US3907642A
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Cholesterol oxidase and method of extracting from nocardia
Assignee: National Research Development Corp UK
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Filing: 1972-09-15
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Granted: 1975-09-23
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Expires: 1992-09-15
Abstract
Abstract Enzyme preparations which will convert cholesterol to Delta 4cholestenone and hydrogen peroxide are obtained from certain Nocardia species belonging to the Mycobacterium rhodocrous group. The preparations have a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and, when in liquid form, a potency of at least 10 2 units/ml. They are prepared by growing the organism and recovering the enzyme preparation, preferably by extracting the harvested cells with a surface active agent such as Triton X-100. The enzyme preparations are used to assay for cholesterol by measuring the amount in which one of the products of the cholesterol oxidase reaction, preferably hydrogen peroxide, is formed or the quantity in which oxygen is used.
Inventors
- 👤 William Richmond
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 22:16
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; C12Q; C12Q1/00; C12Q1/26; C12Q1/60; Y; Y10; Y10S; Y10S210/00; Y10S210/918; Y10S210/922; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/8215; Y10S435/822; Y10S435/863; Y10S435/866; Y10S435/872; Y10S435/911; Y10S435/913; Y10S435/921; Y10S435/923; Y10S435/924; Y10S435/94; Y10S435/942
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
80 words
AN ENZYME PREPARATION USEFUL FOR THE ENZYMATIC DETERMINATION OF THE AMOUNT OF CHOLESTEROL IN A GIVEN SAMPLE, SAID ENZYME PREPARATION BEING DERIVED FROM NOCARDIA SPECIES NCIB 10554 (NRRL 5635) OR NCIB 10555 (NRRL 5636), BY GROWING SAID NOCARDIA SPECIES IN A SUITABLE MEDIUM AND RECOVERING THE ENZYME PREPARATION WHICH IS CHARACTERIZED BY HAVING A CHOLESTEROL OXIDASE SPECIFIC ACTIVITY OF AT LEAST 1 UNIT PER 5 MG OF PROTEIN NITROGEN AND CAPABLE OF OXIDIZING CHLOESTEROL TO $ CHOLESTENONE AND HYDROGEN PEROXIDE.
Claim 2
Dependent Claim
80 words
AN ENZYME PREPARATION USEFUL FOR THE ENZYMATIC DETERMINATION OF THE AMOUNT OF CHOLESTEROL IN A GIVEN SAMPLE, SAID ENZYME PREPARATION BEING DERIVED FROM NOCARDIA SPECIES NCIB 10554 (NRRL 5635) OR NCIB 10555 (NRRL 5636), BY GROWING SAID NOCARDIA SPECIES IN A SUITABLE MEDIUM AND RECOVERING THE ENZYME PREPARATION WHICH IS CHARACTERIZED BY HAVING A CHOLESTEROL OXIDASE SPECIFIC ACTIVITY OF AT LEAST 1 UNIT PER 5 MG OF PROTEIN NITROGEN AND CAPABLE OF OXIDIZING CHLOESTEROL TO $ CHOLESTENONE AND HYDROGEN PEROXIDE.
Claim 3
Dependent Claim
20 words
An enzyme preparation according to claim 1, in liquid form and having a potency of at least 10 2 units/ml.
Claim 4
Dependent Claim
16 words
An enzyme preparation according to claim 2 having a potency of at least 10 1 units/ml.
Claim 5
Dependent Claim
25 words
An enzyme preparation according to claim 1 having a cholesterol oxidase specific activity of at least 1 unit per 50 Mu g of protein nitrogen.
Claim 6
Dependent Claim
19 words
An enzyme preparation according to claim 4, in liquid form and having a potency of at least 0.5 unit/ml.
Claim 7
Dependent Claim
13 words
An enzyme preparation according to claim 4 in freeze-dried or other solid form.
Claim 8
Dependent Claim
24 words
An enzyme preparation according to claim 1 which has a catalase activity of less than 10 1 units per unit of cholesterol oxidase activity.
Claim 9
Dependent Claim
23 words
An enzyme preparation according to claim 7 having a catalase activity of less than 10 2 units per unit of cholesterol oxidase activity.
Claim 10
Dependent Claim
11 words
An enzyme preparation according to claim 8, containing a catalase inhibitor.
Claim 11
Dependent Claim
12 words
An enzyme preparation according to claim 7, which also contains peroxidase activity.
Claim 12
Independent Claim
75 words
A method of preparing an enzyme preparation having cholesterol oxidase activity, which comprises growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), in a suitable medium, harvesting the cells, extracting said cells with a surface active agent and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 13
Independent Claim
75 words
A method of preparing an enzyme preparation having cholesterol oxidase activity, which comprises growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), in a suitable medium, harvesting the cells, extracting said cells with a surface active agent and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 14
Dependent Claim
18 words
A method according to claim 11 in which said surface active ageNt is a non-ionic surface active agent.
Claim 15
Dependent Claim
21 words
A method according to claim 11 in which said surface active agent is iso-octyl-phenoxypoly-ethoxyethanol containing approximately 10 moles of ethylene oxide.
Claim 16
Independent Claim
69 words
A method of preparing an enzyme preparation having cholesterol oxidase activity which comprises growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRRL 5636), harvesting the cells, disrupting said cells, removing cell debris and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 17
Independent Claim
69 words
A method of preparing an enzyme preparation having cholesterol oxidase activity which comprises growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRRL 5636), harvesting the cells, disrupting said cells, removing cell debris and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 18
Independent Claim
80 words
A method of preparing an enzyme preparation having cholesterol oxidase activity, which comprises aerobically growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636) in a medium comprising glycerol as a carbon source, harvesting the cells, extracting said cells with a surface active agent and recovering an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 19
Independent Claim
80 words
A method of preparing an enzyme preparation having cholesterol oxidase activity, which comprises aerobically growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636) in a medium comprising glycerol as a carbon source, harvesting the cells, extracting said cells with a surface active agent and recovering an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and capable of oxidizing cholesterol to Delta 4 cholestenone and hydrogen peroxide.
Claim 20
Dependent Claim
18 words
A method according to claim 15 in which said medium has a pH of from 6 to 8.
Claim 21
Dependent Claim
17 words
A method according to claim 15 in which growth is carried out in the presence of cholesterol.
Claim 22
Dependent Claim
35 words
A method according to claim 15 in which after extraction of said cells with said surface active agent to produce an extract, said extract is further purified by ion exchange chromatography or substrate affinity chromatography.
Claim 23
Dependent Claim
34 words
A method according to claim 15 in which after extraction of said cells with said surface active agent to produce an extract, said extract is further purified by ion exchange chromatography on DEAE cellulose.
Claim 24
Dependent Claim
22 words
A method according to claim 18 in which said extract purified by chromatography is further concentrated by ultrafiltration or reduced pressure evaporation.
Claim 25
Dependent Claim
19 words
A method according to claim 15 which comprises converting said enzyme preparation into the freeze-dried or other solid form.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1978-09-22 | 1979-03-24 | 1979-09-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-09-22 | 1983-03-24 | 1983-09-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-09-22 | 1987-03-24 | 1987-09-23 | $7700.00 | Window Closed / Lapsed |