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Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.

Sojo MM, Bru RR, García-Carmona FF - BMC Biotechnol. (2002)

Bottom Line: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing.Cholesterol oxidase was found mainly in the resulting detergent-rich phase.This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. msojo@um.es

ABSTRACT

Background: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114.

Results: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration.

Conclusions: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

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Related in: MedlinePlus

SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band.
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Figure 4: SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band.

Mentions: The purification was made evident by running samples of COX from cells and culture broth in SDS-PAGE gels. Figure 4 shows that in both cases the detergent-rich phase was enriched in some proteins, including COX, whereas the depleted phase showed other different protein bands.


Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.

Sojo MM, Bru RR, García-Carmona FF - BMC Biotechnol. (2002)

SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101390&req=5

Figure 4: SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band.
Mentions: The purification was made evident by running samples of COX from cells and culture broth in SDS-PAGE gels. Figure 4 shows that in both cases the detergent-rich phase was enriched in some proteins, including COX, whereas the depleted phase showed other different protein bands.

Bottom Line: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing.Cholesterol oxidase was found mainly in the resulting detergent-rich phase.This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. msojo@um.es

ABSTRACT

Background: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114.

Results: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration.

Conclusions: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

Show MeSH
Related in: MedlinePlus