The overexpression system of the active pyridoxine 4-oxidase from Microbacterium luteolum was developed. When chaperonin GroEL/ES genes in plasmid pKY206 were coexpressed, the pyridoxine 4-oxidase gene cloned in the vector pTrc99A was overexpressed in Escherichia coli JM109 cultured in LB medium containing 50 muM riboflavin, the precursor of coenzyme (FAD) of the enzyme, under the cold stress at 23 degreesC. The crude extract from the cotransformant cells showed 88-fold higher specific activity than that from M. luteolum. The chaperonins, cold stress, and the riboflavin cooperatively served to increase the soluble form of the enzyme. A significant correlation between the specific activity and percentage of the soluble form in the total expressed enzyme was found. The overexpressed pyridoxine 4-oxidase was easily purified to homogeneity with two steps of the conventional column chromatography. Published by Elsevier Inc.