CA1301686C - Enzyme reactor with cofactor immobilized on a polymer - Google Patents
Enzyme reactor with cofactor immobilized on a polymerInfo
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- CA1301686C CA1301686C CA000575825A CA575825A CA1301686C CA 1301686 C CA1301686 C CA 1301686C CA 000575825 A CA000575825 A CA 000575825A CA 575825 A CA575825 A CA 575825A CA 1301686 C CA1301686 C CA 1301686C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/098—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer formed in the presence of the enzymes or microbial cells
Abstract
Abstract of the disclosure HOE 87/F 254 An enzyme reactor with cofactor immobilized on a polymer A process for binding cofactors to polymers, in which a cofactor which has aromatic or benzylic amino groups is firstly converted into the corresponding isothiocyanate, and the latter is then attached to a polymer having suit-able functional groups.
Description
13016~6 HOECHST AKTIENGESELLSCHAFT HOE 87/F 254 Dr.KH/sk An enzyme reactor with cofactor immobilized on a polymer The invention relates to an improved process for the immobilization, on polymers, of the cofactors required for enzymatic reactions, and to an enzyme reactor ~hich, be-sides one or more enzymes, contains one or more cofactors immobilized according to the invention on a polymer.
It is known that enzymatic reactions have acquired in-creasing importance, because of their high selectivity and good yields, in the preparation of valuable chemical compounds. This entails enzymes being immobilized by various processes and then used as heterogeneous catalysts.
They can then easily be removed from the reaction mixture and reused.
~here enzymes or enzymatic complexes are active only in the presence of a low molecular weight cofactor, the task has been like~;se to immobilize the cofactor, be-cause other~ise it ~ould have to be continuously addedane~ to the reaction mixture. It would not be possible to remove a readily soluble low molecular ~eight cofactor from the reaction product by simple membrane filtration, ~hich would result in complete loss of the costly cofactor ~ith, at the same time, contamination of the final pro-duct obtained. This is why many investigations into the immobilization of such cofactors on polymers have been described (cf. P.-O. Larsson and K. Mosbach, Biotechnol.
Bioeng. 13 (1971), 393; J. Grun~ald and Th. M.S. Chang, 3~ Biochem. Biophys. Res. Commun. 81 (1978), 565; German Patent 26 31 045; German Patent 28 41 414; German Offen-legungsschrift 29 30 087).
Cofactors having an adenine ring system such as nicotin-amide-adenine dinucleotide (NAD /NADH), nicotinamide-adenine dinucleotide phosphate (NADPI/NADPH), adenosine monophosphate (AMP), adenosine diphosphate ~ADP), adenosine triphosphate (ATP), flavine-adenine dinucleotide ~FAD/
FADH2) and coenzyme A ~hich are covalently bonded to solid supports or to soluble polymers are already used on a large scale. Thus, cofactors bound to solid supports are employed as ligands in affinity chromatography. ~ater-soluble polymers with covalently bonded cofactors are very useful in affinity partition. Cofactors such as NAD /NADH
and NADP+/NADPH can be regenerated and used in enzymatic systems under recycling conditions.
The procedure for the preparation of cofactors which con-tain an adenine ring system and are bound to polymers is such that, firstly, the nitrogen atom in the 6 position is connected to a reactive group which can then in turn react with a functional group of the macromolecule. This entails, firstly, the adenine ring system being reacted on the N(1) atom with an alkylating agent which has another functional group ~hich is intended to allow reac-tion with the macromolecule. Su;table alkylating agents are halogeno carboxylic acids such as iodoacetic acid, epoxides such as 3,4-epoxybutyric acid, lactones such as propionolactone or aziridines such as ethyleneimine.
In the case of AMP, ADP and ATP, the alkylation is fol-lo~ed immediately by a Dimroth rearrangement to give the N(6) form. The Dimroth rearrangement is then followed by reaction with the macromolecule. In the case of NAD+
and NADP+, a reduction is carried out before the Dimroth rearrangement, and a reoxidation of the nicotinamide ring is carried out after the Dimroth rearrangement.
However, it has already been disclosed in German Patent 28 41 414 that these reaction sequences necessary for immobilizing cofactors on polymers are unsatisfactory, because the large number of reactions which are needed reduce the yields, and in this way it ;s possible to immobilize the costly cofactor only in an amount of 12 t~
40% on the polymer. For this reason, it is proposed therein to improve the yield by not subjecting the adenine ~301686 derivatives alkylated in ehe 1 position to a Dimroth re-arrangement until after the addition onto the polymer.
Although it is possible in this ~ay to reduce the losses in yields, the number of reaction stePs ~hich are required S remains the same, and it is still necessary ~ith the NAD+
and NADP derivatives to carry out a reduction and a re-oxidation of the nicotinamide ring.
Hence the object was to simp~ify the process for binding the cofactors to polymers, in order to reduce losses of yields and of activity of the costly cofactors.
This object is achieved according to the invention by co-factors ~hich have aromatic or benzylic amino groups being firstly converted, for example by reaction ~ith thiophos-gene, into the corresponding isothiocyanate, and the latter then being added onto a suitable polymer having functional groups. For example, use of NADH as cofactor and of a polymer having amino groups results in thiourea derivatives of the general formula S
Il NAD - N - C - N - polymer.
H H
Use of a polymer having hydroxyl groups results in the corresponding thiocarbamates. The preparation of thio-ureas and thiocarbamates from isothiocyanates and com-pounds having amino or hydroxyl groups is state of the art (cf. L. Drobnica, P. Krisian, J. Augustin in "The Chem-istry of Functional Groups" ~Patai, Ed.) part 2: "Cyanates and their Derivatives", Wiley, New York, Chapter 22, 1003).
The polymer to be chosen for binding the cofactor ought to be soluble in ~ater, because if the cofactor is bound to a polymer ~hich is insoluble in ~ater it is no longer pos-sible for the bound NAD+ to be reduced completely, and this results in considerable losses of activity ~cf. H.-L. Schm;dt and G. Grenner, Eur. J. 3iochem. 67, 295-302 ~1976)).
l30i.6a6 The polymers preferred for the immob;lization of the co-factors are soluble in water and have primary or secondary amino groups. Copolymers of vinylamine and vinylmethyl-acetamide or vinylmethylamine and vinylmethylacetamide S have proven particularly appropriate, it being possible to vary the ratio of the monomers bet~een 1:99 and 40:60 % by ~eight.
