Specificity of Rhodococcus opacus 1CP cells' responses to benzoate and 3-chlorobenzoate.

Background: Halogenated aromatic compounds are more resistant to microbial degradation than non-halogenated aromatic compounds. Microbial degradation of sodium benzoate in the presence of sodium 3-chlorobenzoate is of interest. The ability to degrade aromatic compounds is largely determined by the substrate specificity of the first enzyme that initiates degradation, namely, benzoate 1,2-dioxygenase for benzoate degradation, and 3-chlorobenzoate 1,2-dioxygenase for 3-chlorobenzoate degradation. In this study, the perspective of immobilized cells of Rhodococcus opacus 1CP actinobacterium for degradation of benzoate and 3-chlorobenzoate was explored.

Methods: The biosensor approach (a membrane microbial sensor based on immobilized cells of Rhodococcus opacus 1CP and the Clark-type oxygen electrode as a transducer) was applied to evaluate the actinobacterial cells' responses to benzoate and 3-chlorobenzoate in the absence of both enzymes, benzoate 1,2-dioxygenase and 3-chlorobenzoate 1,2-dioxygenase, or in the presence of one of the said enzymes.

Results: Data obtained show that 1CP actinobacterium possessed a constitutive system for the transport of benzoate and 3-chlorobenzoate into culture cells. The affinity of the transport system for benzoate was higher than that for 3-chlorobenzoate. Moreover, adaptation to one substrate did not preclude the use of the second substrate. Probably, porins facilitated the penetration of benzoate and 3-chlorobenzoate into 1CP cells. Analyzing V vs. S dependencies, negative cooperativity was found, when benzoate 1,2-dioxygenase bound substrate (3-chlorobenzoate), while positive cooperativity was determined at benzoate binding. The observed difference could be associated with the presence of at least two systems of 3-chlorobenzoate transport into actinobacterial cells and allosteric interaction of active sites of benzoate 1,2-dioxygenase in the presence of 3-chlorobenzoate.

Conclusions: The membrane microbial sensor based on immobilized Rhodococcus opacus 1CP cells could be useful as a perspective tool for comparative evaluation of enzymes of complex structure such as benzoate- and 3-chlorobenzoate 1,2-dioxygenase.