US4247642A
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Enzyme immobilization with pullulan gel
Assignee: Sumitomo Chemical Co Ltd
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Filing: 1978-02-16
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Granted: 1981-01-27
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Expires: 1998-02-16
Abstract
Abstract Enzymes are immobilized by a process wherein the carrier used for immobilizing is a hydrophilic pullulan gel having a three-dimensionally reticulated structure obtained by the reaction between pullulan and a bifunctional compound capable of forming an ether linkage with the hydroxyl group contained in the glucose unit of pullulan, or an ionic pullulan gel obtained by the reaction between said hydrophilic pullulan gel and a compound having at one end an ionizable group and at the other end a functional group capable of forming an ether linkage with the hydroxyl group in the pullulan gel in the presence of an alkaline coumpound. Immobilized enzymes prepared according to this process have a high activity and good retention of the activity.
Inventors
- 👤 Hideo Hirohara
- 👤 Shigeyasu Nabeshima
- 👤 Masanori Fujimoto
- 👤 Tsuneyuki Nagase
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 13:32
CPC Classifications
C; C08; C08B; C08B37/00; C08B37/0006; C08B37/0009; C08B37/0018; C12; C12N; C12N11/00; C12N11/02; C12N11/10
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
61 words
An immobilized enzyme prepared by a process which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the reactivity of hydroxyl groups in said enzyme-immobilizing carrier which is a hydrophilic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by crosslinking pullulan with epichlorohydrin.
Claim 2
Dependent Claim
61 words
An immobilized enzyme prepared by a process which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the reactivity of hydroxyl groups in said enzyme-immobilizing carrier which is a hydrophilic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by crosslinking pullulan with epichlorohydrin.
Claim 3
Dependent Claim
57 words
An immobilized enzyme according to claim 1, wherein the hydrophilic pullulan gel is obtained by dispersing an aqueous pullulan solution containing an alkaline substance in a dispersion medium which is immiscible with said aqueous solution and contains a dispersion stabilizer to form a two-phase system and adding epichlorohydrin to the two-phase system to crosslink pullulan with epichlorohydrin.
Claim 4
Dependent Claim
62 words
An immobilized enzyme according to claims 1 or 2, wherein covalently bonding of the enzyme on the enzyme-immobilizing carrier is effected by attachment by means of a triazinyl derivative using cyanuric chloride, attachment through an azide linkage, attachment by means of diazo linkage, attachment by means of a monohalogenacetyl derivative or attachment by means of the reaction with a chloride of titanium.
Claim 5
Dependent Claim
23 words
An immobilized enzyme according to claims 1 or 2, wherein the molecular weight of the pullulan is 1×10 4 to 1×10 6 .
Claim 6
Dependent Claim
21 words
An immobilized enzyme according to claim 1, wherein crosslinking of pullulan with epichlorohydrin is carried out at 10° to 70° C.
Claim 7
Dependent Claim
19 words
An immobilized enzyme according to claim 2, wherein the alkaline substance is sodium hydroxide, potassium hydroxide or calcium hydroxide.
Claim 8
Dependent Claim
145 words
An immobilized enzyme prepared by a process which comprises adsorbing an enzyme on an enzyme-immobilizing carrier by contacting an aqueous enzyme solution with said enzyme-immobilizing carrier which is an ionic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by reacting in the presence of an alkaline substance said hydrophilic pullulan gel in claims 1 or 2 with a compound represented by the formula, X.sub.1 --R--Z.sub.1 wherein X 1 is a halogen atom or ##STR5## R is a linear alkyl group having 1 to 3 carbon atoms, which may have a hydroxyl group as substituent, and Z 1 is a carboxyl group, sulfonic acid group, ##STR6## (where R 1 , R 2 and R 3 are each hydrogen, methyl group, ethyl group, hydroxyethyl group) or a salt of these compounds.