To prepare these copolymers, N-vinylformamide or N-vinyL-N-methylformamide is reacted with other water-soluble N-vinylamides such as N-vinyl-N-methylacetamide or N-vinyl-pyrrolidone, and the N-vinylformamide or N-vinyl-N-methyl-formamide units which have been incorporated are hydrolyzed with strong acids, preferably hydrochloric acid, in aqueous solution to give N-vinylam;ne or N-vinyl-N-methylamine chain members. The formamides are very readily hydrolyzed, in contrast to other vinylamides, and conse~uently the other N-vinylamides which have been polymerized in are not simultaneously hydrolyzed to a noteworthy extent. Finally, the formic acid which has been eliminated, and the hydro-chloric acid which has been employed, are removed by ion exchange.
The base component can be adjusted as desired by varying the amount of N-vinylformamide or N-vinyl-N-methylform-amide po~ymerized in. If N-vinylformamide is employed for the copolymerization, the final product contains N-vinyla-mine units, and when N-vinyl-N-methylformamide is copoly-merized the final product contains N-vinyl-N-methylamine chain members. However, it is also possible to carry out a terpolymerization, for example of N-vinylformamide, N-vinyl-N-methylformamide and N-vinyl-N-methylacetamide.
Hydrolysis of these polymers permits access to polymeric support substances having primary and ~econdary vinylamine units, that is to say different base reactivities, which are likewise suitable for the immobilization of cofactors.
Very similar polymers are also obta;ned by partial hydroly-sis of poly-N-vinylformamide or poly-N-vinyl-N-130~686 -- 5methylformamide homopolymers. It is likewise possible in ehese cases to adjust the base content as desired by Yarying the amount of acid and the hydrolysis time. It is also possible, by copolymerization of N-~inylformamide and S N-vinyl-N-methylformamide, followed by partial hydrolysis, to synthesize water-soluble polymeric support substances ~ith different base reactivities.
The adjustment of a desired mean molecular size of the basic, water-soluble support substances can be influenced to a large extent ~M~ 10,000 to 500,000) by a number of measures, such as choice of the polymerization tempera-ture, of the solvent and of the initiator concentration.
Experience has likewise been good with the partially alkylamine-substituted ~,~-poly-(2-hydroxyethyl)-D,L-aspartamide.
However, it is also possible to employ other water-soluble polymers which have functional groups reacting with iso-thiocyanates. For example, it is also possible to use polyethyleneimine or water-soluble carbohydrates.
Using the process according to the invention, cofactors are successfully immobilized in virtually quantitative yield, and without loss of activ;ty, on polymers and, moreover, the multistage reaction sequence hitherto regarded as necessary is avoided. The cofactors immobil-ized on polymers in this way can be employed in a variety ; 30 of ways. For example, they can be enclosed together with one or more enzymes in microcapsules. The preparat;on of microcapsules of this type having a semipermeable wall which allows substrate and reaction product to pass through but retains the enzyme(s) and the bound cofactor has long been state of the art ~cf. Th. M.S. Chang, E~io-medical Appl., Immobilized Enzyme Proteins, Vol. 1 ~1977), 69, Plenum, New York 1977).
The enzymes can also be used, together with the cofactor i30~686 immobilized on a polymer, in a membrane reactor equ;pped with an ultrafiltration membrane, which membrane serves to retain the employed enzymes and the cofactor in the reactor but to allow the low molecular weight product and the unre-acted substrate to pass through. Membrane reactors of thistype are also now state of the art and are described, for example, in German Offenlegungsschrift Z9 30 078.
An enzymatic reaction can be carr;ed out continuously, both with semipermeable microcapsules and in an ultrafil-tration cell, if the cofactor(s) catalyze several reactions successively and are, during this, returned to their ini-tial state. For example, NAD+ can convert, with the aid of malic dehydrogenase, malate into oxalacetate. The NADH
produced during this can then reduce, with the aid of alco-hol dehydrogenase, acetaldehyde to ethanol. Thereafter NAD is again present and available for another reduction.
Thus, a system of this type undergoes continual regenera-tion and can in theory carry out this reaction for an un-limited period. However, in practice it is found thatthese systems composed of several enzymes and cofactors gradually lose their activity. As is made clear by the experiments described in detail in the examples, however, no loss of activity has been observed with the enzyme reactors prepared according to the invention. An addi-tional factor is that the cofactor is immobilized accord-ing to the invention in a simple one-pot reaction and in virtually quantitative yield.
Exa-ple 1 Preparation of 6-isothiocyanato-nicotina-ide-adenine dinucleotide ("NAD-NCS") 2 mmol of NADI were dissolved in 10 ml of H20 in a round-bottomed flask with attached bubble-counter, and a little Na2C03 was added. A solution of 20 mmol of thiophosgene in 10 ml of CHCl3 was added with vigorous stirring at 20C.
The pH was checked every 10 minutes and kept between 5.5 and 8.5 by the addition of small amounts of sodium ~301686 carbonate. After 3.5 hours there was no longer any change in the pH, and no further gas ~as evolved. The mixture ~as evaporated to dryness in vacuo and freeze-dr;ed. The sl;ghtly bro~n;sh product m;xture could be pur;f;ed of by-products by column chromatography on silica gel using ~ater/aceton;trile as eluent. However, ;t is advisable to use the product m;xture unpur;fied for the react;on with the polymer (see Example 2), since purif;cat;on by ultra-filtration of the product immobilized on the polymer ;s considerably more straightfor~ard and, moreover, the sen-s;t;v;ty of NAD-NCS results in losses due to dimer;zation and trimerization during purification.
Exa-ple 2 Reaction of vinyla-ine/vinyl-ethylaceta-ide copol~-er ~4.8:95.2X by veight) with 6-isothiocyanato-nicotinamide-adenine dinucleotide (NAD-NCS) 10 ml of a solut;on of 2 mmol of NAD-NCS in 20 ml of water were added to 863 mg of polymer ~ 1 mmol of NH2 groups) in 5 ml of H20 in a 50 ml round-bottomed flask at 20C, the pH ~as adjusted to 8 w;th 1 N NaOH, and the solut;on uas stirred at 20C for 15 hours. The solution ~as then subjected to ultrafiltration through a cellulose acetate membrane (exclusion limit: molecular ~eight 5,000) in an ultrafiltration cell under an excess pressure of 3 bar, making up ~;th 50 ml of water each t;me, and filtering aga;n, 10 t;mes. The retentate and the 10 permeates were measured in a UV spectrometer at ~ = 257 nm (measurement of absorption). The permeates showed an absorption uhich became smaller as ultrafiltration progressed, i.e. a de-creasing NAD content. The retentate contained 70 to 90X
of the NAD employed. This result was confirmed by meas-urement of the absorpt;on of a weighed sample of the freeze-dried retentate d;ssolved in water, and compar;son with a calibration plot constructed with pure NAD+.
