Cholesterol Oxidase Production Research Articles

Optimization and kinetic analysis of cholesterol oxidase production by Rhodococcus equi no. 23 in submerged cultures

The production of cholesterol oxidase (CO; EC 1.1.3.6) by Rhodococcus equi No. 23 was carried out in a 5-l jar fermentor. A central composite experimental design and response surface methodology were employed to derive a statistical model for the effects of the initial cholesterol concentration and cultivation temperature on CO production. An initial concentration of 0.918 g/l cholesterol and a temperature at 39.4°C were found to be optimal for the final enzyme production in batch cultures. The maximum CO production, after 24-h cultivation, predicted from the model was 0.278 U/ml. In addition, kinetic analysis revealed that enzyme production could be classified as the mixed type of growth associated at the optimized conditions. Moreover, the repression or inhibition of 4-cholesten-3-one on enzyme synthesis could be reduced, and CO production increased up to 0.340 U/ml, when the cells were cultivated with a strategy of two-stage culturing temperature (at 37.4°C during the first 8 h and then at 39.4°C till the end of the fermentation) and with the initial cholesterol concentration of 0.918 g/l.

Production of cholesterol oxidase by Rhodococcus equi No. 23 in a jar fermenter.

Rhodococcus equi No. 23 was grown in a batch fermenter. The effects of cultivation temperature, pH of the culture medium, aeration rate and agitation speed on the production of cholesterol oxidase (CholOx) by the test organism were examined. Results revealed that the cultivation temperature, the pH of the medium, the aeration rate and the agitation speed all affected the production of CholOx by R. equi No. 23. Adjusting the operation variables during the cultivation period increased the production of CholOx effectively and prevented the occurrence of overflow of foam during the fermentation period. A maximum CholOx activity of 0.34 unit/ml with a volumetric production rate of 0.011 unit/h per ml could be achieved in 30 h of cultivation at an aeration rate of 5.0 l/min, if the pH of the culture medium, the cultivation temperature and the agitation speed were controlled at 6.5, 39 degrees C and 200 rev. /min respectively during the first 24 h of cultivation, then shifted to 7.5, 37 degrees C and 300 rev./min respectively.

Maximization of cholesterol oxidase production by Rhodococcus equi no. 23 By using response surface methodology.

Medium optimization for the production of cholesterol oxidase (EC 1. 1.3.6) by Rhodococcus equi no. 23 was investigated by using response surface methodology and a central composite design. Results revealed that cholesterol and yeast extract had positive effects and the interaction between any two of three factors had no significant effect on cholesterol oxidase production. The optimized medium was the basal medium with the addition of 2.30 g/l cholesterol, 8.18 g/l yeast extract and 4.10 ml/l Tween 80. The peak cholesterol oxidase production (0.242 unit/ml) after 60-72 h cultivation was approx. 4-fold that in control medium.

Medium improvement by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi No. 23

Medium improvement for the production of cholesterol oxidase (CO, EC 1.1.3.6) by Rhodococcus equi No. 23 was investigated using an orthogonal array design in two steps. Results revealed that yeast extract, Tween 80 and zinc sulphate had positive effects on CO production, but magnesium sulphate had an inhibitory effect. In addition, interaction between cholesterol and sodium chloride also had a significant effect on enzyme production. The improved medium consisted of 2·0 g/litre cholesterol, 8·0 g/litre yeast extract, 1·0 g/litre NH 4Cl, 1·0 g/litre NaCl, 0·50 g/litre KH 2PO 4, 0·25 g/litre Na 2HPO 4, 0·10 g/litre l-valine, 0·15 g/litre l-tyrosine, 0·15 g/litre MgSO 4·7H 2O, 0·01 g/litre ZnSO 4·7H 2O, 0·10 g/litre FeSO 4·7H 2O and 4·0 ml/litre Tween 80. CO production at 60 h (about 0·24 units/ml) was about four-fold greater than with the control medium.

Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli.

A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P(lac)-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 alpha than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0 +/- 0.3) x 10(-3) U/mg and (16.0 +/- 1.0) x 10(-3) U/mg protein equivalent of cell extract in the absence and presence of isopropyl beta-D-thiogalactopyranoside, respectively. The presence of a counter-oriented P(lac) at the 3' end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.

