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US4035237A
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Method for the preparation of cholesterol oxidase
Assignee: Eastman Kodak Co
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Filing: 1975-11-07
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Granted: 1977-07-12
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Expires: 1995-11-07
Abstract
Abstract A method for preparing cholesterol oxidase, comprising the steps of: A. growing a cholesterol oxidase producing microorganism in a medium comprising a nontoxic concentration of nonionic surfactant to yield extracellular cholesterol oxidase; and B. separating the extracellular cholesterol oxidase from the medium. Preferably, cholesterol oxidase is prepared from a growth medium of Nocardia cholesterolicum species NRRL 5767 or 5768 which contains cholesterol, a suitable derivative of cholesterol or a 3-β-hydroxy sterol as a cholesterol oxidase inducer and a nonionic surfactant in a concentration which is not toxic to said bacterium, which does not produce decomposition products in concentrations sufficient to be toxic to said bacterium, and in sufficient concentration to produce extracellular enzyme.
Inventors
- 👤 Prakash S. Masurekar
- 👤 Charles T. Goodhue
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 07:27
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/83; Y10S435/843; Y10S435/863; Y10S435/866; Y10S435/872
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
90 words
A method for the production of cholesterol oxidase comprising the steps of: growing a microorganism that normally produces predominently intracellular cholesterol oxidase in a medium comprising an inducer of cholesterol oxidase and a nonionic surfactant in a concentration which is not toxic to said microorganism and which does not produce decomposition products in sufficient concentrations as to be toxic to said microorganism, said surfactant being present in a concentration of at least about 0.5 g/liter of medium, thereby producing extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 2
Independent Claim
90 words
A method for the production of cholesterol oxidase comprising the steps of: growing a microorganism that normally produces predominently intracellular cholesterol oxidase in a medium comprising an inducer of cholesterol oxidase and a nonionic surfactant in a concentration which is not toxic to said microorganism and which does not produce decomposition products in sufficient concentrations as to be toxic to said microorganism, said surfactant being present in a concentration of at least about 0.5 g/liter of medium, thereby producing extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 3
Dependent Claim
28 words
The method of claim 1 wherein said nonionic surfactant comprises a hydrophilic moiety selected from polyoxyethylene or polyglycidol and a lipophilic moiety comprising at least nine carbon atoms.
Claim 4
Dependent Claim
20 words
The method of claim 2 wherein said lipophilic moiety comprises a fatty acid chain having at least ten carbon atoms.
Claim 5
Dependent Claim
29 words
The method of claim 2 wherein said hydrophilic moiety comprises about twenty polyoxyethylene units and said lipophilic moiety comprises a fatty acid chain having at least sixteen carbon atoms.
Claim 6
Dependent Claim
169 words
The method of claim 1 wherein said nonionic surfactant is selected from the group consisting of: (a) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of palmitic acid; (b) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of stearic acid; (c) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units polyoxyethylene and a lipophilic moiety consisting of one unit of oleic acid; (d) a surfactant comprising a hydrophilic moiety consisting of ten units of polyoxyethylene and a lipophilic moiety consisting of one unit of nonylphenyl; (e) a surfactant comprising a hydrophilic moiety consisting of six units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl; and (f) a surfactant comprising a hydrophilic moiety consisting of ten units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl.
Claim 7
Dependent Claim
25 words
The method of claim 1 wherein said cholesterol producing microorganism is selected from the group consisting of Nocardia cholesterolicum species NRRL 5767 and NRRL 5768.
Claim 8
Independent Claim
106 words
A method for the production of cholesterol oxidase comprising the steps of: growing a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768 in a medium which comprises: (a) an inducer of cholesterol oxidase selected from cholesterol, a derivative of cholesterol and a 3-β-hydroxy sterol, and (b) a nonionic surfactant in a concentration which is not toxic to said bacterium and which does not produce decomposition products in sufficient concentrations as to be toxic to said bacterium wherein said nonionic surfactant is present in a concentration of at least 0.5 g/liter, thereby producing extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 9
Independent Claim
106 words
A method for the production of cholesterol oxidase comprising the steps of: growing a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768 in a medium which comprises: (a) an inducer of cholesterol oxidase selected from cholesterol, a derivative of cholesterol and a 3-β-hydroxy sterol, and (b) a nonionic surfactant in a concentration which is not toxic to said bacterium and which does not produce decomposition products in sufficient concentrations as to be toxic to said bacterium wherein said nonionic surfactant is present in a concentration of at least 0.5 g/liter, thereby producing extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 10
Dependent Claim
28 words
The method of claim 7 wherein said nonionic surfactant comprises a hydrophilic moiety selected from polyoxyethylene or polyglycidol and a lipophilic moiety comprising at least nine carbon atoms.
Claim 11
Dependent Claim
169 words
The method of claim 7 wherein said nonionic surfactant is selected from the group consisting of: (a) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of palmitic acid; (b) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of stearic acid; (c) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units polyoxyethylene and a lipophilic moiety consisting of one unit of oleic acid; (d) a surfactant comprising a hydrophilic moiety consisting of ten units of polyoxyethylene and a lipophilic moiety consisting of one unit of nonylphenyl; (e) a surfactant comprising a hydrophilic moiety consisting of six units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl; and (f) a surfactant comprising a hydrophilic moiety consisting of ten units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl.