Freeze-drying of the retentate yielded 12 g of product (85% based on polymer).
The product was employed in place of NAD+ as cofactor in the standard assay of Boehringer Mannhe;m for the determination of maLate using malic dehydrogenase, of ethanol using alcohol dehydrogenase and of lactate using lactic dehydrogenase. In all cases the enzyme activity, and thus the cofactor activity too, was unchanged when NAD was replaced by an equivalent amount of the NAD~
immobilized on a polymer.
Example 3 Coupling of ,B-poly-(2-hydroxyethyl)-D,L-aspartamide partially substituted vith aminoethyl groups (H2N-PHEA) to NAD-NCS via these a-ino groups The H2N-PHEA contained 10 4 mol/g amino groups. Since, under the conditions used here, it is principally the amino groups which react with the isothiocyanate, NAD-NCS
was employed stoichiometric to this number of amino groups.
1.5 9 of polymer ~1.5 x 10 4 mol of amino groùps) were dissolved in about 25 ml of H20 in a 100 ml roùnd-bottomed flask at 20C, and a solution of 1.4 x 10 4 mol of NAD-NCS
was added. The pH was adjusted to 8 with NaOH. The mixture was stirred at 20C for 20 h and then subjected to ultrafil-tration at 3 bar through a cellulose acetate membrane (exclu-sion limit: molecular weight 5,000), making up with 60 ml of water each time, and filtering again, 6 times. The con-tent of NAD~ in the product was determined by comparing the UV absorption of the permeates and of the retentate at 257 nm. It was 80X of the amount emp~oyed. The retentate was freeze-dried, total yield 1.4 9 (92X), so that the effective ~ield of confugation emerg~s 8S 87 %.
Exaeple 4 Microencapsulation of an enzyme syste- vith N~D i~mobilized on a polymer The enzyme system emPIoyed was malic dehydrogenase (MDH) in conjunction with glutamic-oxalacetic transaminase (GOT), _ 9 _ in analogy to the malate assay marketed by Boehringer Mannheim. ~his entailed L-malate being converted by MDH
with NAD+ to oxalacetate, NADH and H+. In order to displace the equilibrium away from L-malate, the oxal-acetate ~as converted into L-aspartate and ~-ketoglut-arate by GOT and addition of glutamate.
In a typical experiment, 0.01 ml of a 5 mg/ml solution of MDH, 0.01 ml of a 2 mg/ml suspension of GOT tboth in water) and 25.5 mg of the NAD+-polymer (~ 10 mg of NAD~) des-cribed ;n Example 2 were made up to 1.42 ml with water.
0.3 ml of this solution was mixed with 1 g of a 2% strength aqueous solution of alginate 20/60, and the mixture was sprayed using a syringe and needle (internal diameter 0.2 mm) into a 0.4% strength aqueous solution of poly-lysine, average molecular weight 5800. 558 mg of micro-capsules were obtained from 931 mg of sprayed soLution and were removed from the polylysine solution after 15 minutes (decantation) and then washed with 1X strength aqueous NaCl solution. The capsules were then placed for 1 minute in a 12.5X strength glutaraldehyde solution and thus crosslinked. The capsules were placed in water in a 400 ml beaker and agitated by blowing in nitrogen. Aliquots of the supernatant solution ~ere taken after 17 hours, 2 days, 3 days, 6 days and 7 days, and the UV absorption of the solution at 257 nm was measured and compared with a NAD~
calibration plot and thus the NAD content of the super-natant solution (i.e. the NAD lost from the capsules) was determined. The results of this were as follows:
Time NAD ~ost from the capsules 17 h 1.6X ) a few mechanically damaged 2 days 1.7X ) capsules are visible to 3 days 0.8% ) the naked eye 35 4 days 0.8X
; 7 days 0.4X
After 9 days, 44 capsules, corresponding to about 0.7x10 3 mmol of NAD~, were removed and used to convert malate under 1:~01686 the conditions described in the maLate assay of Boehr;nger Mannhe;m. ~he convers;on ach;eved by the m;crocapsules was 0.004 mg from an excess of malate with an equivalent amount of NAD~ w;thin 30 minutes (theoretical conversion S 0.003 mg of L-malate).
Example S
Se-icontinuous enzy-e reactor ~ith NAD i-mobi~ized on a poly-er, ~ith regeneration of the cofactor by recrcling The enzyme system employed for this was formic dehydro-genase (FDH) and lactic dehydrogenase (LDH), with formate being converted by FDH with NAD+ into C02 and NADH, and pyruvate being converted by LDH w;th NADH ;nto lactate.
Pyruvate and formate were used ;n excess as substrates, C2 escaped, and lactate was determined as the only pro-duct of the reaction in solution. The ultrafiltration membrane used for retention of the en2ymes and of the NAD-polymer was composed of cellulose acetate (exclusion limit molecular we;ght 5000). The reaction was carr;ed out in an ultrafiltration cell with the reaction m;xture be;ng under a pressure of 2 bar, and it be;ng possible to determine the lactate ;n the fractions of permeate taken.
NAD-polymer from Example 2 (corresponding to 0.021 mmol of NAD), 0.1 mg of LDH, 4 units of FDH, 0.26 mmol of pyruvate and 0.26 mmol of formate in 3.7 ml of phosphate buffer, pH
7.2, were employed. The permeate fractions were collected every 30 minutes while stirring under an excess pressure of 2 bar of N2. After each fraction, the concentrate was made up to the original 3.7 ml with phosphate buffer, pH
7.2. In addition, after every 4th fraction, 0.26 mmol of pyruvate and 0.26 mmol of formate were added. The per-meates were examined for the lactate content using the lactate determination assay marketed by ~oehr;nger ; Mannheim. The Lactate yield obtained in each permeate fraction (1 ml each) was from 3 to 6x10 4 mmol of Lactate over 22 fractions. After the test had been interrupted and continued after 2 days ~weekend) the lactate content 1301 6~36 in each permeate fract;on remained at the same constant level.