Native Promoter-Plasmid Vector System for Heterologous Cholesterol Oxidase Synthesis in Streptococcus thermophilus

The cholesterol oxidase gene ( choA) of a streptomycete was used as a model for studying heterologous gene expression in Streptococcus thermophilus, an essential bacterium in dairy food fermentations. The vectors pER82 and pER82P were developed from the 2.2-kb indigenous plasmid (pER8) of S. thermophilus ST108, and sP1, a 51-bp synthetic promoter patterned after a chromosomal sequence of S. thermophilus. The presence of sP1 promoter in pER82PbCOb with the choA insert aligned with the cat gene was essential for the intracellular production of cholesterol oxidase. The pER82PbCOb was apparently stable in S. thermophilus with no detectable evidence of deletion mutational events.

An improved method for the entrapment of microbial cells into a thermogel

An improved method was elaborated for the entrapment ofStreptomyces iverini andRhodococcus sp. cells into agar gel using organic solvents as gelation media. Criteria were for the organic solvents the relatively low water-solubility, medium high viscosity, density lower than 1 and 2<logP<4. 2-Ethyl-hexanol proved to be most suitable. The procedure was applied for the “batch” production of mithramycin and cholesterol oxidase.

Incidence of familial hypercholesterolaemia in premature deaths due to coronary heart disease.

Cholesterol estimations were performed on blood collected postmortem from a group of subjects coming to autopsy and showing that they had had a high risk for hypercholesterolaemia, and from an unselected group of subjects dying in hospital. Subjects from the first group had apparently been healthy, aged less than 45 years, had had no known risk factors for hyperlipidaemia, and showed extensive coronary artery atheroma (stenosis greater than 50% by diameter). Eleven cases from 485 consecutive autopsies fulfilled these criteria; three showed considerable hypercholesterolaemia (11.2, 11.8, and 21.6 mmol/L). Family studies confirmed the diagnosis of familial hypercholesterolaemia in one case; the other two remain unproven. Cholesterol measurement by cholesterol oxidase and quinoneimine dye production is subject to interference by haemolysis; provided that serum haemoglobin is less than 200 mg/dl, the cholesterol underestimate is less than 5%. The decline in serum cholesterol in the group of unselected subjects was 1.7 (0.3-4.9) mmol/L, 50.4 (28-84) h postmortem. Results are means and ranges for seven subjects. Measurement of cholesterol in serum obtained postmortem (provided that the sample is not grossly haemolysed) is a valid approximation of antemortem levels: this measurement should be made when autopsy reveals evidence of premature coronary heart disease. If hypercholesterolaemia is discovered, the diagnosis of familial hypercholesterolaemia, a common genetic disorder inherited in an autosomal dominant fashion, should be considered and appropriate family studies instituted.

Characterization of production of cholesterol oxidases in three Rhodococcus strains

The production of cholesterol oxidase in two strains of Rhodococcus equi No. 23 from butter, and Rhodococcus sp. No. 33 from bacon, which had properties on biochemical and physiological tests almost similar to the strains of R. equi, was compared with that of the type strain (ATCC 6939) of R. equi. The intensity of cholesterol oxidase activity, both extracellular and membrane-bound, from the three strains was in the order No. 23, ATCC 6939 and No. 33. More extracellular enzyme was produced by strain No. 23 than by the other two strains. Halo formation on the agar medium containing cholesterol depended on the conversion of cholesterol to 4-cholesten-3-one by the extracellular cholesterol oxidase.

The kinetics of cholesterol oxidase synthesis by Nocardia rhodocrous.

The production of cholesterol oxidase by 3 liter batch cultures of Nocardia rhodocrous growing on a glycerol/yeast extract medium was investigated. Cholesterol was shown to be a good inducer of the enzyme. The optimum time for cholesterol addition and the quantity to be added were determined, resulting in a 15-fold yield increase. Cholesterol oxidase synthesis was influenced by the dissolved oxygen tension. Maximum cholesterol oxidase production was obtained at 30-40% air saturation. The effect of growth conditions on the extraction of cholesterol oxidase by Triton X-100 was investigated. The scale-up of the fermentation to 800 liters in a pilot-plant fermenter is described.