US3650901A
Expired / Lapsed
US
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Polymeric enzyme products
Assignee: Yeda Research and Development Co Ltd
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Filing: 1968-09-27
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Granted: 1972-03-21
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Expires: 1988-09-27
Abstract
Abstract Method of preparing active polymer-enzyme products by reaction or coupling of the polymer with an enzyme derivative in which a reactive group is inactivated and subsequently restoring the activity of the inactivated group, and at least partially inactivated intermediate polymer-enzyme products. Process is conducted with greater facility, and permits higher yields of desired polymer-enzyme product and attainment of higher percentage of activity in polymer-enzyme product. Intermediate polymer-enzyme products thus-produced.
Inventors
- 👤 Ephraim Katchalski
- 👤 Leon Goldstein
- 👤 Yehuda Levin
- 👤 Shmaryahu Blumberg
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 13:13
CPC Classifications
C; C08; C08F; C08F8/00; C12; C12N; C12N11/00; C12N11/02; C12N11/08; C12N11/082; C12N11/087
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
23 words
A product of claim 1, wherein the enzyme is covalently bonded to a polymer through a carboxyl or anhydride group of the polymer.
Claim 2
Dependent Claim
27 words
A product of claim 1, comprising a bivalent metal derivative of an enzyme covalently bonded to a polymer through a carboxyl or anhydride group of the polymer.
Claim 3
Dependent Claim
42 words
A product of claim 1, comprising a metal derivative of an enzyme, having in its native state a sulfhydryl group, as well as an amino and/or hydroxyl group, bonded covalently to a polymer through a carboxyl or anhydride group of the polymer.
Claim 4
Dependent Claim
34 words
A product of claim 1, comprising papain, in which a reactive site has been inactivated but is capable of reactivation, covalently bonded to a polymer through a carboxyl or anhydride group of the polymer.
Claim 5
Dependent Claim
26 words
A product of claim 1, comprising a bivalent metal derivative of papain bonded covalently to a polymer through a carboxyl or anhydride group of the polymer.
Claim 6
Dependent Claim
36 words
A product of claim 1, comprising a bivalent metal derivative of an enzyme, having in its native state a sulfhydryl group, bonded covalently to an EMA-type polymer through a carboxyl or anhydride group of the polymer.
Claim 7
Dependent Claim
26 words
A product of claim 1, comprising a mercury derivative of papain bonded covalently to an EMA-type polymer through a carboxyl or anhydride group of the polymer.
Claim 8
Dependent Claim
34 words
A product of claim 1, comprising a bivalent metal derivative of an enzyme, having in its native state a sulfhydryl group, bonded covalently to EMA through a carboxyl or anhydride group of the polymer.
Claim 9
Dependent Claim
9 words
A product of claim 1, comprising EMA-bivalent metal papain.
Claim 10
Dependent Claim
8 words
A product of claim 1, comprising EMA-zinc papain.
Claim 11
Dependent Claim
7 words
A product of claim 1, comprising EMA-mercuripapain.
Claim 12
Dependent Claim
10 words
A product of claim 1, comprising dialkylaminoalkylamine-modified EMA-bivalent metal papain.
Claim 13
Dependent Claim
11 words
A product of claim 1, comprising dimethylaminopropylamine-modified EMA-mercuripapain or zinc papain.
Claim 14
Dependent Claim
10 words
A product of claim 1, comprising lower-alkylamine-modified EMA-bivalent metal papain.
Claim 15
Dependent Claim
11 words
A product of claim 1, comprising propylamine-modified EMA-mercuripapain or zinc papain.
Claim 16
Dependent Claim
10 words
A product of claim 1, comprising amino-lower-alkanol-modified EMA-bivalent metal papain.
Claim 17
Dependent Claim
11 words
A product of claim 1, comprising aminopropanol-modified EMA-mercuripapain or zinc papain.
Claim 18
Dependent Claim
98 words
A process for the production of an enzymatically-active product comprising an enzyme covalently bonded to a polymer, which comprises the steps of reacting or coupling an enzyme, in which at least one, but not all, active sites have been at least partially inactivated by reaction with a bivalent metal or cysteine, with a polymer adapted to effect covalent bonding to said enzyme through such a remaining reactive group, to produce an at least partially inactivated enzyme-polymer product, and restoring at least one previously-inactivated group in the enzyme moiety of the enzyme-polymer product to produce the desired enzymatically-active product.
Claim 19
Dependent Claim
138 words
A process of claim 19, for the production of an enzymatically-active product comprising an enzyme covalently bonded to a polymer, which comprises the steps of at least partially inactivating an active site in an enzyme having in the molecule, in addition to said active site, another reactive sulfhydryl, hydroxyl, amino, imidazolyl, carboxyl, or disulfide group, to form an enzyme in which an active site has been at least partially inactivated but which enzyme is still capable of reaction, and reacting the thus partially inactivated enzyme with a polymer, adapted to effect covalent bonding to said enzyme through one of said remaining reactive groups, to produce an at least partially inactivated enzyme-polymer product in which the enzyme is bonded covalently to the polymer, and thereafter reactivating the inactivated site of the enzyme to produce the desired enzymatically-active enzyme-polymer product.
Claim 20
Dependent Claim
14 words
A process of claim 20, wherein said polymer has a carboxyl or anhydride group.
Claim 21
Dependent Claim
22 words
A process of claim 21, wherein said enzyme is inactivated by inactivation of at least one sulfhydryl group in the enzyme molecule.
