Eastman-kodak Company

Prakash S Masurekar

Charles T Goodhue

Theodore W Esders

A urease-free creatinine iminohydrolase enzyme preparation obtained from an aerobic soil microorganism. The enzyme of the preparation preferably has a molecular weight of from about 250,000 to 300,000; a maximum activity at a pH between 7 and 8 as measured at 37.degree. C.; a K.sub.m of about 3 to 5 mM for creatinine as measured at 37.degree. C., pH 7.5; and a specific activity for creatinine of at least about 1.0 unit per milligram of protein in the preparation as measured at 37.degree. C., pH 7.5. The preferred enzyme preparation is derived from the aerobic soil microorganism ATCC 31,546. Assay methods, compositions, and elements containing the aforementioned urease-free creatinine iminohydrolase for the determination of creatinine in an aqueous liquid are also disclosed.

Creatinine iminohydrolase free from urease activity

A fermentation process and improved aqueous nutrient medium is used for the production of urease-free creatinine iminohydrolase from an aerobic soil microorganism. In the process, a fresh sample of the microorganism grown on a creatinine-containing maintenance medium is transferred to a microbial growth medium to grow the microorganism, the growing microorganism is then transferred to a production medium to produce microorganism in which creatinine iminohydrolase production has been induced, and the desired enzyme is then extracted from the microorganism. An improved aqueous nutrient medium for use as the aforementioned production medium is disclosed.

Process and nutrient medium for growing microorganism

An improved process for producing .alpha.-glycerophosphate oxidase provides yields of more than 1500 U per liter. The enzyme is produced by growing a member of the family Lactobacillaceae in a medium comprising pyruvate and an inducer for .alpha.-glycerophosphate oxidase. In the preferred embodiment, a medium comprising a mixture of glucose and pyruvate as carbon sources provides a synergistic effect on the production of .alpha.-glycerophosphate oxidase.

Process for the production of .alpha.-glycerophosphate oxidase

An improved fermentation method comprises growing a cholesterol oxidase-producing microorganism 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.

Method for producing cholesterol oxidase in the presence of a nonionic

A method for preparing cholesterol oxidase, comprising the steps of:

Method for the preparation of cholesterol oxidase

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.

Production of cholesterol esterase using Nocardia cholesterolicum

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.

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Webster, NY, United States of America

Prakash S Masurekar