US4950596A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Stabilization of intracellular enzymes
Assignee: Dow Chemical Co
•
Filing: 1987-03-02
•
Granted: 1990-08-21
•
Expires: 2007-03-02
Abstract
Abstract The subject invention concerns a process for stabilizing intact or ruptured microbial cells having glucose isomerase associated therewith. Specifically exemplified is a process for stabilizing glucose isomerase producing cells of a microorganism belonging to the genus Ampullariella. In the invention process the whole or ruptured microbial cells are contacted with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte, for example, a partially carboxymethylated polyethyleneimine to flocculate and stabilize the cells. The flocculated cells are further stabilized by encapsulation with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte. The encapsulation can be done prior to or after the flocculated cells are crosslinked. The net effect is manifested by a dramatic increase in the half-life of the glucose isomerase.
Inventors
- 👤 Roberta C. Cheng
- 👤 Norman G. Moll
- 👤 Robert A. Houtchens
- 👤 Karen M. McCoy
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 23:28
CPC Classifications
C; C12; C12P; C12P19/00; C12P19/24; C12N; C12N1/00; C12N1/04; C12N11/00; C12N11/02; C12N11/04; C12N9/00; C12N9/96
Claims Analysis
Total Claims: 54
Claim 1
Independent Claim
91 words
A process for stabilizing intact or ruptured glucose isomerase-producing microbial cells having high glucose isomerase associated therewith which comprise (1) flocculating said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain flocculated microbial cells, (2) crosslinking said flocculated microbial cells to obtain flocculated-crosslinked microbial cells, (3) encapsulating said flocculated-crosslinked microbial cells with a partially carboxyalkylated-or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain encapsulated microbial cells, and (4) crosslinking said encapsulated microbial cells.
Claim 2
Dependent Claim
91 words
A process for stabilizing intact or ruptured glucose isomerase-producing microbial cells having high glucose isomerase associated therewith which comprise (1) flocculating said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain flocculated microbial cells, (2) crosslinking said flocculated microbial cells to obtain flocculated-crosslinked microbial cells, (3) encapsulating said flocculated-crosslinked microbial cells with a partially carboxyalkylated-or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain encapsulated microbial cells, and (4) crosslinking said encapsulated microbial cells.
Claim 3
Dependent Claim
16 words
The process of claim 1 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 4
Dependent Claim
37 words
The process of claim 2 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, the crosslinking is accomplished using glutaraldehyde as a crosslinking agent, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
Claim 5
Dependent Claim
20 words
The process of claim 1 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 6
Dependent Claim
46 words
The process of claim 1 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 7
Dependent Claim
53 words
The process of claim 4 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine which is carboxymethylated in the range of about 0.1 to about 1.0 equivalents of ethyleneimine when used for flocculation and carboxymethylated in the range of about 0.2 to about 1.0 equivalents of ethyleneimine when used for encapsulation.
Claim 8
Dependent Claim
76 words
A process for stabilizing intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith which comprises (1) flocculating said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain flocculated microbial cells, (2) encapsulating said flocculated microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (3) crosslinking said encapsulated flocculated microbial cells.
Claim 9
Dependent Claim
76 words
A process for stabilizing intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith which comprises (1) flocculating said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom to obtain flocculated microbial cells, (2) encapsulating said flocculated microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (3) crosslinking said encapsulated flocculated microbial cells.
Claim 10
Dependent Claim
16 words
The process of claim 7 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 11
Dependent Claim
37 words
The process of claim 8 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, the crosslinking is accomplished using glutaraldehyde as a crosslinking agent, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
Claim 12
Dependent Claim
20 words
The process of claim 7 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 13
Dependent Claim
46 words
The process of claim 7 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 14
Dependent Claim
53 words
The process of claim 10 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine which is carboxymethylated in the range of about 0.1 to about 1.0 equivalents of ethyleneimine when used for flocculation and carboxymethylated in the range of about 0.2 to about 1.0 equivalents of ethyleneimine when used for encapsulation.
Claim 15
Dependent Claim
44 words
A process for stabilizing intact or ruptured glucose-isomerase producing microbial cells having glucose isomerase associated therewith which comprises flocculating said microbial cells by contacting said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom.
Claim 16
Dependent Claim
44 words
A process for stabilizing intact or ruptured glucose-isomerase producing microbial cells having glucose isomerase associated therewith which comprises flocculating said microbial cells by contacting said microbial cells with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom.
Claim 17
Dependent Claim
16 words
The process of claim 13 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 18
Dependent Claim
27 words
The process of claim 14 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
Claim 19
Dependent Claim
20 words
The process of claim 13 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 20
Dependent Claim
46 words
The process of claim 13 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 21
Dependent Claim
31 words
The process of claim 16 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine which is carboxymethylated in the range of about 0.1 to about 1.0 equivalents of ethyleneimine.
