Screening And Identification For The Strains Of Cholesterol Oxidase(COD)

Reducing cholesterol with biological measures is one of the safest ways. Recent attention has been paid to the study and application of cholesterol oxidase to decrease cholesterol in foods as well as serum cholesterol of human. The main purposes in this paper are as follow: screening the high yield stains of cholesterol oxidase from the simples which were rich of cholesterol; identifying the stains and optimizing the culture conditions of the highest yield strain of cholesterol oxidase.The strains producing cholesterol oxidase were collected from the mixture of rotten egg yolk?lard?sausage and soil.12 strains producing cholesterol oxidase were obtained by enriching and screening culture, and the activity of cholesterol oxidase were detected by colorimetric method. In order to select highest yield ones, the 12 strains were fermented at the same batches to compare the enzyme activity, two highest yield strains were obtained. One was named F-2, its activity was 1433.932U/L; the other was named SP04, its activity was 1385.0987U/L.With physiological and biochemical experiments, the F-2 was considered as bacterium. Because the DNA of the SP04 strain could not be got by the method of bacterium DNA extraction, it was estimated to be a yeast strain. The fragments of 16SrDNA and 26SrDNA were sequenced and submited to NCBI in order to get accession numbers. The F-2's accession number was HQ537785 and SP04's was HQ537786. With the sequences alignments and construction of phylogenetic tree, the F-2 was identified as Pseudomonas aeruginosa, the SP04 was identified as Pichia guilliermondii.Because there were few reports about the yeast which can produce cholesterol oxidase, meanwhile the SP04 strain was a high yield one, the culture condition of SP04 was optimized. The optimal medium was:starch maize 0.9%, yeast extract paste 1.5%, cholesterol 0.2%, tween-801%; the optimal cultrue condition was:pH 7.5, medium volume 75mL in 250mL flask, culturing 48 hours at the tempreture of 37?.Under the optimal cultivation condition, the SP04's activity of cholesterol oxidase was increased to 3510U/L, obviously higher than before.