Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.
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.
Sojo
Mar M
MM
Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Murcia, Spain. msojo@um.es
Bru
Roque R
RR
García-Carmona
Francisco F
FF
D002789:Cholesterol Oxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry; D005285:Fermentation; D007218:Industrial Microbiology / Q000191:economics / Q000379:methods; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology*
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D002789:Cholesterol Oxidase