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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

Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.
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Figure 3: Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.

Mentions: The cell-free extract of Triton X-114 was subjected to phase separation as such. The culture broth was first supplemented with Triton X-114, well dissolved at 4°C and then warmed up to induce detergent phase separation. Figures 2a and 3a show the distribution of enzyme activity in each phase (detergent-depleted and -rich) for each of the COX sources (cells extract and culture broth) respectively.


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)

Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.
Mentions: The cell-free extract of Triton X-114 was subjected to phase separation as such. The culture broth was first supplemented with Triton X-114, well dissolved at 4°C and then warmed up to induce detergent phase separation. Figures 2a and 3a show the distribution of enzyme activity in each phase (detergent-depleted and -rich) for each of the COX sources (cells extract and culture broth) respectively.

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