Claim 22
Dependent Claim
134 words
A process of claim 19, for the production of an enzymatically-active product comprising an enzyme, having in its native state a sulfhydryl group, covalently bonded to a polymer, which comprises the steps of reacting or coupling a bivalent metal derivative of an enzyme, in which a sulfhydryl group is inactivated by reaction with said metal, but having in the enzyme molecule another reactive sulfhydryl, hydroxyl, amino, imidazolyl, carboxyl, or disulfide group which can react, with a polymer adapted to effect covalent bonding to said enzyme through one of said reactive groups, to produce a metal derivative of the enzyme bonded covalently to the polymer, and reacting the product thus produced with a chelating agent to restore the sulfhydryl group in the enzyme moiety of the molecule and produce the desired enzymatically active polymer-enzyme product.
Claim 23
Dependent Claim
14 words
The process of claim 20, wherein the reactivation is carried out under nonoxidizing conditions.
Claim 24
Dependent Claim
12 words
The process of claim 24, wherein said polymer is an EMA-type polymer.
Claim 25
Dependent Claim
23 words
The process of claim 24, wherein said nonoxidizing conditions are effected by carrying out the reactivation in the presence of a mild-reducing agent.
Claim 26
Dependent Claim
13 words
The process of claim 26, wherein said mild-reducing agent is a sulfhydryl-containing compound.
Claim 27
Dependent Claim
10 words
The process of claim 27, wherein said enzyme is papain.
Claim 28
Dependent Claim
117 words
A process of claim 19, for the production of an enzymatically-active product comprising an enzyme, having in its native state a plurality of groups which can be reacted or coupled with an anhydride or carboxyl group, covalently bonded to a polymer having an anhydride or carboxyl group, which comprises the steps of inactivating at least one, but not all, of said reactive groups in said enzyme, reacting the thus-altered enzyme with said polymer having a carboxyl or anhydride group to produce an at least partially inactivated form of the desired polymer-enzyme reaction product, and then restoring the activity of said previously-inactivated group in the enzyme moiety of said Polymer-enzyme reaction product to produce the desired enzymatically-active product.
Claim 29
Dependent Claim
12 words
The process of claim 29, wherein said polymer is an EMA-type polymer.
Claim 30
Dependent Claim
10 words
The process of claim 30, wherein said polymer is EMA.
Claim 31
Dependent Claim
10 words
The process of claim 31, wherein said enzyme is papain.
Claim 32
Dependent Claim
37 words
The process of claim 29, wherein the enzyme contains sulfhydryl groups which are inactivated by conversion to disulfide linkages, and wherein the intermediate inactivated polymer-enzyme product is reactivated by reconversion of the disulfide linkages to sulfhydryl groups.
Claim 33
Dependent Claim
86 words
A process of claim 19, for the production of an enzymatically-active product comprising papain covalently bonded to a polymer, comprising the steps of inactivating at least one, but not all, of the reactive groups of papain, coupling or reacting the thus-altered papain with a polymer, adapted to effect covalent bonding to papain through one of said remaining reactive groups, to produce a desired polymer-papain reaction product, and then restoring the active groups in the papain moiety of the papain-polymeric product to produce the desired enzymatically-active product.
Claim 34
Dependent Claim
47 words
The process of claim 34, wherein papain is converted to a cystine-papain product prior to coupling or reaction with the polymer, and wherein the intermediate polymer-papain product is reactivated by reduction of the cystine moiety to restore sulfhydryl groups in the papain portion of the papain-polymeric product.
Claim 35
Dependent Claim
103 words
A process of claim 19, for the production of an enzymatically-active product comprising papain covalently bonded to a polymer, comprising the steps of coupling or reacting a bivalent metal derivative of papain, wherein said bivalent metal has been attached by reaction with sulfhydryl groups of papain, with a polymer having a carboxyl or anhydride group to produce an at least partially inactivated product comprising a bivalent metal-papain-polymer wherein said bivalent metal papain is covalently bonded to said polymer, and reacting this material with a chelating agent under nonoxidizing conditions to restore sulfhydryl groups of the papain moiety and produce the desired enzymatically-active product.
Claim 36
Dependent Claim
118 words
A process of claim 19, for the production of an enzymatically-active product comprising papain covalently bonded to a polymer, which comprises the steps of at least partially inactivating at least one of the reactive groups of papain, without inactivating all of the reactive groups of papain which are nonessential for the enzymatic activity of papain, coupling or reacting the thus-altered enzyme with a polymer having reactive groups adapted to react with one of said remaining nonessential reactive groups of papain to produce an at least partially inactivated polymer-papain reaction product, and then restoring the activity of the previously inactivated group or groups in the papain moiety of the polymer-enzyme product to produce the desired enzymatically active polymer-papain product.
Claim 37
Dependent Claim
13 words
The process of claim 37, wherein said reactivation is conducted under nonoxidizing conditions.
Claim 38
Dependent Claim
12 words
The method of claim 38, wherein said metal is mercury or zinc.
Claim 39
Dependent Claim
17 words
The method of claim 38, wherein a mercaptan, cysteine, thioglycolic acid, or glutathione is present during reactivation.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1975-03-21 | 1975-09-20 | 1976-03-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1979-03-21 | 1979-09-20 | 1980-03-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1983-03-21 | 1983-09-20 | 1984-03-21 | $7700.00 | Window Closed / Lapsed |