Example 6 A. Preparation of an N-vinylamine/N-vinyl-N-methylaceta-ide copolymer, N-vinyla-ine content ~.8~ by veight 36 g of N-vinyl-N-methylacetamide and 3 9 of N-vinyl-formamide ~ere copolymerized in 40 ml of isopropanol using 400 mg of ~,a'-azoisobutyronitrile as initiator at 80C (24 h).
The isopropanol ~as subsequently removed by distilla-tion, ~ater ~40 ml) simultaneously being added. 40 ml of concentrated hydrochloric acid ~ere added to the resulting aqueous polymer solution, and it ~as heated at 110C for 8 h. The strongly acid reaction mixture ~as diluted with water, and subsequently hydrochloric and formic acids ~ere removed on a strongly basic ion exchanger. Concentration ;n vacuo ~ -100 mbar) resulted in an aqueous solution of a N-vinylamine/N-vinyl-N-nethylacetamide copolymer ~ith an N-vinylamine content of - 4.8% by ueight. The solids content of the polymer solution ~as 33.3Z, the pH was 11.5, and the reduced viscosity of the polymer ~as determined as 0.2~
dl/g (measured on a 1% strength solution in H20 at 25C).
. Preparation of an N-vinyla-ine/N-vinylpyrrolidone copolymer, N-vinylamine content 5.7X by veight.
20 9 of N-vinylpyrrolidone and 2 9 of N-vinylformamide ~ere polymerized in 120 ml of water, to which 1 ml of NH40H solution was added, at 80C under a blanket of argon. Initiator ,'-azoisobutyronitrile 110 mg, polymerization time 24 h. 145 ml of concentrated hydrochloric acid ~ere added to the resulting aqueous copolymer solution, and the mixture ~as refluxed (110C) for 7 h. The mixture was subsequently worked up as described in A using a strongly basic ion ex-changer. 16û 9 of aqueous solution of an N-vinylamine/
130~86 N-vinylpyrrol;done copolymer ~ith an N-vinylamine con-tent of 5.7X by ~eight were then obtained. The solids content of the polymer solution ~as 9.53X, the pH was 10.1, and the reduced viscosity of the polymer ~as S determ;ned as 1.61 dl/g (measured on a 1X strength solution in HzO at 25C).
C. Preparation of N-vinyl-N-methylamine/N-vinyl-N-~ethyl-for-amide copolr-ers (by process B) N-vinyl-N-methyLformamide was polymerized ~ith ~,~'-azo-isobutyronitrile as initiator, and the homopolymer ~as only partially hydrolyzed ~ith hydrochloric acid in aqueous solution at 100C. The results of the partial hydrolysis as a function of the time and of the hydro-chloric acid concentration are to be found in the Table ~hich follo~s:
Hydrolysis Process ~a) Process (b) 1/2 hour 9.5X basic N 13.5X basic N
2 hours 15.7% basic N 20.1% basic N
In process (a) one mol of HCl is employed for one mol of N-vinyl-N-methylformamide, and in process (b) 2.5 mol of HCl are employed for one mol.
The figures for basic nitrogen were determined after re-moval of formic and hydrochloric acids using an ion ex-changer; 10ûX hydrolysis corresponds to a figure of 24.9%
for basic nitrogen. An N-vinyl-N-methylamine/N-vinyl-N-methylformamide copolymer ~ith 9.0X basic nitrogen, forexample, has an N-vinyl-N-methylamine content of 36.0X by ~eight.
It is known that enzymatic reactions have acquired in-creasing importance, because of their high selectivity and good yields, in the preparation of valuable chemical compounds. This entails enzymes being immobilized by various processes and then used as heterogeneous catalysts.
They can then easily be removed from the reaction mixture and reused.
~here enzymes or enzymatic complexes are active only in the presence of a low molecular weight cofactor, the task has been like~;se to immobilize the cofactor, be-cause other~ise it ~ould have to be continuously addedane~ to the reaction mixture. It would not be possible to remove a readily soluble low molecular ~eight cofactor from the reaction product by simple membrane filtration, ~hich would result in complete loss of the costly cofactor ~ith, at the same time, contamination of the final pro-duct obtained. This is why many investigations into the immobilization of such cofactors on polymers have been described (cf. P.-O. Larsson and K. Mosbach, Biotechnol.
Bioeng. 13 (1971), 393; J. Grun~ald and Th. M.S. Chang, 3~ Biochem. Biophys. Res. Commun. 81 (1978), 565; German Patent 26 31 045; German Patent 28 41 414; German Offen-legungsschrift 29 30 087).
Cofactors having an adenine ring system such as nicotin-amide-adenine dinucleotide (NAD /NADH), nicotinamide-adenine dinucleotide phosphate (NADPI/NADPH), adenosine monophosphate (AMP), adenosine diphosphate ~ADP), adenosine triphosphate (ATP), flavine-adenine dinucleotide ~FAD/
FADH2) and coenzyme A ~hich are covalently bonded to solid supports or to soluble polymers are already used on a large scale. Thus, cofactors bound to solid supports are employed as ligands in affinity chromatography. ~ater-soluble polymers with covalently bonded cofactors are very useful in affinity partition. Cofactors such as NAD /NADH
and NADP+/NADPH can be regenerated and used in enzymatic systems under recycling conditions.
The procedure for the preparation of cofactors which con-tain an adenine ring system and are bound to polymers is such that, firstly, the nitrogen atom in the 6 position is connected to a reactive group which can then in turn react with a functional group of the macromolecule. This entails, firstly, the adenine ring system being reacted on the N(1) atom with an alkylating agent which has another functional group ~hich is intended to allow reac-tion with the macromolecule. Su;table alkylating agents are halogeno carboxylic acids such as iodoacetic acid, epoxides such as 3,4-epoxybutyric acid, lactones such as propionolactone or aziridines such as ethyleneimine.
In the case of AMP, ADP and ATP, the alkylation is fol-lo~ed immediately by a Dimroth rearrangement to give the N(6) form. The Dimroth rearrangement is then followed by reaction with the macromolecule. In the case of NAD+
and NADP+, a reduction is carried out before the Dimroth rearrangement, and a reoxidation of the nicotinamide ring is carried out after the Dimroth rearrangement.
