Overexpression and purification of the recombinant 4-hydroxy-2-oxovalerate aldolase and 2-hydroxypent-2,4-dienoate hydratase

Abstract

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. In Sphingomonas sp. DJ77, the genes for the lower pathway were previously found to be clustered as phnDEGHIJKL and their nucleotide sequences were determined. Through a nucleotide sequence analysis, the phnJ gene is thought to encode a 4-hydroxy-2-oxovalerate aldolase and the phnH gene is thought to encode a 2-hydroxypent-2,4-dienoate hydratase. The phnJ and phnH gene were cloned into expression vectors, pET and pMAL, and expressed gene products in E. coli. The proteins expressed from the pET system were mostly insoluble but those from the pMAL system were soluble. The soluble proteins were purified by an affinity chromatography. 0The enzymatic analysis with the purified proteins indicated that the phnJ gene encodes a functional 4-hydroxy-2-oxovalerate aldolase. The assay system established in this study can be used in further modification of these enzymes through protein engineering.