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-.beta.-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.

Abstract: An improved fermentation method comprises growing Nocardia cholesterolicum in a medium comprising a carbon source, such as glycerol, yeast extract, a nonionic surfactant, an auxiliary carbon source which is also an inducer of cholesterol oxidase, and trace salts. Yields of cholesterol oxidase up to about 900 U. (international units) per liter have been obtained using the improved method of the present invention in contrast with the yields of 1-3 U. per liter obtained in the prior art. Proper selection of environmental conditions also enables the coproduction of cholesterol esterase.

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; andB. 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-.beta.-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.

Abstract: An integral analytical element for analysis of liquids for their cholesterol content is disclosed. The element is of the type which comprises at least two superposed layers including a spreading layer and a reagent layer in fluid contact and, optionally, a support. Cholesterol oxidase and a composition for the hydrolysis of cholesterol esters comprising lipase having cholesterol esterase activity and protease are included in the element such that cholesterol esters contained in a sample applied to the spreading layer are saponified to free cholesterol and free cholesterol is decomposed in the presence of cholesterol oxidase to produce a detectable change related to the total cholesterol content of the sample.