However, it has already been disclosed in German Patent 28 41 414 that these reaction sequences necessary for immobilizing cofactors on polymers are unsatisfactory, because the large number of reactions which are needed reduce the yields, and in this way it ;s possible to immobilize the costly cofactor only in an amount of 12 t~
40% on the polymer. For this reason, it is proposed therein to improve the yield by not subjecting the adenine ~301686 derivatives alkylated in ehe 1 position to a Dimroth re-arrangement until after the addition onto the polymer.
Although it is possible in this ~ay to reduce the losses in yields, the number of reaction stePs ~hich are required S remains the same, and it is still necessary ~ith the NAD+
and NADP derivatives to carry out a reduction and a re-oxidation of the nicotinamide ring.
Hence the object was to simp~ify the process for binding the cofactors to polymers, in order to reduce losses of yields and of activity of the costly cofactors.
This object is achieved according to the invention by co-factors ~hich have aromatic or benzylic amino groups being firstly converted, for example by reaction ~ith thiophos-gene, into the corresponding isothiocyanate, and the latter then being added onto a suitable polymer having functional groups. For example, use of NADH as cofactor and of a polymer having amino groups results in thiourea derivatives of the general formula S
Il NAD - N - C - N - polymer.
H H
Use of a polymer having hydroxyl groups results in the corresponding thiocarbamates. The preparation of thio-ureas and thiocarbamates from isothiocyanates and com-pounds having amino or hydroxyl groups is state of the art (cf. L. Drobnica, P. Krisian, J. Augustin in "The Chem-istry of Functional Groups" ~Patai, Ed.) part 2: "Cyanates and their Derivatives", Wiley, New York, Chapter 22, 1003).
The polymer to be chosen for binding the cofactor ought to be soluble in ~ater, because if the cofactor is bound to a polymer ~hich is insoluble in ~ater it is no longer pos-sible for the bound NAD+ to be reduced completely, and this results in considerable losses of activity ~cf. H.-L. Schm;dt and G. Grenner, Eur. J. 3iochem. 67, 295-302 ~1976)).
l30i.6a6 The polymers preferred for the immob;lization of the co-factors are soluble in water and have primary or secondary amino groups. Copolymers of vinylamine and vinylmethyl-acetamide or vinylmethylamine and vinylmethylacetamide S have proven particularly appropriate, it being possible to vary the ratio of the monomers bet~een 1:99 and 40:60 % by ~eight.
To prepare these copolymers, N-vinylformamide or N-vinyL-N-methylformamide is reacted with other water-soluble N-vinylamides such as N-vinyl-N-methylacetamide or N-vinyl-pyrrolidone, and the N-vinylformamide or N-vinyl-N-methyl-formamide units which have been incorporated are hydrolyzed with strong acids, preferably hydrochloric acid, in aqueous solution to give N-vinylam;ne or N-vinyl-N-methylamine chain members. The formamides are very readily hydrolyzed, in contrast to other vinylamides, and conse~uently the other N-vinylamides which have been polymerized in are not simultaneously hydrolyzed to a noteworthy extent. Finally, the formic acid which has been eliminated, and the hydro-chloric acid which has been employed, are removed by ion exchange.
The base component can be adjusted as desired by varying the amount of N-vinylformamide or N-vinyl-N-methylform-amide po~ymerized in. If N-vinylformamide is employed for the copolymerization, the final product contains N-vinyla-mine units, and when N-vinyl-N-methylformamide is copoly-merized the final product contains N-vinyl-N-methylamine chain members. However, it is also possible to carry out a terpolymerization, for example of N-vinylformamide, N-vinyl-N-methylformamide and N-vinyl-N-methylacetamide.
Hydrolysis of these polymers permits access to polymeric support substances having primary and ~econdary vinylamine units, that is to say different base reactivities, which are likewise suitable for the immobilization of cofactors.
Very similar polymers are also obta;ned by partial hydroly-sis of poly-N-vinylformamide or poly-N-vinyl-N-130~686 -- 5methylformamide homopolymers. It is likewise possible in ehese cases to adjust the base content as desired by Yarying the amount of acid and the hydrolysis time. It is also possible, by copolymerization of N-~inylformamide and S N-vinyl-N-methylformamide, followed by partial hydrolysis, to synthesize water-soluble polymeric support substances ~ith different base reactivities.
The adjustment of a desired mean molecular size of the basic, water-soluble support substances can be influenced to a large extent ~M~ 10,000 to 500,000) by a number of measures, such as choice of the polymerization tempera-ture, of the solvent and of the initiator concentration.
Experience has likewise been good with the partially alkylamine-substituted ~,~-poly-(2-hydroxyethyl)-D,L-aspartamide.
However, it is also possible to employ other water-soluble polymers which have functional groups reacting with iso-thiocyanates. For example, it is also possible to use polyethyleneimine or water-soluble carbohydrates.
Using the process according to the invention, cofactors are successfully immobilized in virtually quantitative yield, and without loss of activ;ty, on polymers and, moreover, the multistage reaction sequence hitherto regarded as necessary is avoided. The cofactors immobil-ized on polymers in this way can be employed in a variety ; 30 of ways. For example, they can be enclosed together with one or more enzymes in microcapsules. The preparat;on of microcapsules of this type having a semipermeable wall which allows substrate and reaction product to pass through but retains the enzyme(s) and the bound cofactor has long been state of the art ~cf. Th. M.S. Chang, E~io-medical Appl., Immobilized Enzyme Proteins, Vol. 1 ~1977), 69, Plenum, New York 1977).
The enzymes can also be used, together with the cofactor i30~686 immobilized on a polymer, in a membrane reactor equ;pped with an ultrafiltration membrane, which membrane serves to retain the employed enzymes and the cofactor in the reactor but to allow the low molecular weight product and the unre-acted substrate to pass through. Membrane reactors of thistype are also now state of the art and are described, for example, in German Offenlegungsschrift Z9 30 078.
An enzymatic reaction can be carr;ed out continuously, both with semipermeable microcapsules and in an ultrafil-tration cell, if the cofactor(s) catalyze several reactions successively and are, during this, returned to their ini-tial state. For example, NAD+ can convert, with the aid of malic dehydrogenase, malate into oxalacetate. The NADH
produced during this can then reduce, with the aid of alco-hol dehydrogenase, acetaldehyde to ethanol. Thereafter NAD is again present and available for another reduction.
