polyurethane according to U.S.P. 3,672,955. The enzyme tested was amyloglucosidase.

The enzyme bound foam according to the invention was prepared as in Example 3 above. An unfoamed amyloglucosidase - polyurethane was prepared as described in Example 1 of U.S. Patent 3,672,955. Enzyme activity was determined first for the immediate product, then after the product had been washed once in water, and then for extensive water washing of the product. Results were as follows: Amount of Initial Soluble Enzyme Activity Bound Enzyme Recovered in: Unwashed dry bound enzyme Wash Water (first) Extensively washed Dry Bound Enzyme Prior art 54% 17% 1% This invention 100% 50% 10% The significance of these results is as follows: Starting with the figure of assay of the total product shows that as the enzyme originally put therein. 54%, this means an it is 54% as active The material was assayed in toto and it therefore contains 100% of the enzyme put into the binding reaction. There 46% of the enzyme has been inactivated but it is still present in the unwashed polyurethane product of O. S. Patent 3,672,955 The 54% refers to the activity displayed by this enzyme, not to an assay of amount of enzyme on a protein basis.

The 100% figure given for the foam shows that no enzyme inactivation has taken place in the preparation of the immobilizing bound enzyme by the process of this invention.

Going on now to the second column, the 17% figure for the unfoamed product shows that 17% of the activity of the enzyme appears as enzyme activity in the first wash water. Inactive enzyme may also appear in the wash water but again this is an activity measurement and not a protein measurement. The 50% figure for the product of the present invention refers to the same thing, an activity assay.

None of the data so far indicate any binding of enzyme on polyurethane product. This information comes in the third column.

Looking now at the figure of less than 1%, this means that the bound activity which cannot be washed out is less than 1% of the activity originally present in the enzyme used in the binding step. Column 1 showed that 46% of the activity originally used in the binding step of U.S. Patent 3,672,955 was denatured and the activity was lost. Whether it was bound and inactivated or unbound-:--—_ and inactivated, is not known. Of the 54% of the initial activity which remained after the binding step 17% was removed in subsequent washings, leaving 36% (54% - 17% - 1%) was removed in subsequent washings, leaving less than 1% of the initial activity present in the final product in immobilized form. In comparison the present process lost no activity in the reacting or binding step. 50% of the activity was washed out in the first wash water. Since the final product contained 10% of the activity originally used in the reacting stage, an additional 40% washed out during additional washing steps. (50% - 40%).

To state the above in another way, in the present process, starting with 100 grams of enzyme, one could conclude with 10 grams bound and active. This would compare with the prior art's binding a maximum of one gram as active enzyme, giving a product which could contain anywhere from zero to 46% grams of enzyme in inactive form.

To compare the two processes (1) the present process achieves greater than ten times the amount of bound activity in the final product; (2) no inactivation results from the present process, therefore, the enzyme recovered from the wash water could be dried and reused in subsequent binding reactions.

Claims (17)

1. WE CLAIM:

1. Catalyst for a biochemical reaction comprising a polyurethane foam in which there is chemically bound to previously free isocyanate groups 5 in a polyurethane molecule a residue of cellulase, pectinase, papain, bromelain, chymotrypsin, trypsin, ficin, lysozyme, lactose enzyme (lactase), amyloglucosidase, penicillin amidase, glucose isomerase, alpha amylase, amino acid acylase, amino acid oxidase, asparaginase, . 10 glucose oxidase, invertase, peroxidase, pullulanase or rennin.

2. A catalyst according to claim 1 in the form of pieces of foam.

3. A catalyst according to claim 1 or 2 wherein 15 the polyurethane molecules contain urethane portions derivable from the reaction of a toluene diisocyanate with a polyhydroxy compound selected from polybutylene oxide, ethylene glycol, diethylene glycol, polyethylene oxide, pentaerythritol, glycerol, trimethylolpropane 20 and polypropylene oxide.

4. A catalyst according to claim 1 substantially as described in any one of the Examples.

5. A method of preparing a catalyst claimed in any preceding claim which comprises contacting a polyurethane 25 prepolymer having on average at least two isocyanate groups per molecule with an aqueous dispersion of an enzyme specified in claim 1, under foam-forming conditions, whereby the prepolymer foams and the enzyme becomes chemically bound to the thus formed polyurethane. 23 4 3 608

6. A method according to claim 5 wherein the molar ratio of water to -NCO groups of the prepolymer present during the foam-forming reaction is from 6.5:1 to 390:1.

7. A method according to claim 5 or 6 in which the resulting polyurethane foam is washed to remove unbound enzymes and to hydrolyze any unreaeted isocyanate groups.

8. A method according to claim 5, 6 or 7 in which the prepolymer is an isocyanate-capped polyoxyethylene polyol.

9. A method according to claim 5,6, 7 or 8 wherein the prepolymer is added as such, without first adding a water-immiscible diluent or solvent to it, to the aqueous dispersion of the enzyme.

10. A method according to claim 5, substantially as described in any one of the Examples.

11. A catalyst prepared by a method claimed in any one of claims 5 to 10.

12. A method of effecting a biochemical reaction of the type which is catalyzed by ce.Llulase, pectinase, papain, bromelain, chymotrypsin, trypsin, ficin, lysozyme, lactose enzyme (lactase), amyloglucosidase, penicillin amidase, glucose isomerase, alpha amylase, amino acid acylase, amino acid oxidase, asparaginase, glucose oxidase, invertase, peroxidase, pullulanase or rennin, which comprises effecting said reaction in the presence of a catalyst comprising a polyurethane foam containing therein a residue of the appropriate enzyme bound to the polyurethane molecule. 24 42608

13. A method according to claim 12 for hydrolyzing starch to sugars, which method comprises contacting an aqueous dispersion of starch under amyloglucosidase enzyme hydrolysis conditions with a catalyst according 5 to any of claims 1 to 4 and 11 wherein the enzyme is amyloglucosidase.

14. A method according to claim 12 for converting glucose to fructose, which method comprises contacting an aqueous solution of glucose under glucose isomerase Ιθ enzyme reaction conditions with a catalyst according to any of claims 1 to 4 and 11 wherein the enzyme is glucose isomerase.

15. A method according to claim 12 for converting lactose to glucose which comprises contacting an aqueous 15 solution of lactose under lactase enzyme reaction conditions with a catalyst according to any of claims 1 to 4 and 11 wherein the enzyme is lactase.

16. A method according to claim 12 for converting penicillin G to 6-amino-penicillanic acid which comprises 20 contacting an aqueous solution of penicillin G under penicillin amidase enzyme reaction conditions with a catalyst according to any of claims 1 to 4 and 11 wherein the enzyme is penicillin amidase.

17. A foamable composition comprising: 25 an isocyanate-capped polyoxyethylene polyol having a reaction functionality of greater than two or, when the mixture also comprises a cross-linking agent itself having a reaction functionality greater than two, a reaction functionality of at least two; - i an aqueous comjjonent comprising water and an enzyme selected from cellulase, pectinase, papain, bromelain, chymotrypsin, trypsin, ficin, lysozyme, lactose enzyme (lactase), amyloglucosidase, penicillin 5 amidase, glucose isomerase, alpha amylase, amino acid acylase, amino acid oxidase, asparaginase, glucose oxidase, invertase, peroxidase, pullulanase and rennin; wherein the mole ratio of water to moles of NCO groups in the isocyanate-capped polyoxyethylene ln polyol is from 6.5:1 to 390:1.