Claim 9
Dependent Claim
156 words
An immobilized enzyme prepared by a process which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the reactivity of hydroxyl groups, primary amino group, secondary amino group or carboxyl group in said enzyme-immobilizing carrier which is an ionic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by reacting in the presence of an alkaline substance said hydrophilic pullulan gel in claims 1 or 2 with a compound represented by the formula, X.sub.1 --R--Z.sub.1 wherein X 1 is a halogen atom or ##STR7## R is a linear alkyl group having 1 to 3 carbon atoms, which may have a hydroxyl group as substituent and Z 1 is a carboxyl group, sulfonic acid group, ##STR8## (wherein R 1 , R 2 and R 3 are each hydrogen, methyl group, ethyl group, hydroxyethyl group) or a salt of these compounds.
Claim 10
Dependent Claim
50 words
An immobilized enzyme according to claim 7, wherein the preparation of the ionic pullulan gel by reacting said hydrophilic pullulan gel in claims 1 or 2 with the compound represented by the formula, X 1 --R--Z 1 , is carried out in an aqueous solvent at 5° to 100° C.
Claim 11
Dependent Claim
50 words
An immobilized enzyme according to claim 8, wherein the preparation of the ionic pullulan gel by reacting said hydrophilic pullulan gel in claims 1 or 2 with the compound represented by the formula, X 1 --R--Z 1 , is carried out in an aqueous solvent at 5° to 100° C.
Claim 12
Dependent Claim
19 words
An immobilized enzyme according to claim 7, wherein the alkaline substance is sodium hydroxide, potassium hydroxide or calcium hydroxide.
Claim 13
Dependent Claim
19 words
An immobilized enzyme according to claim 8, wherein the alkaline substance is sodium hydroxide, potassium hydroxide or calcium hydroxide.
Claim 14
Dependent Claim
37 words
An immobilized enzyme according to claim 7, wherein the compound represented by the formula, X 1 --R--Z 1 , is 2-dimethylaminoethyl chloride, 2-diethylaminoethyl chloride, 3-diethylamino-1,2-epoxypropane, γ-chloro-β-hydroxypropyltrimethylammonium chloride, β,γ-epoxypropyltriethylammonium chloride, 2-chloroethylamine, monochloroacetic acid, 2-bromoethanesulfonic acid or salts thereof.
Claim 15
Dependent Claim
37 words
An immobilized enzyme according to claim 8, wherein the compound represented by the formula, X 1 --R--Z 1 , is dimethylaminoethyl chloride, 2-diethylaminoethyl chloride, 3-diethylamino-1,2-epoxypropane, γ-chloro-β-hydroxypropyltrimethylammonium chloride, β,γ-epoxypropyltriethylammonium chloride, 2-chloroethylamine, monochloroacetic acid, 2-bromoethanesulfonic acid or salts thereof.
Claim 16
Dependent Claim
31 words
An immobilized enzyme according to claim 13, wherein the compound represented by the formula, X 1 --R--Z 1 , is 2-diethylaminoethyl chloride, γ-chloro-β-hydroxypropyltrimethylammonium chloride, β,γ-epoxypropyltriethylammonium chloride, monochloroacetic acid or salts thereof.
Claim 17
Dependent Claim
31 words
An immobilized enzyme according to claim 14, wherein the compound represented by the formula, X 1 --R--Z 1 , is 2-diethylaminoethyl chloride, γ-chloro-β-hydroxypropyltrimethylammonium chloride, β,γ-epoxypropyltriethylammonium chloride, monochloroacetic acid or salts thereof.
Claim 18
Dependent Claim
61 words
An immobilized enzyme according to claim 8, wherein covalently bonding of the enzyme on the enzyme-immobilizing carrier is effected by attachment by means of triazinyl derivative by use of cyanuric chloride, attachment through an azide linkage, attachment by means of diazo linkage, attachment by means of a monohalogenacetyl derivative, attachment by use of carbodiimide derivative or attachment by use of glutaraldehyde.
Claim 19
Dependent Claim
39 words
An immobilized enzyme according to claim 1, wherein said enzyme is trypsin, chymotrypsin, lipase, microbial protease, esterase, urease, bromelain, ribonuclease, deoxyribonuclease, penicillin amidase, aminoacylase, β-galactosidase, glucose isomelase, glucose oxidase, creatine kinase, papain, invertase, pepsin, β-amylase, isoamylase, maltase or uricase.