Thus, a system of this type undergoes continual regenera-tion and can in theory carry out this reaction for an un-limited period. However, in practice it is found thatthese systems composed of several enzymes and cofactors gradually lose their activity. As is made clear by the experiments described in detail in the examples, however, no loss of activity has been observed with the enzyme reactors prepared according to the invention. An addi-tional factor is that the cofactor is immobilized accord-ing to the invention in a simple one-pot reaction and in virtually quantitative yield.
Exa-ple 1 Preparation of 6-isothiocyanato-nicotina-ide-adenine dinucleotide ("NAD-NCS") 2 mmol of NADI were dissolved in 10 ml of H20 in a round-bottomed flask with attached bubble-counter, and a little Na2C03 was added. A solution of 20 mmol of thiophosgene in 10 ml of CHCl3 was added with vigorous stirring at 20C.
The pH was checked every 10 minutes and kept between 5.5 and 8.5 by the addition of small amounts of sodium ~301686 carbonate. After 3.5 hours there was no longer any change in the pH, and no further gas ~as evolved. The mixture ~as evaporated to dryness in vacuo and freeze-dr;ed. The sl;ghtly bro~n;sh product m;xture could be pur;f;ed of by-products by column chromatography on silica gel using ~ater/aceton;trile as eluent. However, ;t is advisable to use the product m;xture unpur;fied for the react;on with the polymer (see Example 2), since purif;cat;on by ultra-filtration of the product immobilized on the polymer ;s considerably more straightfor~ard and, moreover, the sen-s;t;v;ty of NAD-NCS results in losses due to dimer;zation and trimerization during purification.
Exa-ple 2 Reaction of vinyla-ine/vinyl-ethylaceta-ide copol~-er ~4.8:95.2X by veight) with 6-isothiocyanato-nicotinamide-adenine dinucleotide (NAD-NCS) 10 ml of a solut;on of 2 mmol of NAD-NCS in 20 ml of water were added to 863 mg of polymer ~ 1 mmol of NH2 groups) in 5 ml of H20 in a 50 ml round-bottomed flask at 20C, the pH ~as adjusted to 8 w;th 1 N NaOH, and the solut;on uas stirred at 20C for 15 hours. The solution ~as then subjected to ultrafiltration through a cellulose acetate membrane (exclusion limit: molecular ~eight 5,000) in an ultrafiltration cell under an excess pressure of 3 bar, making up ~;th 50 ml of water each t;me, and filtering aga;n, 10 t;mes. The retentate and the 10 permeates were measured in a UV spectrometer at ~ = 257 nm (measurement of absorption). The permeates showed an absorption uhich became smaller as ultrafiltration progressed, i.e. a de-creasing NAD content. The retentate contained 70 to 90X
of the NAD employed. This result was confirmed by meas-urement of the absorpt;on of a weighed sample of the freeze-dried retentate d;ssolved in water, and compar;son with a calibration plot constructed with pure NAD+.
Freeze-drying of the retentate yielded 12 g of product (85% based on polymer).
The product was employed in place of NAD+ as cofactor in the standard assay of Boehringer Mannhe;m for the determination of maLate using malic dehydrogenase, of ethanol using alcohol dehydrogenase and of lactate using lactic dehydrogenase. In all cases the enzyme activity, and thus the cofactor activity too, was unchanged when NAD was replaced by an equivalent amount of the NAD~
immobilized on a polymer.
Example 3 Coupling of ,B-poly-(2-hydroxyethyl)-D,L-aspartamide partially substituted vith aminoethyl groups (H2N-PHEA) to NAD-NCS via these a-ino groups The H2N-PHEA contained 10 4 mol/g amino groups. Since, under the conditions used here, it is principally the amino groups which react with the isothiocyanate, NAD-NCS
was employed stoichiometric to this number of amino groups.
1.5 9 of polymer ~1.5 x 10 4 mol of amino groùps) were dissolved in about 25 ml of H20 in a 100 ml roùnd-bottomed flask at 20C, and a solution of 1.4 x 10 4 mol of NAD-NCS
was added. The pH was adjusted to 8 with NaOH. The mixture was stirred at 20C for 20 h and then subjected to ultrafil-tration at 3 bar through a cellulose acetate membrane (exclu-sion limit: molecular weight 5,000), making up with 60 ml of water each time, and filtering again, 6 times. The con-tent of NAD~ in the product was determined by comparing the UV absorption of the permeates and of the retentate at 257 nm. It was 80X of the amount emp~oyed. The retentate was freeze-dried, total yield 1.4 9 (92X), so that the effective ~ield of confugation emerg~s 8S 87 %.
Exaeple 4 Microencapsulation of an enzyme syste- vith N~D i~mobilized on a polymer The enzyme system emPIoyed was malic dehydrogenase (MDH) in conjunction with glutamic-oxalacetic transaminase (GOT), _ 9 _ in analogy to the malate assay marketed by Boehringer Mannheim. ~his entailed L-malate being converted by MDH
with NAD+ to oxalacetate, NADH and H+. In order to displace the equilibrium away from L-malate, the oxal-acetate ~as converted into L-aspartate and ~-ketoglut-arate by GOT and addition of glutamate.
In a typical experiment, 0.01 ml of a 5 mg/ml solution of MDH, 0.01 ml of a 2 mg/ml suspension of GOT tboth in water) and 25.5 mg of the NAD+-polymer (~ 10 mg of NAD~) des-cribed ;n Example 2 were made up to 1.42 ml with water.
0.3 ml of this solution was mixed with 1 g of a 2% strength aqueous solution of alginate 20/60, and the mixture was sprayed using a syringe and needle (internal diameter 0.2 mm) into a 0.4% strength aqueous solution of poly-lysine, average molecular weight 5800. 558 mg of micro-capsules were obtained from 931 mg of sprayed soLution and were removed from the polylysine solution after 15 minutes (decantation) and then washed with 1X strength aqueous NaCl solution. The capsules were then placed for 1 minute in a 12.5X strength glutaraldehyde solution and thus crosslinked. The capsules were placed in water in a 400 ml beaker and agitated by blowing in nitrogen. Aliquots of the supernatant solution ~ere taken after 17 hours, 2 days, 3 days, 6 days and 7 days, and the UV absorption of the solution at 257 nm was measured and compared with a NAD~
calibration plot and thus the NAD content of the super-natant solution (i.e. the NAD lost from the capsules) was determined. The results of this were as follows:
Time NAD ~ost from the capsules 17 h 1.6X ) a few mechanically damaged 2 days 1.7X ) capsules are visible to 3 days 0.8% ) the naked eye 35 4 days 0.8X
; 7 days 0.4X
After 9 days, 44 capsules, corresponding to about 0.7x10 3 mmol of NAD~, were removed and used to convert malate under 1:~01686 the conditions described in the maLate assay of Boehr;nger Mannhe;m. ~he convers;on ach;eved by the m;crocapsules was 0.004 mg from an excess of malate with an equivalent amount of NAD~ w;thin 30 minutes (theoretical conversion S 0.003 mg of L-malate).
