Abstract

Bacterial degradation of biphenyl and some polychlorinated biphenyls is initiated by and dependent on the ability of biphenyl 2,3-dioxygenase to dihydroxylate substrate. Biphenyl 2,3-dioxygenase is a multi-component enzyme requiring reductase (BphG) and ferredoxin (BphF) components that transport electrons from NADH to the terminal dioxygenase. DNA shuffling of bphAE , using various techniques to create random fragments, resulted either in a very low yield of reassembled product and a high level of non-specific recombination, or in reassembly of wild type enzymes. Sequencing of parts of 13 randomly chosen active (8) or inactive (5) shuffled bphAE genes revealed only two recombination events. Inability to shuffle both subunits Ì and Ý together may be caused by the highly variable inter-genic region, which may not allow homologous recombination to occur between DNA templates. A second strategy was DNA shuffling of the Ý-subunit alone, as this subunit has been shown to affect enzyme specificity