Claim 20
Dependent Claim
39 words
An immobilized enzyme according to claim 7, wherein said enzyme is trypsin, chymotrypsin, lipase, microbial protease, esterase, urease, bromelain, ribonuclease, deoxyribonuclease, penicillin amidase, aminoacylase, β-galactosidase, glucose isomelase, glucose oxidase, creatine kinase, papain, invertase, pepsin, β-amylase, isoamylase, maltase or uricase.
Claim 21
Dependent Claim
39 words
An immobilized enzyme according to claim 8, wherein said enzyme is trypsin, chymotrypsin, lipase, microbial protease, esterase, urease, bromelain, ribonuclease, deoxyribonuclease, penicillin amidase, aminocylase, β-galactosidase, glucose isomelase, glucose oxidase, creatine kinase, papain, invertase, pepsin, β-amylase, isoamylase, maltase or uricase.
Claim 22
Dependent Claim
61 words
A process for preparing an immobilized enzyme which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the reactivity of hydroxyl groups in said enzyme-immobilizing carrier which is a hydrophilic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by crosslinking pullulan with epichlorohydrin.
Claim 23
Dependent Claim
61 words
A process for preparing an immobilized enzyme which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the reactivity of hydroxyl groups in said enzyme-immobilizing carrier which is a hydrophilic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by crosslinking pullulan with epichlorohydrin.
Claim 24
Dependent Claim
61 words
A process for preparing an immobilized enzyme according to claim 21, wherein the hydrophilic pullulan gel is obtained by dispersing an aqueous pullulan solution containing an alkaline substance in a dispersion medium which is immiscible with said aqueous solution and contains a dispersion stabilizer to form a two-phase system and adding epichlorohydrin to the two-phase system to crosslink pullulan with epichlorohydrin.
Claim 25
Dependent Claim
66 words
A process for preparing an immobilized enzyme according to claims 21 or 22, wherein covalently bonding of the enzyme on the enzyme-immobilizing carrier is effected by attachment by means of a triazinyl derivative using cyanuric chloride, attachment through an azide linkage, attachment by means of diazo linkage, attachment by means of a monohalogenacetyl derivative or attachment by means of the reaction with a chloride of titanium.
Claim 26
Dependent Claim
145 words
A process for preparing an immobilized enzyme which comprises adsorbing an enzyme on an enzyme-immobilizing carrier by contacting an aqueous enzyme solution with said enzyme-immobilizing carrier which is an ionic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by reacting in the presence of an alkaline substance said hydrophilic pullulan gel in claims 1 or 2 with a compound represented by the formula, X.sub.1 --R--Z.sub.1 wherein X 1 is a halogen atom or ##STR9## R is a linear alkyl group having 1 to 3 carbon atoms, which may have a hydroxyl group as substituent, and Z 1 is a carboxyl group, sulfonic acid group, ##STR10## (where R 1 , R 2 and R 3 are each hydrogen, methyl group, ethyl group, hydroxyethyl group) or a salt of these compounds.
Claim 27
Dependent Claim
157 words
A process for preparing an immobilized enzyme which comprises covalently bonding an enzyme on an enzyme-immobilizing carrier by utilizing the ractivity of hydroxyl groups, primary amino group, secondary amino group or carboxyl group in said enzyme-immobilizing carrier which is an ionic pullulan gel in spherical bead form of 10 to 500μ in diameter having a water regain of 1 to 50 g/g which is obtained by reacting in the presence of an alkaline substance said hydrophilic pullulan gel in claims 1 or 2 with a compound represented by the formula, X.sub.1 --R--Z.sub.1 wherein X 1 is a halogen atom or ##STR11## R is a linear alkyl group having to 1 to 3 carbon atoms, which may have a hydroxyl group as substituent and Z 1 is a carboxyl group, sulfonic acid group, ##STR12## (where R 1 , R 2 and R 3 are each hydrogen, methyl group, ethyl group, hydroxyethyl group) or a salt of these compounds.
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) | 1984-01-27 | 1984-07-28 | 1985-01-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-01-27 | 1988-07-28 | 1989-01-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-01-27 | 1992-07-28 | 1993-01-27 | $7700.00 | Window Closed / Lapsed |