Example S
Se-icontinuous enzy-e reactor ~ith NAD i-mobi~ized on a poly-er, ~ith regeneration of the cofactor by recrcling The enzyme system employed for this was formic dehydro-genase (FDH) and lactic dehydrogenase (LDH), with formate being converted by FDH with NAD+ into C02 and NADH, and pyruvate being converted by LDH w;th NADH ;nto lactate.
Pyruvate and formate were used ;n excess as substrates, C2 escaped, and lactate was determined as the only pro-duct of the reaction in solution. The ultrafiltration membrane used for retention of the en2ymes and of the NAD-polymer was composed of cellulose acetate (exclusion limit molecular we;ght 5000). The reaction was carr;ed out in an ultrafiltration cell with the reaction m;xture be;ng under a pressure of 2 bar, and it be;ng possible to determine the lactate ;n the fractions of permeate taken.
NAD-polymer from Example 2 (corresponding to 0.021 mmol of NAD), 0.1 mg of LDH, 4 units of FDH, 0.26 mmol of pyruvate and 0.26 mmol of formate in 3.7 ml of phosphate buffer, pH
7.2, were employed. The permeate fractions were collected every 30 minutes while stirring under an excess pressure of 2 bar of N2. After each fraction, the concentrate was made up to the original 3.7 ml with phosphate buffer, pH
7.2. In addition, after every 4th fraction, 0.26 mmol of pyruvate and 0.26 mmol of formate were added. The per-meates were examined for the lactate content using the lactate determination assay marketed by ~oehr;nger ; Mannheim. The Lactate yield obtained in each permeate fraction (1 ml each) was from 3 to 6x10 4 mmol of Lactate over 22 fractions. After the test had been interrupted and continued after 2 days ~weekend) the lactate content 1301 6~36 in each permeate fract;on remained at the same constant level.
Example 6 A. Preparation of an N-vinylamine/N-vinyl-N-methylaceta-ide copolymer, N-vinyla-ine content ~.8~ by veight 36 g of N-vinyl-N-methylacetamide and 3 9 of N-vinyl-formamide ~ere copolymerized in 40 ml of isopropanol using 400 mg of ~,a'-azoisobutyronitrile as initiator at 80C (24 h).
The isopropanol ~as subsequently removed by distilla-tion, ~ater ~40 ml) simultaneously being added. 40 ml of concentrated hydrochloric acid ~ere added to the resulting aqueous polymer solution, and it ~as heated at 110C for 8 h. The strongly acid reaction mixture ~as diluted with water, and subsequently hydrochloric and formic acids ~ere removed on a strongly basic ion exchanger. Concentration ;n vacuo ~ -100 mbar) resulted in an aqueous solution of a N-vinylamine/N-vinyl-N-nethylacetamide copolymer ~ith an N-vinylamine content of - 4.8% by ueight. The solids content of the polymer solution ~as 33.3Z, the pH was 11.5, and the reduced viscosity of the polymer ~as determined as 0.2~
dl/g (measured on a 1% strength solution in H20 at 25C).
. Preparation of an N-vinyla-ine/N-vinylpyrrolidone copolymer, N-vinylamine content 5.7X by veight.
20 9 of N-vinylpyrrolidone and 2 9 of N-vinylformamide ~ere polymerized in 120 ml of water, to which 1 ml of NH40H solution was added, at 80C under a blanket of argon. Initiator ,'-azoisobutyronitrile 110 mg, polymerization time 24 h. 145 ml of concentrated hydrochloric acid ~ere added to the resulting aqueous copolymer solution, and the mixture ~as refluxed (110C) for 7 h. The mixture was subsequently worked up as described in A using a strongly basic ion ex-changer. 16û 9 of aqueous solution of an N-vinylamine/
130~86 N-vinylpyrrol;done copolymer ~ith an N-vinylamine con-tent of 5.7X by ~eight were then obtained. The solids content of the polymer solution ~as 9.53X, the pH was 10.1, and the reduced viscosity of the polymer ~as S determ;ned as 1.61 dl/g (measured on a 1X strength solution in HzO at 25C).
C. Preparation of N-vinyl-N-methylamine/N-vinyl-N-~ethyl-for-amide copolr-ers (by process B) N-vinyl-N-methyLformamide was polymerized ~ith ~,~'-azo-isobutyronitrile as initiator, and the homopolymer ~as only partially hydrolyzed ~ith hydrochloric acid in aqueous solution at 100C. The results of the partial hydrolysis as a function of the time and of the hydro-chloric acid concentration are to be found in the Table ~hich follo~s:
Hydrolysis Process ~a) Process (b) 1/2 hour 9.5X basic N 13.5X basic N
2 hours 15.7% basic N 20.1% basic N
In process (a) one mol of HCl is employed for one mol of N-vinyl-N-methylformamide, and in process (b) 2.5 mol of HCl are employed for one mol.
The figures for basic nitrogen were determined after re-moval of formic and hydrochloric acids using an ion ex-changer; 10ûX hydrolysis corresponds to a figure of 24.9%
for basic nitrogen. An N-vinyl-N-methylamine/N-vinyl-N-methylformamide copolymer ~ith 9.0X basic nitrogen, forexample, has an N-vinyl-N-methylamine content of 36.0X by ~eight.
Claims (10)
1. A process for binding cofactors to polymers, which com-prises a cofactor which has aromatic or benzylic amino groups firstly being converted into the corresponding isothiocyanate, and the latter then being attached to a polymer having suitable functional groups.
