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US4593004A
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Process for the immobilization of cyclodextrine glycosyltransferase enzyme
Assignee: Reanal Finomvegyszergyar Rt
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Filing: 1983-08-24
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Granted: 1986-06-03
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Expires: 2003-08-24
Abstract
Abstract The invention relates to a process for the preparation of a cyclodextrine glycosyltransferase enzyme. The immobilization can be carried out by two methods. According to the first method a cyclodextrine glycosyltransferase enzyme is activated with a solution of a carbodiimide compound and the thus treated enzyme is applied in a solution having a pH value of 4.5-8.5 onto a polysaccharide derivative comprising at least 0.1 m-equiv./g of free amino group. According to the second method the enzyme is applied in a solution having a pH value of 4.5-8.5 onto a polymer produced from an acrylic acid and/or methacrylic acid or acrylic amide and/or methacrylic amide monomer by means of a cross-linking agent of the acrylic or allylic type which polymer comprises at least 0.1 m-equiv./g of functional carboxy group, activating the polymer with a solution of a carbodiimide compound and washing and if desired drying the product thus obtained. The advantage of the process of the present invention is that the thus immobilized enzyme--contrary to the dissolved enzyme--is suitable for longlasting application and can be readily used on industrial scale continuous production.
Inventors
- 👤 Laszlo Boross
- 👤 Ivan Daroczi
- 👤 Katalin Ivony nee Kaldive
- 👤 Gabor Seres
- 👤 Bela Szanjani
- 👤 Jozsef Szejtli
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 23:26
CPC Classifications
C; C12; C12P; C12P19/00; C12P19/18; C12N; C12N11/00; C12N11/02; C12N11/08; C12N11/082; C12N11/087; C12N11/10
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
121 words
A process for the immobilization of cyclodextrin glycosyl transferase enzyme which comprises the steps of: (a) activating a cyclodextrin glycosyl transferase enzyme either in an aqueous solution with a carbodiimide compound that is water-soluble, at a temperature of 0° to 4° C. or in an organic solvent with a carbodiimide compount that is soluble in an organic solvent, at a temperature less than 0° C; and (b) immobilizing the enzyme by applying the enzyme treated according to step (a) in an aqueous buffer solution having a pH value between 4.5 and 8.5 onto a carrier which is a derivative of a polysaccharide compound comprising at least 0.1 m. equiv. of NH 2 groups per gram of carrier, and washing the product.
Claim 2
Dependent Claim
121 words
A process for the immobilization of cyclodextrin glycosyl transferase enzyme which comprises the steps of: (a) activating a cyclodextrin glycosyl transferase enzyme either in an aqueous solution with a carbodiimide compound that is water-soluble, at a temperature of 0° to 4° C. or in an organic solvent with a carbodiimide compount that is soluble in an organic solvent, at a temperature less than 0° C; and (b) immobilizing the enzyme by applying the enzyme treated according to step (a) in an aqueous buffer solution having a pH value between 4.5 and 8.5 onto a carrier which is a derivative of a polysaccharide compound comprising at least 0.1 m. equiv. of NH 2 groups per gram of carrier, and washing the product.
Claim 3
Dependent Claim
25 words
The process defined in claim 1, wherein the carrier in step (b) which contains --NH 2 groups is an aminopropyl derivative of cellulose or dextran.
Claim 4
Dependent Claim
18 words
The process defined in claim 1, wherein in step (a) the carbodiimide compound is N-cyclohexyl-N'-[2-(4-morpholinyl)-ethyl]-carbodiimide-methyl-p-toluene-sulfonate or N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
Claim 5
Dependent Claim
31 words
The process defined in claim 1, wherein in step (b) the immobilization is carried out in a 0.05 to 0.1 molar sodium acetate buffer solution having a pH value of 6.5.
Claim 6
Dependent Claim
18 words
The process defined in claim 1, wherein in step (b) the immobilization is carried out for 45-55 hours.
Claim 7
Dependent Claim
36 words
The process defined in claim 1, wherein in step (b) the immobilization enzyme is first washed with a sodium acetate buffer, then with a sodium acetate buffer also containing sodium chloride, and then with distilled water.
Claim 8
Dependent Claim
141 words
A process for the immobilization of cyclodextrin glycosyl transferase enzyme which comprises the steps of: (a) activating a polymer carrier comprising at least 0.1 m. equiv. of functional --COOH groups per gram of carrier, said polymer carrier having been produced from an acrylic acid and/or methacrylic acid, or acrylic amide and/or methacrylic amide monomer, and a cross-linking agent of the acrylic or allylic type either in an aqueous solution with a carbodiimide compound that is water soluble, at a temperature of 0° to 4° C. or in an organic solvent with a carbodiimide compound that is soluble in an organic solvent, at a temperature less than 0° C; and (b) applying a cyclodextrin glycosyl transferase enzyme in an aqueous buffer solution having a pH value of 4 to 9 onto the polymer carrier to immobilize the enzyme, and washing the product.
Claim 9
Dependent Claim
141 words
A process for the immobilization of cyclodextrin glycosyl transferase enzyme which comprises the steps of: (a) activating a polymer carrier comprising at least 0.1 m. equiv. of functional --COOH groups per gram of carrier, said polymer carrier having been produced from an acrylic acid and/or methacrylic acid, or acrylic amide and/or methacrylic amide monomer, and a cross-linking agent of the acrylic or allylic type either in an aqueous solution with a carbodiimide compound that is water soluble, at a temperature of 0° to 4° C. or in an organic solvent with a carbodiimide compound that is soluble in an organic solvent, at a temperature less than 0° C; and (b) applying a cyclodextrin glycosyl transferase enzyme in an aqueous buffer solution having a pH value of 4 to 9 onto the polymer carrier to immobilize the enzyme, and washing the product.
Claim 10
Dependent Claim
22 words
The process defined in claim 9, wherein the polymer carrier in step (a) comprising functional --COOH groups is an acrylamide-N,N'-methylene-bis-acrylamide-acrylic acid copolymer.
Claim 11
Dependent Claim
19 words
The process defined in claim 7, wherein in step (a), the carbodiimide compound is N-cyclohexyl-N'-[2-(4-morpholinyl)-ethyl]-carbodiimide-methyl-p-toluene-sulfonate or N-ethyl-N' (3-dimethylaminopropyl)-carbodiimide hydrochloride.
Claim 12
Dependent Claim
31 words
The process defined in claim 7, wherein in step (b) the immobilization is carried out in a 0.05 to 0.1 molar sodium acetate buffer solution having a pH value of 6.5.
Claim 13
Dependent Claim
18 words
The process defined in claim 7, wherein in step (b) the immobilization is carried out for 45-55 hours.
Claim 14
Dependent Claim
36 words
The process defined in claim 7, wherein in step (b), the immobilized enzyme is first washed with a sodium acetate buffer, then with a sodium acetate buffer also containing sodium chloride, and then with distilled water.
References Cited (Backward Citations)
US4338398A
Cited
US4525457A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-06-02 | 1989-12-02 | 1990-06-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-06-02 | 1993-12-02 | 1994-06-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-06-02 | 1997-12-02 | 1998-06-03 | $7700.00 | Window Closed / Lapsed |