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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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Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.
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Figure 5: Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.

Mentions: Results are shown in Figure 5. Concerning the enzyme extracted from cells, partition coefficient reaches an optimum at 3% w/v detergent, decreasing at higher detergent concentrations. We do not have direct evidence to explain this decrease but a reason for it could be a change in the composition of the rich phase that may contain more components of the bacterial cell wall extracted at high detergent concentration, thus interfering in the partitioning of COX. In the case of the extracellular COX the higher the detergent concentration the larger the partition coefficient. As seen in table 4, the rich phase is quite more compact after phase separation in the culture broth than in the cell-extract and its relative volume is not proportional to the detergent concentration. Thus the rich phase is less hydrated, that is, more hydrophobic, with the culture broth than with the cell-extract and the more hydrophobic the higher the detergent concentration. As a result the partition coefficient for COX in the culture broth increases with the detergent concentration. Phase separation of Triton X-114 is affected by the presence of other surfactants such as Triton X-45 [28] and polyols such as glycerol [29], thus bacterial surfactants extracted during detergent treatment may affect phase separation as well. Natural surfactants may be also present in the broth but at a much lower concentration, therefore having less influence on the phase separation.


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)

Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.
Mentions: Results are shown in Figure 5. Concerning the enzyme extracted from cells, partition coefficient reaches an optimum at 3% w/v detergent, decreasing at higher detergent concentrations. We do not have direct evidence to explain this decrease but a reason for it could be a change in the composition of the rich phase that may contain more components of the bacterial cell wall extracted at high detergent concentration, thus interfering in the partitioning of COX. In the case of the extracellular COX the higher the detergent concentration the larger the partition coefficient. As seen in table 4, the rich phase is quite more compact after phase separation in the culture broth than in the cell-extract and its relative volume is not proportional to the detergent concentration. Thus the rich phase is less hydrated, that is, more hydrophobic, with the culture broth than with the cell-extract and the more hydrophobic the higher the detergent concentration. As a result the partition coefficient for COX in the culture broth increases with the detergent concentration. Phase separation of Triton X-114 is affected by the presence of other surfactants such as Triton X-45 [28] and polyols such as glycerol [29], thus bacterial surfactants extracted during detergent treatment may affect phase separation as well. Natural surfactants may be also present in the broth but at a much lower concentration, therefore having less influence on the phase separation.

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