2. The process as claimed in claim 1, wherein the polymer is soluble in water.
3. The process as claimed in claim 2, wherein the witer-soluble polymer has primary or secondary amino groups.
4. The process as claimed in claim 3, wherein the water-soluble polymer is a copolymer of vinylamine and vinyl-methylacetamide or a copolymer of vinylmethylamine and vinylmethylacetamide, it being possible for the ratio of the monomers to be between 1:99 and 40:60% by weight.
5. The process as claimed in claim 3, wherein the water-soluble polymer is a partially alkylamine-substituted .alpha.,.beta.-poly-t2-hydroxyethyl)-D,L-aspartamide.
6. The process as claimed in claim 2, wherein the water-soluble polymer is polyethyleneimine or a water-soluble carbohydrate.
7. A cofactor which is bound via an aromatic or benzylic amino group to a water-soluble polymer by means of a thio-urea or thiocarbamate bridge.
8. An enzyme reactor which contains a cofactor bound to a polymer as claimed in claim 7, and one or more enzymes.
9. An enzyme reactor as claimed in claim 8, wherein the co-factor bound to the polymer, and the enzymes, are contained in microcapsules.
10. An enzyme reactor as claimed in claim 8, wherein the cofactor bound to the polymer, and the enzymes, are con-tained in a membrane reactor equipped with an ultrafil-tration membrane.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3728772.9 | 1987-08-28 | ||
| DE19873728772 DE3728772A1 (en) | 1987-08-28 | 1987-08-28 | ENZYME REACTOR WITH POLYMER-FIXED COFACTOR |
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| Publication Number | Publication Date |
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| CA1301686C true CA1301686C (en) | 1992-05-26 |
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| CA000575825A Expired - Fee Related CA1301686C (en) | 1987-08-28 | 1988-08-26 | Enzyme reactor with cofactor immobilized on a polymer |
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|---|---|
| US (1) | US5106740A (en) |
| EP (1) | EP0304781A3 (en) |
| JP (1) | JPH01100193A (en) |
| CA (1) | CA1301686C (en) |
| DE (1) | DE3728772A1 (en) |
| DK (1) | DK480088A (en) |
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| DE3905448C1 (en) * | 1989-02-22 | 1990-10-18 | Berthold Dr. 7940 Emeringen De Singruen | Process for the synthesis of macromolecules of specific chain length, structure and sequence |
| JP2712892B2 (en) * | 1991-07-05 | 1998-02-16 | ヤマハ株式会社 | Music control device |
| US6004534A (en) * | 1993-07-23 | 1999-12-21 | Massachusetts Institute Of Technology | Targeted polymerized liposomes for improved drug delivery |
| WO1995003035A1 (en) * | 1993-07-23 | 1995-02-02 | Massachusetts Institute Of Technology | Polymerized liposomes with enhanced stability for oral delivery |
| US6060082A (en) * | 1997-04-18 | 2000-05-09 | Massachusetts Institute Of Technology | Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery |
| US7202070B2 (en) * | 2000-10-31 | 2007-04-10 | Biocatalytics, Inc. | Method for reductive amination of a ketone using a mutated enzyme |
| JP5442624B2 (en) | 2007-10-29 | 2014-03-12 | シーエスアイアール | Emulsion-derived particles |
| JP2009158458A (en) * | 2007-12-06 | 2009-07-16 | Sony Corp | Fuel cell, method of manufacturing fuel cell, electronic apparatus, enzyme immobilization electrode, biosensor, bioreactor, energy conversion element, and enzyme reaction utilization device |
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| IT1017564B (en) * | 1974-04-30 | 1977-08-10 | Snam Progetti | PROCESS FOR THE PREPARATION OF FUNCTIONALIZED ADENINE DERIVATIVES AND PRODUCTS THUS OBTAINED |
| IT1039756B (en) * | 1975-07-10 | 1979-12-10 | Snam Progetti | PROCEDURE TO IMPROVE THE ACTIVITY OF OXIDOREDUCTASE ENZYMES ENCLOSED IN FILAMENT STRUCTURES |
| DE2708018A1 (en) * | 1977-02-24 | 1978-09-07 | Boehringer Mannheim Gmbh | BIOLOGICALLY ACTIVE PROTEIN FIXED TO POLYAMIDE AND THE PROCESS FOR ITS PRODUCTION |
| US4250267A (en) * | 1977-11-10 | 1981-02-10 | W. R. Grace & Co. | Immobilized biological material |
| DE2841414C2 (en) * | 1978-09-22 | 1980-04-03 | Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf), 3300 Braunschweig | Process for the production of coenzymes bound to macromolecules and containing an adenine ring system |
| DE2930087A1 (en) * | 1979-07-25 | 1981-02-26 | Biotechnolog Forschung Gmbh | METHOD FOR THE CONTINUOUS ENCYMATIC CONVERSION OF WATER-SOLUBLE ALPHA KETOCARBONIC ACIDS INTO THE CORRESPONDING ALPHA HYDROXYCARBONIC ACIDS |
| US4818542A (en) * | 1983-11-14 | 1989-04-04 | The University Of Kentucky Research Foundation | Porous microspheres for drug delivery and methods for making same |
| US4713333A (en) * | 1985-09-23 | 1987-12-15 | Miles Laboratories, Inc. | Immobilization of biocatalysts on granular diatomaceous earth |
| DE3617535A1 (en) * | 1986-05-24 | 1987-11-26 | Biotechnolog Forschung Gmbh | METHOD FOR PRODUCING N (ARROW UP) 6 (ARROW UP) SUBSTITUTED NAD OR NADP |
-
1987
- 1987-08-28 DE DE19873728772 patent/DE3728772A1/en not_active Withdrawn
-
1988
- 1988-08-17 EP EP19880113319 patent/EP0304781A3/en not_active Withdrawn
- 1988-08-26 DK DK480088A patent/DK480088A/en not_active Application Discontinuation
- 1988-08-26 CA CA000575825A patent/CA1301686C/en not_active Expired - Fee Related
- 1988-08-26 JP JP63210858A patent/JPH01100193A/en active Pending
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| JPH01100193A (en) | 1989-04-18 |
| EP0304781A3 (en) | 1991-06-12 |
| EP0304781A2 (en) | 1989-03-01 |
| DK480088D0 (en) | 1988-08-26 |
| DE3728772A1 (en) | 1989-03-09 |
| DK480088A (en) | 1989-03-01 |
| US5106740A (en) | 1992-04-21 |
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