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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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Characterization of the R. erythropolis fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates.
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Figure 1: Characterization of the R. erythropolis fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates.

Mentions: Results from a typical batch fermentation are shown in Figure 1. Three stages can be differentiated during the fermentation process, (i) A first stage (0–16 h), in which 02 consumption increases continuously and HCl is consumed to keep the pH constant to 6.75. Buckland et al. [12] found that pH of the culture rose by 0.5 units in the first part of growth, and then fell. An exponential increase of cell mass is observed and low levels of COX activity appear linked to cells, (ii) The second stage (16–45 h) is characterized by a strong 02 consumption and a consumption of base. In this phase aerobic metabolism drives the cell growth but the growth rate is certainly limited by the O2 availability – note pO2 is nearly zero under continuous stirring and air supply-. This stage is also characterized by a high rate of COX production of both types, cell-linked and extracellular, (iii) The third stage (45 h to the end of the process) a second phase of consumption of acid was recorded whereas pO2 increased again to reach saturating levels. The greatest increase of cell-linked COX production was observed in this stage whilst extracellular COX production stopped.


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)

Characterization of the R. erythropolis fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Characterization of the R. erythropolis fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates.
Mentions: Results from a typical batch fermentation are shown in Figure 1. Three stages can be differentiated during the fermentation process, (i) A first stage (0–16 h), in which 02 consumption increases continuously and HCl is consumed to keep the pH constant to 6.75. Buckland et al. [12] found that pH of the culture rose by 0.5 units in the first part of growth, and then fell. An exponential increase of cell mass is observed and low levels of COX activity appear linked to cells, (ii) The second stage (16–45 h) is characterized by a strong 02 consumption and a consumption of base. In this phase aerobic metabolism drives the cell growth but the growth rate is certainly limited by the O2 availability – note pO2 is nearly zero under continuous stirring and air supply-. This stage is also characterized by a high rate of COX production of both types, cell-linked and extracellular, (iii) The third stage (45 h to the end of the process) a second phase of consumption of acid was recorded whereas pO2 increased again to reach saturating levels. The greatest increase of cell-linked COX production was observed in this stage whilst extracellular COX production stopped.

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