US4042461A
Expired / Lapsed
US
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Method for purifying cholesterol esterase
Assignee: Genzyme Corp
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Filing: 1976-09-10
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Granted: 1977-08-16
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Expires: 1996-09-10
Abstract
Abstract Cholesterol esterase is purified by removal of the separable lipase in an enzyme preparation containing both cholesterol esterase and lipase activities by treatment using a hydrophobic chromatography column comprising carboxylated polysaccharide material having lower alkylamine with less than 8 carbon atoms attached thereto.
Inventors
- 👤 Theodore Walter Esders
- 👤 James Robert Schaeffer
- 👤 Harry Wayne Harris
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 15:29
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/16; C12N9/18; C12N9/20; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/8215; Y10S435/822; Y10S435/826; Y10S435/872; Y10S435/886; Y10S435/893; Y10S435/911; Y10S435/921; Y10S435/922
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
56 words
A method for purifying a microbial enzyme preparation having separable cholesterol esterase and lipase activities, said method comprising: treating the enzyme preparation by hydrophobic chromatography using carboxylated polysaccharide material having attached thereto lower alkylamine with less than 8 carbon atoms to remove the separable lipase from the enzyme preparation to yield a purified cholesterol esterase preparation.
Claim 2
Independent Claim
56 words
A method for purifying a microbial enzyme preparation having separable cholesterol esterase and lipase activities, said method comprising: treating the enzyme preparation by hydrophobic chromatography using carboxylated polysaccharide material having attached thereto lower alkylamine with less than 8 carbon atoms to remove the separable lipase from the enzyme preparation to yield a purified cholesterol esterase preparation.
Claim 3
Independent Claim
59 words
A method for purifying a microbial cholesterol esterase preparation having separable lipase activity, said method comprising: a. treating the cholesterol esterase preparation by hydrophobic chromatography using a column comprising carboxylated polysaccharide material having attached thereto lower alkylamine with less than 8 carbon atoms; and b. eluting cholesterol esterase separately from the separable lipase activity using a stepwise salt gradient.
Claim 4
Independent Claim
59 words
A method for purifying a microbial cholesterol esterase preparation having separable lipase activity, said method comprising: a. treating the cholesterol esterase preparation by hydrophobic chromatography using a column comprising carboxylated polysaccharide material having attached thereto lower alkylamine with less than 8 carbon atoms; and b. eluting cholesterol esterase separately from the separable lipase activity using a stepwise salt gradient.
Claim 5
Dependent Claim
23 words
The method as described in claim 2 wherein the carboxylated polysaccharide material is cyanogen bromide-activated agarose having a free carboxyl-containing group attached thereto.
Claim 6
Dependent Claim
25 words
The method as described in claim 2 wherein the carboxylated polysaccharide material comprises the reaction product of a cyanogen bromide-activated polysaccharide and an aminoalkanoic acid.
Claim 7
Dependent Claim
20 words
The method as described in claim 4 wherein the aminoalkanoic acid has from about 4 to about 30 carbon atoms.
Claim 8
Dependent Claim
14 words
The method as described in claim 4 wherein the aminoalkanoic acid is aminohexanoic acid.
Claim 9
Dependent Claim
12 words
The method as described in claim 2 wherein the alkylamine is butylamine.
Claim 10
Independent Claim
76 words
A method for purifying a microbial cholesterol esterase preparation by removing separable lipase activity, said method comprising: a. applying the cholesterol esterase preparation to a hydrophobic chromatography column comprising a carbodiimide reaction product of (1) alkylamine having less than 8 carbon atoms and (2) cyanogen bromide-activated agarose having free carboxyl-containing group attached thereto; and b. eluting the cholesterol esterase with a stepwise salt gradient to remove separable lipase activity thereby obtaining a purified cholesterol esterase preparation.
Claim 11
Independent Claim
76 words
A method for purifying a microbial cholesterol esterase preparation by removing separable lipase activity, said method comprising: a. applying the cholesterol esterase preparation to a hydrophobic chromatography column comprising a carbodiimide reaction product of (1) alkylamine having less than 8 carbon atoms and (2) cyanogen bromide-activated agarose having free carboxyl-containing group attached thereto; and b. eluting the cholesterol esterase with a stepwise salt gradient to remove separable lipase activity thereby obtaining a purified cholesterol esterase preparation.
Claim 12
Dependent Claim
25 words
The method as described in claim 8 wherein the alkylamine is butylamine and the aminoalkanoic acid has from about 4 to about 30 carbon atoms.
Claim 13
Independent Claim
75 words
A method for removing separable lipase from an enzyme preparation having cholesterol esterase activity obtained by growing Candida rugosa, said method comprising: a. applying the enzyme preparation to a hydrophobic chromatography column comprising a reaction product of (1) butylamine and (2) cyanogen bromide-activated agarose having aminohexanoic acid attached thereto; and b. eluting the cholesterol esterase activity using a stepwise salt gradient to obtain a purified enzyme preparation having cholesterol esterase activity with separable lipase removed.
Claim 14
Independent Claim
75 words
A method for removing separable lipase from an enzyme preparation having cholesterol esterase activity obtained by growing Candida rugosa, said method comprising: a. applying the enzyme preparation to a hydrophobic chromatography column comprising a reaction product of (1) butylamine and (2) cyanogen bromide-activated agarose having aminohexanoic acid attached thereto; and b. eluting the cholesterol esterase activity using a stepwise salt gradient to obtain a purified enzyme preparation having cholesterol esterase activity with separable lipase removed.
References Cited (Backward Citations)
GB1429526A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-08-15 | 1981-02-14 | 1981-08-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-08-15 | 1985-02-14 | 1985-08-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-08-15 | 1989-02-14 | 1989-08-16 | $7700.00 | Window Closed / Lapsed |