Claim 22
Dependent Claim
45 words
Immobilized glucose isomerase comprising flocculated and crosslinked intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith wherein said microbial cells are flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom and then crosslinked.
Claim 23
Dependent Claim
45 words
Immobilized glucose isomerase comprising flocculated and crosslinked intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith wherein said microbial cells are flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom and then crosslinked.
Claim 24
Dependent Claim
21 words
Immobilized glucose isomerase of claim 19 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 25
Dependent Claim
17 words
Immobilized glucose isomerase of claim 20 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine.
Claim 26
Dependent Claim
17 words
Immobilized glucose isomerase of claim 21 wherein the crosslinking is accomplished using glutaraldehyde as a crosslinking agent.
Claim 27
Dependent Claim
22 words
Immobilized glucose isomerase of claim 19 wherein the flocculated and crosslinked microbial cells are encapsulated with a partially carboxymethylated polyethyleneimine and crosslinked.
Claim 28
Dependent Claim
38 words
Immobilized glucose isomerase comprising flocculated intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom and then crosslinked.
Claim 29
Dependent Claim
38 words
Immobilized glucose isomerase comprising flocculated intact or ruptured glucose isomerase-producing microbial cells having glucose isomerase associated therewith encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom and then crosslinked.
Claim 30
Dependent Claim
21 words
Immobilized glucose isomerase of claim 24 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 31
Dependent Claim
17 words
Immobilized glucose isomerase of claim 25 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine.
Claim 32
Dependent Claim
17 words
Immobilized glucose isomerase of claim 26 wherein the crosslinking is accomplished using glutaraldehyde as a crosslinking agent.
Claim 33
Dependent Claim
31 words
Immobilized glucose isomerase of claim 24 wherein said glucose isomerase is produced by microbial cells belonging to the genus Ampullariella, and the microbial cells are flocculated with a partially carboxymethylated polyethyleneimine.
Claim 34
Dependent Claim
44 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (2) crosslinked.
Claim 35
Dependent Claim
44 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (2) crosslinked.
Claim 36
Dependent Claim
20 words
The process of claim 29 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 37
Dependent Claim
46 words
The process of claim 29 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 38
Dependent Claim
16 words
The process of claim 30 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine.
Claim 39
Dependent Claim
16 words
The process of claim 29 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 40
Dependent Claim
37 words
The process of claim 33 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, the crosslinking is accomplished using glutaraldehyde as a crosslinking agent, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
Claim 41
Dependent Claim
65 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, (2) crosslinked, (3) encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (4) crosslinked.
Claim 42
Dependent Claim
65 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) flocculated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, (2) crosslinked, (3) encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (4) crosslinked.
Claim 43
Dependent Claim
20 words
The process of claim 35 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 44
Dependent Claim
46 words
The process of claim 35 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 45
Dependent Claim
16 words
The process of claim 36 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine.
Claim 46
Dependent Claim
16 words
The process of claim 35 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 47
Dependent Claim
37 words
The process of claim 39 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, the crosslinking is accomplished using glutaraldehyde as a crosslinking agent, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
Claim 48
Dependent Claim
44 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (2) crosslinked.
Claim 49
Dependent Claim
44 words
A process for preparing D-fructose from D-glucose which comprises contacting D-glucose with glucose isomerase-producing intact or ruptured microbial cells which have been (1) encapsulated with a partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte having a positive charge carried by a nitrogen atom, and (2) crosslinked.
Claim 50
Dependent Claim
20 words
The process of claim 41 wherein said partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is a partially carboxyalkylated- or partially phosphonoalkylated-polyamine.
Claim 51
Dependent Claim
46 words
The process of claim 41 wherein the alkyl of the carboxyalkyl or phosphonoalkyl groups of the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is --(CH 2 ) n -- wherein n=1-3, or --(CHR)--(CH 2 ) n -- wherein R=methyl, ethyl, propyl or isopropyl, and n=1 or 2.
Claim 52
Dependent Claim
16 words
The process of claim 42 wherein said partially carboxyalkylated- or partially phosphonoalkylated-polyamine is partially carboxymethylated polyethyleneimine.
Claim 53
Dependent Claim
16 words
The process of claim 41 wherein said glucose isomerase-producing microbial cells belong to the genus Ampullariella.
Claim 54
Dependent Claim
37 words
The process of claim 45 wherein said Ampullariella glucose isomerase-producing cells are Ampullariella species 3876, the crosslinking is accomplished using glutaraldehyde as a crosslinking agent, and the partially carboxyalkylated- or partially phosphonoalkylated-cationic polyelectrolyte is partially carboxymethylated polyethyleneimine.
References Cited (Backward Citations)
US4355105A
Cited
US4675292A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-08-20 | 1994-02-19 | 1994-08-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-08-20 | 1998-02-19 | 1998-08-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-08-20 | 2002-02-19 | 2002-08-21 | $7700.00 | Window Closed / Lapsed |