Subcloning and Partial Purification of Perchlorate Reductase from Dechloromonas aromatica RCB

The chlorooxide anions form a class of highly water-soluble, toxic species. Perchlorate reductase from Dechloromonas aromatica RCB is responsible for the enzymatic reduction of perchlorate to chlorate and chlorate to chlorite in two, two electron transfers.

Two heterologous expression systems for perchlorate reductase were constructed in this work. One system was based on the expression of active nitrate reductase and the second system utilized polycistronic coexpression. Gel documentation of enzymatic digests indicated successful molecular subcloning of both constructs. However, the lack of IPTG-inducible overexpression of the above constructs supported the supposition that the presence of several rare codons halted DNA replication. This finding suggested the use of an E .coli strain genetically modified to replicate such rare codons is necessary for the desired overexpression to occur.

The experimentally confirmed high isoelectic point of said enzyme (8.20-9.6) led to the construction of a three-part purification scheme. Utilizing DEAE (diethylaminoethyl) reverse anion exchange as a first step resulted in an increase of perchlorate reductase specific activity from 0.0759 U/mg to 0.218 U/mg. While further downstream steps (Carboxymethyl (CM) Sepharose Fast Flow and Sephacryl S-200 High Resolution gel filtration) were able to increase specific activity to 2.29 U/mg, total activity decreased over the course of purification and complete separation from cellular proteins (mainly chlorite dismutase) was not achieved. Thus, a summary of the partial purification achieved is presented rather than the complete purification of perchlorate reductase from D. aromtica.