Claim 12
Independent Claim
120 words
A method for the production of cholesterol oxidase comprising the steps of: inoculating a medium with an inoculum culture of a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768, said medium comprising: (i) yeast extract, (ii) an inducer of cholesterol oxidase selected from the group consisting of cholesterol, β-sitosterol, ground mixed soy sterols, 5α-cholestan-3β-ol, cholest-4-en-3-one, and cholesterol esters, and (iii) a nonionic surfactant in a concentration which is not toxic to said bacterium and which does not produce decomposition products in sufficient concentrations as to be toxic to said bacterium, said surfactant concentration being at least 0.5 g/liter; growing the bacterium in the medium thereby yielding extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 13
Independent Claim
120 words
A method for the production of cholesterol oxidase comprising the steps of: inoculating a medium with an inoculum culture of a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768, said medium comprising: (i) yeast extract, (ii) an inducer of cholesterol oxidase selected from the group consisting of cholesterol, β-sitosterol, ground mixed soy sterols, 5α-cholestan-3β-ol, cholest-4-en-3-one, and cholesterol esters, and (iii) a nonionic surfactant in a concentration which is not toxic to said bacterium and which does not produce decomposition products in sufficient concentrations as to be toxic to said bacterium, said surfactant concentration being at least 0.5 g/liter; growing the bacterium in the medium thereby yielding extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 14
Dependent Claim
22 words
The method of claim 10 wherein the growing step includes oxygenating the medium with at least 0.6 volume of air/volume of medium/minute.
Claim 15
Dependent Claim
28 words
The method of claim 10 wherein said nonionic surfactant comprises a hydrophilic moiety selected from polyoxyethylene or polyglycidol and a lipophilic moiety comprising at least nine carbon atoms.
Claim 16
Dependent Claim
170 words
The method of claim 10 wherein said nonionic surfactant is selected from the group consisting of: (a) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of palmitic acid; one (b) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of stearic acid; (c) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units polyoxyethylene and a lipophilic moiety consisting of one unit of oleic acid; (d) a surfactant comprising a hydrophilic moiety consisting of ten units of polyoxyethylene and a lipophilic moiety consisting of one unit of nonylphenyl; (e) a surfactant comprising a hydrophilic moiety consisting of six units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl; and (f) a surfactant comprising a hydrophilic moiety consisting of ten units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl.
Claim 17
Independent Claim
260 words
A method for the production of cholesterol oxidase comprising the steps of: growing a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768 in a medium comprising: (i) at least about 8 g/liter of Nutrient broth, (ii) about 1.0 to about 5.0 g/liter of yeast extract, (iii) about 0.5 to about 5.0 g/liter of an inducer of cholesterol oxidase selected from the group consisting of cholesterol, cholesterol linoleate, cholesterol oleate, cholesterol hexonate, and cholesterol propionate, and (iv) at least about 0.5 g/liter of a nonionic surfactant selected from the group consisting of: (a) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of palmitic acid; (b) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of stearic acid; (c) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units polyoxyethylene and a lipophilic moiety consisting of one unit of oleic acid; (d) a surfactant comprising a hydrophilic moiety consisting of ten units of polyoxyethylene and a lipophilic moiety consisting of one unit of nonylphenyl; (e) a surfactant comprising a hydrophilic moiety consisting of six units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl; and (f) a surfactant comprising a hydrophilic moiety consisting of ten units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl, thereby yielding extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
Claim 18
Independent Claim
260 words
A method for the production of cholesterol oxidase comprising the steps of: growing a bacterium selected from Nocardia cholesterolicum species NRRL 5767 and NRRL 5768 in a medium comprising: (i) at least about 8 g/liter of Nutrient broth, (ii) about 1.0 to about 5.0 g/liter of yeast extract, (iii) about 0.5 to about 5.0 g/liter of an inducer of cholesterol oxidase selected from the group consisting of cholesterol, cholesterol linoleate, cholesterol oleate, cholesterol hexonate, and cholesterol propionate, and (iv) at least about 0.5 g/liter of a nonionic surfactant selected from the group consisting of: (a) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of palmitic acid; (b) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units of polyoxyethylene and a lipophilic moiety consisting of one unit of stearic acid; (c) a surfactant comprising a hydrophilic moiety consisting of one unit sorbitan and 20 units polyoxyethylene and a lipophilic moiety consisting of one unit of oleic acid; (d) a surfactant comprising a hydrophilic moiety consisting of ten units of polyoxyethylene and a lipophilic moiety consisting of one unit of nonylphenyl; (e) a surfactant comprising a hydrophilic moiety consisting of six units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl; and (f) a surfactant comprising a hydrophilic moiety consisting of ten units of polyglycidol and a lipophilic moiety consisting of one unit of nonylphenyl, thereby yielding extracellular cholesterol oxidase; and separating the cholesterol oxidase from the medium.
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) | 1980-07-11 | 1981-01-10 | 1981-07-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-07-11 | 1985-01-10 | 1985-07-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-07-11 | 1989-01-10 | 1989-07-12 | $7700.00 | Window Closed / Lapsed |